There have been considerable interests in the relationship between hormone secreting endocrine cells and microvessel network patterns in human pituitary gland. However, its 3D microcirculatory networks have not been studied extensively. Therefore, this study was designed to visualize hormone secreting endcrine cells and microvessels in 3D by CLSM and to reveal relationship between hormone secretion and microvessel networks in the rat pituitary gland.

CLSM has been an exciting new apparatus because of its more powerful resolution than conventional light microscopy and the high-performance on 3D optical sectioning. The images taken with CLSM can be digitized and made susceptible to image analysis manipulation and computer-assisted 3D reconstructions.

The female normal adult Wistar rats were used in this study. To demonstrate microvessels, fluorescent probe( 30 mg/ml)-conjugated gelatin solution was injected to left ventricle of the heart After complete perfusion, the pituitary glands were resected and immediately fixed in cold graded PFA(0.5- 8 %) containing 15 % picric acid solutions (4 °C. GH, ACTH cells or S100 protein positive fcfliculostellate (FS) cells were demonstrated by indirect TRTTC-labeled or HRP labeled antibody methods.

We have previously shown that the spatial dynamics of GFP-tagged proteins can be investigated by locally enhancing the fluorescence of GFP using a short UV-laser pulse. 1 We have since developed photobleaching and photoenhancement methods to study binding parameters of GFP-tagged protein domains in intact cells. Here we present an analysis procedure which uses either photobleaching or photoenhancement measurements to determine simultaneously (1) the lateral diffusion coefficient and (2) the dissociation time constant defined for a plasma membrane associated, GFP-tagged protein. The dissociation time constant is defined as the time required for the dissociation of 63% of bound protein and is a functionally relevant parameter related to the binding strength. High affinity interactions are associated with a slow dissociation time constant and low affinity interactions with a fast dissociation time constant. The dissociation time constant of a signaling protein also imposes a critical limit on the cellular response time for signal transduction.

We have investigated the infection process of Green Fluorescence Protein marked Autographa Californica multiple nucleopolyhedrovirusCAcMNPV-GFP) under both light and electron microscopes. Enveloped virus particles were observed in the nucleus of columnar epithelial cells of the midgut 24 hours after feeding Trichoplusia ni larvae with AcMNPV-GFP infected freeze-dried larvae. by this time enveloped virus particles were also observed in the plasma membrane reticular system(PMRS) of midgut epithelial cells and nuclei of the tracheoblasts that are present in the connective tissue of the midgut. by 60 hours post feeding, tissues such as fat body and epidermis were showing enveloped virus particles in their nuclei while the the midgut epithelial cells were now largely devoid of virus.

Based on these results we propose that enveloped virions released from occlusion bodies in the lumen enter columnar epithelial cells and replicate in the nucleus. At the same time some of the virions enter the PMRS and get quick access to tracheoblasts present in the midgut connective tissue and thereafter to hemocytes present in the haemolymph.

Formation of the chick heart is initiated when paired regions of the mesoderm migrate to a specific region in the primitive head. Certain cells eventually form a pair of endocardiac tubes that become the lining of the heart, while the remaining precardiac mesoderm ultimately differentiates into the myocardium. Fibronectin is suspected as a crucial component of the migratory process. Very few investigations have been conducted on the relationship between cell motility and microgravity.

The primary objectives of this investigation were to determine the effect of altered gravity on the production and distribution of fibronectin, an extracellular matrix glycoprotein, during embryonic development of the chick heart, and to ascertain the extent to which these alterations affect myogenesis or morphogenesis. A microgravity bioreactor vessel, originally designed by NASA as a low-shear culture system, was employed to culture the cellular expiants in a simulated microgravity environment. Fertile, White-Leghorn chicken eggs were incubated at 38°C for 32 hours in a humidified egg incubator.

