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Gap junctions in intestinal smooth muscle and interstitial cells of Cajal

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MICROSCOPY RESEARCH AND TECHNIQUE 47:291–301 (1999)
5TH California Microscopy Colloquium
The California State University & Northern California Society
for Microscopy
Saturday, October 2, 1999
ALEXANDER, C.G.
Department of Biology, San Francisco State University, San
Francisco CA 94132.
Parasites as Art
I began photographing parasites when I was working on shark tapeworms for my doctorate thesis at
UCLA in 1953. After I began teaching Parasitology in
1955 at San Francisco State I developed a system of
preparing color-slides using a photomicrographic outfit obtained with a grant from the National Institutes
of Health. I now have several thousand slides of prepared and stained specimens of virtually every species
of parasite from the lab studies of my class and from
my own research. I put together a show using these
slides initially to introduce the students on the first
day of class to a consideration of parasites not as something to be viewed with distaste and horror but as one
integral part of the whole of “creation,” each with its
own beauty and fascination of complexity. One cannot
help but wonder - why is such intricate design of structure developed simply to grasp and hang on to the villus of a shark’s intestine?
ANTIPA G.A.
Department of Biology, San Francisco State University, San
Francisco CA 94132.
Hi Res/Lo Tech computer imaging for the
classroom
I have found that scanning micrographs, either
prints or 35mm slides, at reasonably high resolution
(i.e., 2–5 Mb/image) provide image files that can be
“zoomed-in” during a class session. This allows the
opportunity to select parts of an image and magnify
just that section for the class. The continuity between
ALL low magnification and high magnification images
adds significantly to clarity for all students, a clarity
not generally available in textbooks or standard slide
presentations. That’s the good news. Unfortunately,
most existing slides or prints were not produced with
© 1999 WILEY-LISS, INC.
that objective in mind. As a consequence, they are
either not in proper focus or with sufficient resolution
to be used in the zoomed-in mode. Of approximately
1000 slides of micrographs of three-dimensional
objects that I use routinely in classroom presentations,
perhaps as few as five (5) met the criteria for zoom
presentation; new images must be produced. One of
the most effective presentations of these images
makes use of a digital to analog converter, a standard
classroom TV (available routinely in many classrooms), and any version of Photoshop™. Photoshop™
automatically centers the feature selected to magnify
or demagnify. The TV presentation is very effective for
images but ineffective for text. TV presentation is
especially functional as an enhancement for class sizes
below thirty. A computer projector while also effective
(especially for larger classes) is seldom as convenient
or available.
BANISH, N., WEISS BIZZOCO, R. L. W., LU, M., AND
SAAVEDRA, S.
Department of Biology, San Diego State University, San Diego
CA 92182-0057.
DAPI based discovery of a new group of
extremophiles: hyperthermal acidophilic rods
We report here on the discovery of a new group of
rod shaped hyperthermal acidophiles living in nature
at temperatures of 70–80°C at pH 2–3. At pH 2 the
maximum temperature at which these organisms
were observed was 88°C. That these rods are organisms is supported by DNA specific DAPI staining and
combined phase contrast fluorescence microscopy. In
most acid hot springs examined, these rods were the
predominant organisms above 70°C. They were found
from 65–88°C, pH 2–3. Two types of rod shaped cells
exist, one with a cell wall structure that resembles the
Sulfolobus wall and others that have a multilayered
Gram negative type cell wall. 24 hour slide immersion
studies at 65–75°C showed rods were the only
attached entities, other than sulfur.
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5TH CALIFORNIA MICROSCOPY COLLOQUIUM
BARANSKI, M.1, BERDOUGO, E.1,3, SANDLER, J.S.1,3,
DARNELL, D. K.2, AND BURRUS, L.W.1,4
Department of Biology, San Francisco State University, 1600
Holloway Avenue, San Francisco CA 94132
Department of Biology, Lake Forest College, 555 N. Sheridan
Road, Lake Forest, IL 60045
3
These two authors contributed equally to the work.
4
Corresponding author: LBurrus@sfsu.edu
1
2
The dynamic expression pattern of frzb-1
suggests multiple roles in chick development
The Wnt family of secreted proteins has been shown
to have multiple roles in embryonic development. Frzb
proteins have been shown to inhibit Wnt signaling. In
order to gain a better understanding of the potential
roles of Frzb-1 in chick development, we utilized the
polymerase chain reaction (PCR) to isolate a partial
cDNA of the chick orthologue of frzb-1 and compared its
expression pattern to that of Wnt-1 and Wnt-5a, by
whole mount in situ hybridization. The earliest expression of cfrzb-1 is in cells fated to become neural ectoderm in streak stage embryos. After primitive streak
stages, cfrzb-1 expression is gradually attenuated in
the closing neural tube of the trunk and is concomitantly up-regulated in neural crest cells. Finally, cfrzb1 appears in the condensing mesenchyme of the bones
in both the limb and the trunk. Expression patterns for
Wnt-1 and Wnt-5a suggest possible antagonistic relationships between cFrzb-1 and these Wnt proteins.
BARLOW, S.B.
Electron Microscope Facility, San Diego State University, San
Diego CA 92182-4614.
Education outreach: resources for microscopy
in education
Microscopy is a powerful tool for studying and comprehending structure. It is now possible to operate several different kinds of microscopes with a desktop computer and the appropriate software, regardless of
whether the microscope is adjacent to that computer
or located at a distance. With high-speed Internet
access and the increasing sophistication of both computer hardware and software, students and
researchers can remotely access instruments not
owned by their own laboratory. However, simply making the transmission electron microscope, the scanning
electron microscope, the atomic force microscope, and
the confocal light microscope remotely accessible does
not insure the incorporation of these tools into the precollege and college academic curriculum. Several steps
can be taken toward this end. First, instructors need to
write laboratory exercises that can be incorporated
into student laboratories or daily lesson plans. These
exercises would emphasize student involvement in
planning and preparing samples to be viewed either in
local or remotely accessible instruments. Once written, these exercises would be collated and disseminated. Second, students can use computer simulations
that mimic digital microscope operation. Third, microscopists can make available collections of microscope
images that can be used as part of the lesson plan.
These three activities are being carried out by the
Microscopy Society of America sub-committee on
Education Outreach. Further descriptions of
microscopy outreach resources are available at
http://www.sci.sdsu.edu/emfacility/outreach.html
BART, K.M.1, AND BAILEY, D.G.2
Department of Biology, Hamilton College, Clinton NY 13323
Department of Geology, Hamilton College, Clinton NY 13323.
1
2
Enhancing undergraduate education through
remote operation of an SEM/ EDS system
At Hamilton College we have tried to broaden and
diversify the pool of students that are able to use the
EM facilities. We have done this by providing remote
access to the SEM/EDS system in large undergraduate courses. Recent advances in digital technologies
have provided new opportunities for dramatically
increasing the number of students exposed to SEM
and X-ray microanalytical systems. Recently, students in a 200-level Geology course (Mineralogy)
have used the system by remote operation from their
normal classroom. During class, approximately 25
students are able to examine and investigate geological samples in real time. It is one of the best ways to
teach students about the chemistry and structure of
complex, polyphase inorganic materials. The hardware and software needed to control the system from
a remote computer terminal are relatively inexpensive and simple to configure. The main requirements
are: 1) a 10 BaseT Ethernet network, 2) computer
with remote access software (e.g. LapLink™); and 3)
a data projection system. Use of the EM facilities has
expanded enormously. Most significantly, many more
students are now using the instrument for senior
research projects. We believe that remote operation
of microscopy facilities will become one of the most
efficient and effective ways to teach important scientific techniques and concepts to large numbers of
students.
BIZZOCO, R.L.W., AND DIAZ, R.G.
Molecular Biology Institute, San Diego State University, San
Diego CA 92182.
Effect of carbohydrate on the function of the
contractile vacuole in Chlamydomonas
reinhardtii
When Chlamydomonas reinhardtii 406 (mt-), a
wall-less, flagella-less mutant is incubated in low concentrations of sorbitol (10 to 70 mM), the time of the
contractile vacuole cycle (filling to emptying) is
increased. We have examined this response using both
phase-contrast and electron microscopy. In normal
control cells filling starts when small vesicles fuse into
a large vacuole. Water expulsion begins with a rapid
vesiculation of the contractile vacuole. This causes a
reduction in vacuole volume and an abrupt collapse.
During volume reduction water is somehow forced out
of the cell at the vacuole-plasma membrane juncture.
After sorbitol treatment the contractile vacuole cycle
time increases and pulsation of the two vacuoles
5TH CALIFORNIA MICROSCOPY COLLOQUIUM
becomes asynchronous, i.e. they fill at the same time.
The contractile vacuole in normal control cells has a
vectoral tendency towards a permanent attachment
site on the inner plasma membrane surface. We
observed this vectoral membrane flow during vacuole
collapse both by video microscopy and gluteraldehydeosmium tetroxide fixed cells. We present micrographs
that depict these chemically fixed cells and cells prepared by rapid freezing in liquid propane, followed by
freeze infiltration in osmium tetroxide. Cells exposed
to hypertonic medium, such as sorbitol, would ordinarily be expected to lose water and crenate as a consequence of high osmotic strength in the external
medium. These cells do not crenate. Instead, the contractile vacuole behavior becomes abnormal. Although
it is unclear why the contractile vacuole changes, we
offer a contractile vacuole pumping model.
BLAKE, D.
NASA Ames Research Center, Moffett Field CA 94035-1000.
Life on Mars, and life in the Mars meteorite.
Has the latter taken on a life of its own?
In 1996, McKay and co-authors announced the discovery of evidence of ancient life in a meteorite from
Mars. The data consist of five lines of evidence which,
while individually weak, according to the authors,
together provided compelling evidence of ancient bacterial life on Mars. Since the original proposal was
made, three of the lines of evidence have been shown
to be either inconclusive, or to have no bearing on life.
The two remaining lines of evidence are difficult to
either prove or disprove, but are, in the opinion of
many researchers, not persuasive. Regardless of the
resolution of the Mars meteorite controversy, many
believe that Mars could have harbored early life.
Indeed, the search for evidence of life is one of the central themes of NASA’s Mars exploration program.
BLINK, A. 1,2, KWONG, A.1,2, ROZENFELD, S.1,
LARGMAN, C.1, AND KOMUVES, L.G.2
Molecular Hematopoiesis Laboratory
Microscopy and Molecular Histology Laboratory, V. A. Medical
Center, UCSF, San Francisco CA 94121.
1
2
The localization of PRX2, a homeobox gene in
developing human skin
PRX2 is a homeobox protein thought to regulate
nuclear transport of interacting homeobox proteins.
Although earlier we established the spatial and temporal changes in the expression of a set of homeobox
genes during fetal skin development, the role of PRX2
in skin is not known. The aim of this investigation was
to determine PRX2 localization in developing human
skin. At 10 weeks both the fetal epidermis and periderm stained strongly for PRX2. However, at 17 weeks
the basal cells stained only weakly whereas the
suprabasal cells continued to display a strong staining
for PRX2. Moreover, whereas PRX2 clearly localized to
the cytoplasm of basal cells, both cytoplasmic and
nuclear localization was seen in suprabasal cells. The
anlangens of the forming hair follicles and cells in the
293
dermal papillae did not stain for PRX2. At 21 weeks
epidermal staining was similar to that observed at 17
weeks. In the fully formed hair follicles strong staining
was observed in the outer root sheath and the bulge.
No staining was seen in the dermal papillae and
fibroblasts. In adult skin a decreased cytoplasmic
PRX2 staining was seen in the spinous and granular
layers. However, intense staining persisted in the hair
follicles and sweat glands.
BUCHANAN, J., AND SMITH, S.J.
Molecular and Cellular Physiology, Stanford University School
of Medicine, Stanford CA 94305.
Observations on the fine structure of cell
cultures prepared by microwave processing
Microwave processing is a rapid and reliable method
used to prepare monolayer cell cultures for E. M. studies. We have studied hippocampal neurons and MDCK
cells by combining confocal and correlative electron
microscopy. Glass coverslips can be processed in less
than two hours using conventional fixatives and resin
mixtures. The ultrastructure of these cultures is similar
to bench processed tissue. Observations on early contacts between developing neurons reveal the presence of
synaptic vesicles and dendritic filopodia.
CHANG, E. AND ANTIPA, G.A.
Department of Biology, San Francisco State University, San
Francisco CA 94132.
Morphogenesis in the ciliate Conchophthirus:
structural detail of thigmotactic field
formation
The ciliature and infraciliature of the thigmotactic
field of C. curtus (Scuticociliatida) is distinctive.
Morphogenesis was studied by Antipa and
Hatzidimitriou (J. Protozool. 28:206-214). Here we
report further study by TEM and provide additional
developmental detail. Within the nine stages of morphogenesis previously described by Antipa and
Hatzidimitriou, we have distinguished two major
phases of thigmotactic field development. Phase 1
includes stages 1 through 5 during which the replication zone is formed by duplication of basal bodies within all kinetal rows. Monokinetids initially replicate to
produce dikinetids, and then trikinetids, and finally
quadkinetids. This results in a band dense with basal
bodies that encircles the cell and demarcates the presumptive proter from the opisthe. All of these new
basal bodies become ciliated. Phase 2 begins at stage
6, in which the linear arrangement of the replication
zone basal bodies becomes remodeled. On the left side
of the organism, in the area of the opisthe’s developing
thigmotactic field, there is one more round of basal
body proliferation. This produces one additional basal
body for each basal body already present and establishes a zig-zag arrangement of basal bodies characteristic of the thigmotactic field. Each new basal body
becomes ciliated at the end of stage 6, and the thigmotactic field of the opisthe is complete. The proter inherits the parental thigmotactic field.
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5TH CALIFORNIA MICROSCOPY COLLOQUIUM
CISNEROS-MORENO, Y., AND BIZZOCO, R.L.W.
Department of Biology, San Diego State University, San Diego
CA 92182-0057.
Contractile vacuole function in
Chlamydomonas
This study defines the membrane events in the contractile vacuole cycle of Chlamydomonas reinhardtii, a
single cell green alga. It provides a morphological and
temporal basis for the study of membrane fusion and
fluid transport across membranes in a cell favorable for
genetic analysis. The contractile vacuole (CV) is an
organelle that serves primarily as a pump to remove
excess water entering the cell by osmosis from dilute
solutions. It has been reported that fluid discharge
from contractile vacuoles is caused by the systolic pressure and that the rate of discharge is higher when the
vacuole size at the start of discharge (systole) is
greater. Our study stands in contrast to previously published work in Chlamydomonas which offers a fusion
model (CV-plasma membrane) for CV discharge. A loss
of both water and CV membrane is postulated to occur
as a result of CV-plasma membrane fusion. Our data
show no such loss and that CV membrane persists in
the cytoplasm throughout the pumping cycle. This provides evidence against a fusion mechanism. We also
show continuous attachment of the main CV membrane to the plasma membrane. No previous study has
shown this. This study defines the different stages of
the contractile vacuole cycle by correlating observations made by video- and electron microscopy. The
results we present indicate that the complete cycle consists of slow filling and rapid emptying and that the CV
may act as a pump, releasing water through a pore at
a plasma membrane attached site.
CONTI, N., AND ANTIPA, G.A.
Department of Biology, San Francisco State University, San
Francisco CA 94132.
Buccal ultrastructure of Conchophthirus
curtus: Implications in the arrangement of
vestibular basal body fibrillar structures
The buccal ultrastructure and infraciliature of C.
curtus has been examined by transmission electron
microscopy. Dikinetal vestibular rows have zigzag ciliated basal bodies where both have kinetodesmal fiber,
transverse fiber, transverse and postciliary microtubular ribbons. Monokinetal vestibular rows have ciliated
basal bodies each with kinetodesmal fiber, transverse
fibers, transverse and postciliary microtubular ribbons.
Three polykineties contain ciliated basal bodies in three
main kinetal rows with a fourth offset. These basal bodies have kinetodesmal and transverse fibers and postciliary microtubular ribbons. Haplokinety has zigzag
ciliated basal bodies with transverse fibers and postciliary microtubular ribbons. DKU has monokinetal unciliated basal bodies with postciliary microtubular ribbons. Oral rib ridge has three cytoplasmic layers and
membrane junctions with four and two microtubules
apiece. Cytopharynx has numerous stacked microtubular ribbons along one side. Previous studies show that
basal bodies of the locomotor region are monokinetal
and each has a kinetodesmal fiber, transverse fiber,
transverse and postciliary microtubular ribbons; those
of the thigmotactic region are dikinetal, zigzag and have
posterior basal body only with kinetodesmal fiber,
transverse fiber and postciliary microtubular ribbons.
Comparisons of dikinetal rows with these latter regions
suggest that dikinetal vestibular rows (which are
somatic in origin) probably receive some cellular signal
that makes the dikinetal rows have a characteristic
paired zigzag arrangement and imposes locomotor
infraciliature over both basal bodies.
CORTES, P., AND YOUNGBLOM, J.
Department of Biology, California State University, Stanislaus,
Turlock CA 95382.
The effect of cigarette tar extract on sister
chromatid exchanges in Leber’s Hereditary
Optic Neuropathy
Leber’s Hereditary Optic Neuropathy (LHON) is a
maternally inherited disorder that involves various
genes associated with the electron transport system in
the mitochondria. The primary clinical manifestation
associated with this condition is loss of vision, usually
bilaterally. Several environmental factors have been
implicated in exacerbating the onset of the symptoms.
Two frequently cited factors are excessive cigarette
smoking and alcohol consumption. Despite these speculations, there have been no reported systematic studies to examine the role of these factors in the LHON
disorder. In this study, we investigated the effect of
varying doses of aqueous cigarette tar (ACT) extract
on the level of sister chromatid exchanges in one
LHON patient and one control subject. The sister chromatid exchange (SCE) assay is a highly sensitive
method for detecting DNA damage to cells upon exposure to various agents. The more damaging the agent,
the higher the level of sister chromatid exchanges.
Lymphocyte cultures were prepared from whole blood
samples obtained from the LHON and control individuals. The cells were exposed to varying doses of ACT
added at the initiation of the cell cultures. To visualize
the SCEs, the chromosomes were stained with propidium iodide and analyzed with a confocal microscope.
Using the Mann-Whitney test, the results showed a
significantly higher level of SCEs in the LHON patient
following exposure to ACT as compared with the control individual. This study provides the first direct evidence that specific components of cigarette smoke
have a more deleterious effect on LHON cells than
normal cells.
DEMAREE, R.S., AND FOX, N.E.
Department of Biological Sciences, California State University,
Chico, Chico CA 95929.
One hour microwave sample preparation for
SEM.
The use of microwave-assisted sample preparation
for TEM is slowly gaining acceptance as a routine
method of tissue processing (Giberson, Demaree and
Nordhousen, 1997. J. Vet. Diagn. Invest. 9:61-67). We
5TH CALIFORNIA MICROSCOPY COLLOQUIUM
have recently reported a one hour microwave enhanced
procedure for preparing bacteria for SEM (Fox and
Demaree, 1999. Microsc. Res. Tech. 46:338–339, 1999).
We have extended these studies and now report that we
can successfully prepare eukaryotic cells for SEM in
under one hour with microwave enhancement.
Fixatives successfully employed include Parducz’ fixative, glutaraldehyde and osmium tetroxide. Either
ethanol or acetone may be used to dehydrate cells.
Drying was most rapid using hexamethyldisilazane in a
convection oven, but critical point drying can also be
used. We have prepared HeLa cells, human lymphocytes, Peranema sp., Spirostomum sp., as well as various plant and animal cells. Examination by field emission SEM at magnifications up to 100,000× revealed no
preparation artifacts.
EVANS, K. L.
Department of Biological Science, California State University,
Hayward CA 94542.
Using scanning electron microscopy to
characterize previously unreported freshwater
sponges from the California Delta
The California Delta forms an inland coastal environment and is one of the most extensive bodies of
water in California. Sponges previously unreported
were found there year round, mainly growing on the
pondweed Egeria but also on the side of certain covered marina floats. Scanning electron microscope
(S.E.M.) techniques were used to identify the most
common small gray sponges based on structure and
asexual reproductive stages. In freshwater sponges
one method of asexual reproduction is by gemmules,
which have a resistant coat containing unique gemmoscleres made of silica. Such silica spicules interfered with additional transmission electron
microscopy. Gemmosclere rotules (“teeth”) were detected, which numbered fewer than 12 and were 20mm in
length, suggesting the species Ephydatia muelleri
(Lieberkuhn). One problem with this identification is
the presence of a microsclere as part of the gemmule
coat; these are not reported to be present in this
species. A similar sponge is Spongilla, also reported in
much of North America. Invertebrates growing over
the sponge surface also were examined, indicating the
bryozoan Plumatella, growing within and upon some
sponges. The ecological role of such animals is hypothesized as patchy but active filters of very small particles, occupying a niche very different from other animals. Keywords: California Delta, freshwater sponges,
Ephydatia muelleri, gemmules, gemmoscleres, rotules,
bryozoan.
GAERTIG, J.
Department of Cellular Biology, University of Georgia, Athens
GA 30602-2607.
Molecular and microscopic analysis of
organelle assembly in Tetrahymena
Microtubules form structural frameworks of cells
and organelles which are essential for maintaining
295
shape and play major roles in cell movement, intracellular transport and cell division. Different microtubule
systems (e.g. cytoplasmic network, axonemes, centrioles, and mitotic spindle) contain MTs that have distinct lengths, spatial arrangement, and dynamics. Our
goal is to uncover the molecular mechanisms which
regulate the assembly and maintenance of distinct
microtubular organelles. The ciliated protozoan
Tetrahymena thermophila maintains at least 17 distinct microtubule structures in a single cytoplasm.
Our studies implicate two mechanisms in the assembly and maintenance of distinct microtubular systems
1) targeting of specific motor proteins to specific microtubules and 2) post-translational modifications of a
subset of microtubules. We found that kinesin-II, a
microtubular motor protein, is preferentially targeted
to cilia and plays an essential role in ciliary assembly
and maintenance. Although disruption of kinesin-II
genes gave a complex phenotype including paralysis,
cell division arrest and lethality, all these changes
appear to be induced by the loss of cilia. Distinct
microtubules are subjected to unique combinations of
several types of postranslational modifications. We
show that polyglycylation, a postranslational modification specific to cells with axonemes, is essential for
survival and required for normal functions of multiple
types of microtubules.
GRIFFITH, E.
Boston Technology Center, FEI Company, 66A Concord Street,
Wilmington MA 01887-2185.
New types of imaging and analytical data
available from an ESEM™
In an SEM, the ability to image materials without a
conductive coating is the starting point for enhancing
the level of imaging information available from the
material. Additionally, the ability to image materials
in an SEM in different states and the ability to image
materials as they change state, provides opportunities
for collecting new sample information. The
Environmental Scanning Electron Microscope
(ESEM™) has a specimen chamber operating mode
that allows for a low pressure range of up to 20 torr of
operator specified gases. These gases can be inert,
reducing, or oxidizing. Water vapor is also a gas that
can be used in ESEM™;. The use of sample stages
with temperature control, in combination with an
operator specified gas, provides for dynamic, in-situ,
sample experimentation. Examples of imaging materials wet and hot will be explored in this session.
GRIZZI, F.1, INTERNATI, C.M.2, AND DIOGUARDI, N. 3
1
Researcher of Scientific Direction, Istituto Clinico Humanitas,
Rozzano, Milano, Italy,
2
Myeloma and Transplantation Research Center, Arkansas
Cancer Research Center, UAMS, Little Rock, Arkansas,
3
Scientific Director of Istituto Clinico Humanitas, Rozzano,
Milan, Italy; President Centro Medicina Teoretica, University of
Study, Milan, Italy
Functional and morphological quantitative
evaluation of human myeloma dendritic cells
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5TH CALIFORNIA MICROSCOPY COLLOQUIUM
Dendritic cells are considered the most important
professional antigen presenting cells, distinguished by
their potency and ability to initiate primary T cell
responses in vitro and in vivo. The most common characteristic of these cells is the irregular morphology
which is strictly linked to their immunological function. Despite the qualitative visual perception of this
morphological complexity, is not available a quantitative method for measuring the irregular shape of these
cells for comparing this quality with the specific
immunological function of these cells. In fact, the dendritiform morphology cannot be correctly measured by
conventional Euclidean geometry which is limited to
regular structures, practically unknown in nature.
Instead, natural forms can be measured by the fractal
dimension, which represents the space-filling property
of an irregular object. In this paper, we describe a fractal system, for the quantitative analysis of structural
changes during dendritic cell differentiation from
blood monocytes, obtained by human myeloma blood.
Fractal dimension was compared with the expression
of MHC surface antigens and adhesion/costimulation
molecules. Moreover, our study was undertaken to
clarify the morphometric estimations by an image
analysis system, of various cytological features that
characterize these cells. The fractal and morphometric
analyses provided data (p < 0.0005) which suggest
that the early development of irregular dendritiform
morphology reflects high modifications in immunological properties of the cell. Furthermore, the study of
the fractal properties of dendritic cells is likely to
reveal more about its morphology as well as the kinetics of expression of several immunologically important
surfaces antigens during the monocyte differentiation
pathway to dendritic cells.
INGMIRE, P.D., LALLY, D., AND HE, Z.H.
Department of Biology, San Francisco State University, San
Francisco CA 94132.
Tissue and cellular characterization of cell
Wall-Associated receptor protein Kinases (WAK)
in transgenic plants
The plant cell wall plays a dynamic role and functions not only as a mechanical structure but as an
important component in signaling pathways as well.
The members of a family of transmembrane proteins,
called cell Wall-Associated receptor protein Kinases
(WAK), are tightly associated with the cell wall and
play a critical role in mediating signals between the
extracellular environment and the cytoplasm. The five
members of the WAK family exist as a highly conserved
gene family clustered together as a 30 kb region on
chromosome one in Arabidopsis thaliana. They are
characterized as having a cytoplasmic serine/threonine
kinase which shares high homology among the members, a single transmembrane spanning region, and
extracellular domains unique to each of the members
that could serve to bind ligands specific for each receptor. Genetic manipulation of members of the WAK fam-
ily results in severe morphological alterations in the
plant. Transgenic plants show stunted growth,
advanced senescence, and necrosis. Microscopic analysis of leaves stained with lactophenol trypan blue and
examined using a dissecting microscope confirms cell
death in manipulated transgenic plants. Using scanning electron microscopy (SEM), it was also shown that
manipulated dwarf plants do not show markedly fewer
cell numbers but rather reduced cell size, suggesting
that disruption of members of the WAK family affects
normal cell expansion. SEM analysis also shows developmental disruption of trichome formation on young
leaves, and reduced root hair and lateral root formation
on affected plants. This work was supported by NIHMBRS grant 2 S06 GM52588-04 and NIH-RIMI grant
(Z.-H. H).
ISAACSON, M.
Cornell University, Ithaca NY 14853.
Nanoscopy: A new paradigm for microscopy in
the next century
The last decade has seen a veritable explosion of
new microscopies aimed at the exploration of inner
space. In the past however, the tools used for this
exploration were large scale complex instruments,
scanning transmission and scanning electron microscopes for example. In fact, the smaller the object to be
explored, the larger and more complex was the instrument. This has changed with advent of scanned tip
instruments; not necessarily the complexity, but the
size scale of the “interrogator.” As we look at the next
century there will be this paradigm shift in how we go
about the exploration of inner space. No longer will the
observing tools be orders of magnitude larger than the
object to be viewed, but rather on the same order of
magnitude in size. This shift has occurred largely thru
the ability to use large scale tools to create small scale
devices. In this talk, we will explore this paradigm
shift, and review the micro observational tools we have
at hand and predict what we might have as we begin
the new millennium.
KOMUVES, L. G.
Microscopy and Molecular Histology Laboratory, University of
California, San Francisco, V. A. Medical Center (190), San
Francisco CA 94121.
Activation of peroxisome proliferator-activated
receptor-α regulates keratinocyte
differentiation
Ligands of peroxisome proliferator-activated
receptor-α (PPARα) stimulate keratinocyte differentiation and accelerate epidermal development in fetal
rat skin explants (Komuves et al., J. Invest.
Dermatol. 1998, 111: 429-433). The aim of the present
study was to determine if topically applied PPARα
ligands (clofibrate and Wy-14,643) could regulate
keratinocyte differentiation in adult epidermis.
Topical treatment with PPARα activators resulted in
a decreased epidermal thickness. The expression of
5TH CALIFORNIA MICROSCOPY COLLOQUIUM
structural proteins of spinous and granular layers
(involucrin, filaggrin, loricrin) increased following
topical treatment with PPARα activators. This treatment also decreased cell proliferation and increased
apoptosis in the epidermis. Furthermore, topical
treatment with PPARα activators resulted in rapid
normalization of both subacute (induced by repeated
barrier abrogation) and chronic (caused by essential
fatty acid deficiency) hyperproliferative skin conditions. Topical treatment with PPARα activators
resulted in a decrease in epidermal hyperplasia in
both models of hyperproliferation. Following topical
clofibrate treatment, PCNA-expressing cells were
restricted to the basal layer, similar to normal epidermis. In hyperproliferative epidermis the expression of involucrin, filaggrin, and loricrin was
decreased. Following topical treatment with PPARα
activators staining for these mRNAs and proteins
increased towards normal levels. Finally, topically
applied clofibrate also increased apoptosis. The present study demonstrates that topical PPARα activators have profound effects on gene expression, cell
proliferation and apoptosis both in normal and
hyperproliferative epidermis. This study identifies
PPARα activators as novel potential skin therapeutic
agents.
KWONG, A.1,2, ROZENFELD, S.1, LARGMAN, C.1, AND
KOMUVES, L. G.2
Molecular Hematopoiesis Laboratory
Microscopy and Molecular Histology Laboratory, V. A. Medical
Center, UCSF, San Francisco CA 94121.
1
2
Immunohistochemical localization of HoxB13
during fetal mouse development
Homeobox genes regulate embryonic development, including organogenesis. To understand the
role of HoxB13 in mouse development (E13-E17) we
analyzed HoxB13 localization by immunohistochemistry. Strong HoxB13 staining was seen in fetal epidermis but not in hair follicles and dermis.
Interestingly, the stratified keratinizing epithelium
of the tongue did not stain for HoxB13 whereas
strong staining was seen in the oral mucosa. No
HoxB13 staining was found in developing teeth.
Striated muscle cells, including skeletal muscles, the
diaphragm, the musculature of the tongue, heart
muscles stained strongly for HoxB13 whereas no
staining was seen in smooth muscle. The liver
parenchyma did not stain for HoxB13 during E1315, but was weakly positive later. The thymus and
haemopoietic tissues (liver and bone marrow) did
not stain for HoxB13. Similarly, no staining was seen
in the pancreas and in the salivary glands. In the
lung HoxB13 staining was restricted to the airways,
no staining was seen in the respiratory epithelium.
Weak staining was found in the collecting ducts in
kidney. No HoxB13 staining was observed in the
intestinal tract (stomach, small intestine, large
intestine). Whereas the ependymal cells in the
297
choroid plexus displayed intense HoxB13 staining,
no staining was seen in the central nervous system
(brain and spinal cord). Therefore, HoxB13 has complex expression pattern during organogenesis in
fetal mouse. It is mainly expressed in organs of ectodermal and mesodermal origin (such as epidemis,
striated muscles), whereas it is not present in organs
originating from the endoderm (such as the intestinal tract and digestive glands).
LAU, B.1,2,3, KWONG, A.1,2, LARGMAN, C.1, AND
KOMUVES, L.G.2
1
Molecular Hematopoiesis Laboratory, V. A. Medical Center,
UCSF, San Francisco CA 94121
2
Microscopy and Molecular Histology Laboratory, V. A. Medical
Center, UCSF, San Francisco CA 94121.
3
B. Lau (A. Lincoln High School) was supported by the UCSF
Science and Health Education Partnership Summer Internship
Program.
Double immunofluorescent staining with
tyramide-mediated signal amplification using
mouse monoclonal antibodies
Fluorescence-based immunohistochemical detection methods are essential in double-labeling studies
where information about the spatial relationship
between different proteins is sought. The feasibility of
double-labeling experiments, however, is often compromised by the limited availability of primary antibodies
raised in different species. Our goal was to determine
whether mouse monoclonal antibodies could be used
for double immunofluorescence with tyramide-mediated signal amplification (TSA). To establish doublelabeling TSA on paraffin-embedded sections, we used
monoclonal antibodies against human keratin 14 and
filaggrin. These proteins were specifically detected in
single-labeling TSA, using an HRP-conjugated antimouse IgG as a bridge reagent. In the double-labeling
experiments the antigens were detected sequentially,
with FITC- or Cy3-labeled tyramide reagents. A set of
controls established that TSA during the localization
of the first antigen did not interfere with TSA detection of the second antigen. Moreover, TSA prevented
the non-specific deposition of the bridge antibody to
the sections during the detection of the second antigen. Using this technique we were able to identify a
novel cell in dysplastic epidermis of basal cell carcinomas. These solitary cells are wedged between basal
cells and possess an unusual phenotype, expressing
both early (keratin 14) and late markers (filaggrin) of
keratinocyte differentiation.
LAUZON, C.R., AND POTTER, S. .
Department of Biological Sciences, California State University,
Hayward, Hayward CA 94546.
Nitrogen utilization by bacteria in the gut of
two tephritid species.
Tephritid flies consume a variety of microorganisms
while feeding on natural food sources such as bird
feces, plant structures, and aphid honeydew. Despite
the consumption of an array of microbial species, only
two bacterial species are consistently recovered from
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5TH CALIFORNIA MICROSCOPY COLLOQUIUM
their intestines. This selection suggests that these bacteria contribute to the welfare of the insects. Although
the exact mechanism(s) for selection of these bacterial
species remains to be defined, we present evidence
that the bacteria participate jointly in nitrogen cycling
for the fly using biochemical assays alone and coupled
with electron microscopy.
LIM, J.J., AND KOCH, R.A.
Department of Biological Science, California State University,
Fullerton, Fullerton CA 92834.
Integrins and cadherins as cell adhesion
molecules in ascidian sperm mitochondrial
translocation.
Mitochondrial translocation in the sea squirt,
Ascidia ceratodes, is the movement of the sperm mitochondrion from the head to tail during fertilization
and is an actin:myosin- dependent process that defines
sperm activation and provides the force for penetration (Lambert & Lambert, Dev. Biol. 106:307, 1984).
Activation of myosin filament formation has been previously supported by the work of Koch et al. (Dev. Biol.,
submitted). Actin polymerization, a process not well
studied in ascidian sperm, requires the appropriate
actin binding proteins and multi-protein complexes
that anchor species-selective cell adhesion molecules
to the sperm cell plasma membrane for stability and
initiation of signal transduction. These cell adhesion
molecules mediate the association of actin with egg
surface anchor proteins to form multi-protein complexes that recruit signaling molecules and activate
actin-myosin dependent motility (Porter & Hogg,
Trends in Cell Biol., 8:10, 1998). I hypothesize that
integrins, a family of transmembrane glycoproteins
that consist of noncovalent heterodimers, are present
in A. ceratodes sperm and are responsible for anchoring and initiating mitochondrial translocation during
fertilization. However, integrins have not yet been
identified in the ascidian sperm. To test this hypothesis, I plan to use fluorescently tagged and colloidal
gold tagged antibodies to detect the presence of integrin in A. ceratodes sperm using light and electron
microscopy. Preliminary evidence at the light microscope level using anti-integrin monoclonal antibodies
(Transduction Labs, #141720) supports the hypothesis. The alternative hypothesis that adhesion is via
cadherin molecules is currently being tested. Electron
microscopy will follow. (Supported by CSUF Biology;
NIH R15HD36500.)
MATEY, V.E., DEVAUX, C.A., AND WALSH, T.A.
da is similar in both sexes and formed by two large
lips. Each lip is armed with tripartite tooth, external
tooth and numerous lateral teeth. Two knob-like papillae, anterior sensory amphid, and three sensitive
porous structures are placed on the lip surface. Dilated
cuticle surrounding cephalic end forms the collarette
with two papillae. Posterior end of male and female
worms is different. Male: caudal alae is well developed
and supported by four pairs of pedunculated papillae.
Cloaca is located in the upper part of tail, number of
spicules varies. Two spicules were found in four males,
one spicule in three males and none in two males.
Seven button-like papillae are associated with cloaca,
another three pairs are situated below it. Posterior
receptors, phasmids, are placed before the second pair
of papillae. Ventral surface of the male tail shows
three different patterns of ornamentation specific for
this parasite. Female: vulva is located in the anterior
part of body. Conical posterior end bears crescent anal
opening and few phasmids.
MILLER, J.J., AND ANTIPA, G.A.
San Francisco State University, San Francisco CA 94132.
Ultrastructure of Fernandez-Galiano stained
Conchophthirus curtus
The Fernandez-Galiano ammoniacal silver carbonate method is frequently used for viewing the
macronucleus and the kinetal rows of ciliates by
means of light microscopy. Light micrographs of
Conchophthirus curtus indicate that ciliary tips, basal
bodies, kinetodesmal fibers, and the macronucleus are
stained. The DKU (a putative group of submerged
basal bodies) does not appear to stain. This could be
due to differences in the fiber system of the DKU when
compared to other kinetal rows. Transmission electron
micrographs confirm that ciliary tips, basal bodies
(particularly the transition zone and the cartwheel
region), kinetodesmal fibers, and the macronucleus are
stained. In addition, the transverse fiber appears to
pick up the stain. Staining of the transverse fiber and
the ciliary tips is a novel finding. It is possible that
some feature of the cilium confers thigmotactic ability
to C. curtus which is also responsible for the argentophilicity of the tips. However, cilia in all areas of the
cell stained in the same way. Could it be that all of the
cilia of C. curtus are potentially thigmotactic and the
density of cilia in the thigmotactic region as compared
to the other regions makes this region sticky?
MORGAN, C.L.1, LAUZON, C.R.2, AND SMITH, N. R.2
Department of Biology, San Diego State University, San Diego
CA 92182.
Department of Mathematics and Computer Science;
Department of Biological Sciences, California State University,
Hayward CA 94542.
Surface ultrastructure of parasitic nematoda Physaloptera turgida
For the first time, SEM was used to study key taxonomic characters of Physaloptera turgida Rud., 1819
(Nematoda: Spiruroidaea) from the opossums
Didelphis virginiana. 10 male and 10 female worms
from stomach of 5 infected animals trapped in San
Diego County were examined. Cephalic end of P. turgi-
Development of a standard model for remote
control of scientific instrumentation
Faculty and staff of the Microscope and Graphic
Imaging Center (MAGIC) at California State
University, Hayward (CSUH) have been involved for
several years in developing software for a standardized architecture for remote control of scientific instrumentation. Commercial software and freeware are
1
2
5TH CALIFORNIA MICROSCOPY COLLOQUIUM
available for connecting computers to control and communicate with scientific instruments, but they do not
afford the flexibility, the security, and the scalability
that custom software can offer. When we acquired our
Philips (FEI) XL-40 scanning electron microscope, the
challenge to develop a software system to gain remote
users a standardized Internet-accessible system was
undertaken. Using the remote control software developed by faculty and students in the Department of
Mathematics and Computer Science, the SEM has
been controlled locally, nationally and internationally.
The SEM has been used by sister CSU campuses, local
community colleges and high schools, and through inhome use by staff and faculty. An adjunct project
involving HPLC remote instrumentation control incorporates the same basic computer-system architectural
design. This model system can be used to control any
digital visual-output instrumentation.
MORIMUNE, M.1, 2, 3, KWONG, A.2, ROZENFELD, S.1,
LARGMAN, C.1, AND KOMUVES, L.G.2
Molecular Hematopoiesis Laboratory
Microscopy and Molecular Histology Laboratory, V. A. Medical
Center, UCSF, San Francisco CA 94121.
3
M. Morimune (A. Lincoln High School) was supported by UCSF
Science and Health Education Partnership Summer Internship
Program.
1
2
Immunohistochemical localization of HOXB4
during fetal human skin development
Our previous studies have established the spatial
and temporal changes in the expression of several
homeobox genes during fetal skin development
(Differentiation 62:33-41, 1997; J. Invest. Dermatol.
110:110-115, 1998). To expand these studies, we
report here the developmental changes in HOXB4
expression in fetal human skin. Whereas at 10
weeks only weak staining was seen in the epidermis,
at 17 weeks the basal cells strongly stained for
HOXB4. Moreover, several cells stained in the core of
the forming hair follicles. No staining was seen in
the dermal papillae. At 21 weeks, in the stratified
epidermis the basal cells (which are proliferating
cells) stained strongly for HOXB4. Moderate staining was also observed in the suprabasal cells. In the
fully formed hair follicles the cells in the outer root
sheath, in the bulge (where the epidermal stem cells
are located), and in sebaceous glands stained for
HOXB4. However, no staining was seen in the dermal papillae and fibroblasts. This expression pattern
(presence of HOXB4 in proliferating cells and in
stem cells) suggests that HOXB4 plays a role in the
regulation of cell proliferation and/or stem cell
renewal in developing fetal human epidermis.
MURPHY, J. A.
San Joaquin Delta College, Microscopy Technology Center, 5151
Pacific Ave., Stockton CA 95207. jmurphy@sjdccd.cc.ca.us.
The Challenges of Teaching Research
Microscopy at a Community College
San Joaquin Delta College has a unique 2 yr
Microscopy Certificate Program which successfully
places 95% of its students either in the job market
299
directly or as transfers to other 4 yr institutions.
Microscopy, in general, is research oriented in that it
provides tools to solve problems. The program has
advanced project courses which train the student to do
a research project from the very beginning stages of
experimental design through data collection and
analysis. There are however, many challenges for
teaching research microscopy at the community college level, e.g., library resources, how class logistics
affect experiments, equipment necessary, cost, staff,
and space required. Although research is not the mission of most Community Colleges, in order to provide
high technology skills required in today’s job market,
they need to address the elements of scientific method,
e.g., critical thinking, problem solving, organization,
and team work. Instrument based programs need
more financial and space support than traditional programs
and
absolutely
need
District
and
Administrative support at both the local and state
level minimally. In addition, funding agencies need to
re-think how they distribute funds, i.e., funding of
research training activities such as for supplies,
instrumentation, and buildings, regardless if it is a 2
yr, 4 yr, or graduate institution. We must meet these
challenges in innovative ways as in the end, it all
reflects on our society’s commitment to our young people, to make them the best that they can be. They are,
after all, our future.
PARVIN, B.1, TAYLOR, J.1, AND O’KEEFE, M.A.2
ICSD, Lawrence Berkeley Laboratory, Berkeley CA 94720
MSD, Lawrence Berkeley Laboratory, Berkeley CA 94720.
1
2
Deepview: A channel for distributed
microscopy.
The Materials Microcharacterization Collaboratory
(MMC), a pilot project of DOE2000, unites DOE user
facilities at LBNL, ANL, ORNL, the University of
Illinois and NIST. The MMC offers materials scientists
access to an on-line virtual laboratory that collectively houses the nation’s most advanced electron
microscopes, expert microscopists, and other
microcharacterization tools. As an integral part of
the MMC, we have developed a generalized
“Microscopy Channel” over the wide area network. A
microscopy channel advertises a listing of available
online microscopes, where users can seamlessly participate in an experiment, acquire expert opinions,
collect and process data, and store this information
in their electronic notebooks. This channel is a collaborative problem-solving environment (CPSE)
that allows for both synchronous and asynchronous
collaboration. Deepview, provides a layer of abstraction for controlling any type of microscope, a common
set of utilities for information management and
transaction, and the analytical capabilities needed
for online microscopy. With Deepview, we have provided remote users with collaborative access to electron microscopes in Berkeley (LBNL) and Tennessee
(ORNL) from several materials science laboratories
sited at locations distributed across the country.
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5TH CALIFORNIA MICROSCOPY COLLOQUIUM
POTTER, S.E., AND LAUZON, C.R.
Department of Biological Sciences, California State University,
Hayward, Hayward CA 94542.
Bacterial assemblages in the gut of two
tephritid species.
The true fruit flies are destructive insect pests of economically-important agricultural crops such as coffee,
citrus, and nuts. Current control methods for these
insects include the use of pesticides that affect nontarget species, pollute the environment, and are expensive.
These are incentives for researchers to find new control
methods that are species specific. In order to achieve
this, the biology of the insect must be more completely
understood. We have looked closely at the bacteria that
reside in the intestines of two tephritid pests. It has
been hypothesized that bacteria are important in the
life cycle of these insects. We have found a close association exists between the insect gut tissue and bacteria.
Bacteria attach to the peritrophic matrix and form a
biofilm. The biofilm includes packets of bacteria, an
architecture conducive to the flow of nutrients in and
the excretion of waste products out of the gut. These
data provide evidence that physical factors may be
involved in selection of bacteria within the fly gut.
SADREDDIN, A., LUONG, J., LEE, R., CARMEN, G.,
AND BRECKLER, J.
Department of Biology, San Francisco State University, San
Francisco CA 94132.
Immunolocalization of cytoskeletal elements in
retinal pigment epithelium (RPE) during
phagocytosis
RPE are phagocytic cells in the vertebrate eye that
engulf rod outer segments (ROS) that are shed with
light on-set. Phagocytosis is an actin and microtubuledependent process. Therefore we are studying actin
and microtubule cytoskeleton during RPE phagocytosis. We are particularly interested in ADF (actin
depolymerizing factor). ADF depolymerizes actin filaments in the cytoskeleton and allows them to perform
and rearrange for phagocytosis. Immunocytochemistry
is used to study the distribution of ADF using antibodies and secondary fluorescent detection methods.
In addition, we are using Rhodamine-Phalloidin staining method to localize the F-actin in the cell and compare this localization pattern to the ADF pattern. We
hope to answer the following questions: Where is ADF
more concentrated, and does it localize in areas where
phagocytosis is taking place? Does ADF co-localize
with F-actin? In order to follow microtubules during
phagocytosis, we are using anti-tubulin. We also plan
to use anti-myosin VI to see if this particular type of
myosin is involved. Two sets of experiments are in
progress. One is on Sunfish and the other is on albino
(unpigmented) rat eyes. In order to look at the presence of ADF in cells that are actively phagocytizing, we
examined different time points. We performed rat eye
dissection one hour after light on-set (8 am), at which
there is a burst of ROS shedding and most of phagocytosis is taking place. Another experiment is the late
afternoon dissection (approximately 8 hrs. after light
on-set). In our study, we are comparing and contrasting these time points in order to determine any shift in
distribution of ADF, actin, microtubules and myosin VI
during several stages of engulfment in vitro.
SMITH, N.R.
Microscope and Graphic Imaging Center (MAGIC), California
State University, Hayward CA 94542.
Teaching Microscopy at CSUH: Then and Now
Electron microscopy classes were first taught at
CSUH in 1971. The classes were small in size with a
maximum of six students for the transmission electron
microscopy course and as many as 14 for the scanning
microscopy course. Then, as now, students were
required to produce a finished project at the end of each
term. However, now rather than spending many hours
in the darkroom, students are producing images on
their computers. The development of remote microscopy
has changed our perspective on how microscopy courses
are taught and has reduced the limitations on class size.
The one instructor, one student approach to teaching
microscopy is elevated to another level and it can now
be done in a lighted room, thanks to digital microscopy.
Using telemicroscopy methods we can bring images into
classrooms on our campus and outreach to local community colleges and high schools through the Internet
thus serving many more students than in the past.
More students can be exposed to the direct experience
of all types of digital microscopy. Digital output on
TEMs and SEMs has saved students a tremendous
amount of time in producing micrographs and has
reduced costs to the facilities. Students are able to save
images on disks and output them from their homes or
schools and incorporate them into imaging enhancing
and analytical software.
THOMPSON, J.M.
Biology Department, California State University, San
Bernardino CA 92407.
Remote Microscopy - current status and future
promise
Remote microscopy can take several forms. On a simple level, it is nothing more than sending a micrograph
to a colleague. This is a technology which has existed for
years, but which limits the immediate interaction and
value to both participants. New technologies now allow
for real-time interaction between microscopists and colleagues who are at remote, often distant, sites. This
interaction can take the form of person to person communication using videoconferencing hardware and software through internet connections, and multiple remote
observers can also participate with the appropriate software. If the microscope can be controlled by a computer,
the interaction may also occur by remote control of the
microscope by the colleague at the remote site.
Limitations in existing hardware and software include
slow rates of internet transmission, low resolution
images, small screen size of images, and often a lack of
audio capabilities. In this symposium, we will discuss
the hardware and software currently available, as well
5TH CALIFORNIA MICROSCOPY COLLOQUIUM
as future developments to enable these remote interactions. Demonstrations of current remote microscopy
from several sites will also be presented.
TIFFANY, M.A.
Department of Biology, San Diego State University. San Diego
CA 92128.
Studies on the skeletal development of
Hermesinum adriaticum Zacharias, a flagellate
from the Salton Sea, California.
Hermesinum adriaticum is a rarely reported unicellular biflagellated organism of uncertain classification. Unlike silicoflagellates it has a solid internal
siliceous skeleton. It was found in cyanobacterial mats
and in the sediments of the Salton Sea, a salt lake in
California, USA. Stages of the developing skeleton
were studied with SEM and the progression from
small tetraxial daughter skeletons to complete asymmetrical adult skeletons is presented.
VANDIVER, A. AND MENON, J.
Department of Biology, William Paterson University of NJ,
Wayne NJ 07470.
Retinoic acid and apoptosis
The thyroid hormone-induced tissue remodeling
that occurs during metamorphosis is generally accomplished by the death of larval cells (via apoptosis) and
the growth and differentiation of stem cells into an
adult organ. Retinoids (include retinol, retinal, trans
and cis form of retinoic acid) have been linked to the
induction of apoptosis in several in vivo and in vitro
models. Thyroid hormone receptors and retinoic acid
receptors belong to the same family of nuclear receptors. In Xenopus laevis it is shown that thyroid hormone receptors (TRs) and retinoic acid X receptors
(RXRs) function together in vivo to regulate T3
response genes that are normally expressed during
metamorphosis. Whole tails of Xenopus laevis were
treated in vivo to regulate T3 response genes that are
normally expressed during metamorphosis. Whole
tails of Xenopus laevis were treated in vitro with
retinol palmitate (2IU/ml). The tail epidermis was
processed for scanning and transmission electron
microscopy. Under SEM, retinol treated tail epidermis
showed a loss of intercellular connections as well as
microvilli at their apical surface. Also there was pitting on epidermal surface of retinol treated tail. In
contrast, microvilli were clearly visible in epidermal
cells was observed following retinol treatment. Muscle
fibers were also affected: muscle bundles were less
numerous and more degenerated in retinol treated tissue than in the control. Role of retinoids in inducing
apoptosis will be discussed.
WRIGHTING, D., AGNEW, B. AND BRECKLER, J.
Department of Biology San Francisco State University, San
Francisco CA 94132.
Involvement of the actin cytoskeleton in the
phagocytosis of rod outer segment (ROS) disks
by retinal pigment epithelial (RPE) cells
The Retinal Pigment Epithelium (RPE) consists of
supportive cells beneath the vertebrate retina. One of
301
the primary functions of the RPE is to phagocytose
rod outer segment (ROS) disks shed daily from the
distal tips of the photoreceptors. The function of RPE
phagocytosis is critical to retinal integrity, adhesion
and survival. Striking evidence has been collected for
the role of actin cytoskeletal elements during the
process of phagocytosis and specifically during
phagocytic cup formation. The initial purpose of our
study is to directly visualize the dynamics of rapid
actin filament turnover during RPE phagocytosis.
Once G-actin addition involved in cup formation in
normal cells has been characterized, we will study
the specific roles of actin depolymerizing factor
(ADF) in this process by transfecting cells with adenoviral vectors containing mutant ADF isoforms. In
our studies, the use of the RPE-J cells, an immortalized RPE rat cell line, has proved invaluable. The
RPE-J cells possess the important aspects of RPE
morphology and function, including the ability to recognize and phagocytose ROS in vitro. We have developed an assay that incorporates exogenous fluorescent and biotinylated G-actin into RPE-J cells to
visualize actin rearrangements during phagocytosis
by both light and electron microscopy. Support for
this research provided by NIH-RIMI #RR11805 to JB
and NIH-BRIDGES to DW.
YOUNGBLOM, J.H., WILKINSON, J., AND
YOUNGBLOM, J.J./
Department of Biology, California State University, Stanislaus,
Turlock CA 95382.
Remote access confocal microscopy.
In recent years, there has been a growing interest
in the development of remote access capabilities to a
variety of microscopy systems. While certain types of
microscopes, such as electron microscopes and scanning probe microscopes have been well established for
telepresence microscopy, there have been no reports of
similar developments for the confocal microscope. At
California State University-Stanislaus, home of the
CSUPERB (California State University Program for
Education and Research in Biotechnology) Confocal
Microscope Core Facility, we have established a remote
access confocal laser scanning microscope facility that
allows users with virtually any type of computer platform to connect to our system. Our Leica TCS NT confocal system, with an interchangeable upright and
inverted microscope set up, is accessible to any authorized user via the Internet by using a free software
program called VNC (Virtual Network Computing).
Once connectivity is established, users are able to control virtually all the functions to conduct real time
image analysis and quantitative assessments of their
specimen. The ease of connectivity to the microscope
system and the relatively rapid speed of image transfer allow accessibility of this highly sophisticated
instrument to the larger global community. A shared
resource facility of this type, without geographic constraints, helps to catalyze the interaction of individuals with diverse interests and expertise, and promotes
a center for academic collaboration and innovation.
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