Transforming growth factor beta (TGFß) genes are linked to a variety of developmental processes and are the subject of in vivo and in vitro transgene research studies. We are evaluating TGFß-2 effects on mouse inner ear development, with emphasis on the cochlear duct (CD), by comparing plastic sections of intact inner ears from developmental day (D) 16.5,18.5 and 19.5 littermates with wildtype (+/+), heterozygous (+/−) and mutant (−/−) TGFß-2 genotypes as determined by polymerase chain reaction analysis of tail digests. Auditory and vestibular organs of all D16.5 mice appear similar: membranous labyrinth epithelium varies from simple cuboidal/low columnar to pseudostratified/stratified columnar. Surrounding mesenchyme varies in cell density regionally, the most cellular mesenchyme underlies areas of sensory epithelium. Sparse mesenchymal cell distribution in the vestibule and basal CD indicates sites of perilymph channel formation. The spiral and vestibular ganglia and their unmyelinated fibers are prominent. Otoconia and hair cells are present in the utricle (U) and saccule (S) maculae; hair cells are less easily identifiable in the CD.

Infertility has been reported to be a significant consequence of cryptorchidism. Testicular cancer has been increasing since the beginning of the century (2). It has been reported in men with unilateral cryptorchidism who develop cancer that 75% of the cases are in the undescended testis. In earlier reports, investigators found that almost half of the cryptorchid testis that developed testicular cancer were abdominal (3). The extent of the degenerative changes are more extensive in the abdominal testis than the inguinal testis (1). Heat shock proteins have been detected in normal as well as in heat treated testes (4, 7). It has been stated that unchecked heat shock protein leads to irreversible damage and ultimately cell death (6). Heat shock protein is used in this study as a marker. The long range objective of this study is to determine the sequence of the ultrastructural degenerative damages of the testis over specific periods in rats as a model and relate these changes to a comparable human age.

Just after neural fold fusion to form the neural tube, neural crest cells detach from the neural crest, a transient structure located in the dorsal region of the neural tube. Neural crest cells migrate and differentiate into many structures and cells. But the underlying controls of this detachment and initiation of emigration are unknown. Neural crest cells are usually not morphologically distinct from the adjacent neural epithelium (neural tube) and epidermal ectoderm (epiblast) flanking them. We are combining morphological and immunohistochemical approaches to study neural crest cells in their early stage of detachment from the neural crest.

Hamburger and Hamilton (1951) stages 9 to 12 White Leghorn chick embryos. Fixation in 2.5% glutaraldehyde - 0.5% tanic acid and postfixation in 1% osmium tetroxide. Embryos contrasted in bloc using uranyl acetate and embedded in araldite. Semithin transversal sections stained with toluidine blue for light microscopy. Ultrathin sections contrasted with lead citrate.

Somite disintegration begins with the disruption and dispersion of cells in its ventromedial region, that upon detaching from the somite form a defined group of cells called “sclerotome”. The sclerotome is formed by scleroblasts that originate paraxial segments. Scleroblasts disperse and migrate forming the “sclerotomal mesenchyma”. This sclerotomal mesenchyma, in its evolution and cell differentiation, will give rise to skeletal tissues. One of the first features of the scleroblast is the production of extracellular matrix rich in hialuronic acid that facilitates the spreading of filopodia and the migration of the sclerotome. Sclerotome development is closely associated to the microenvironment created by the extracellular matrix in which scleroblasts are located. Hialuronic acid is found in the ample intercellular spaces occupied by the hydrated matrix.

White Leghorn chick embryos at H.H. stages 9 to 12 were fixed in 2.5% glutaraldehyde, postfixed in 1 % osmium tetroxide and embedded in araldite. Semithin transversal sections were stained with toluidine blue and visualized under the light microscope to locate the sclerotome.

Previously, we have shown the apparent development of synapses utilizing immunohistochemistry (IHC) for synapse-associated proteins in the facial and hypoglossal motor nuclei of the Brazilian opossum. This study suggests that synaptogenesis is delayed in the facial motor nucleus (FMN) as compared to the hypoglossal motor nucleus (HMN). In the present study we plan to confirm and extend these findings at the electron microscopic (EM) level.

We have examined the ultrastructure of the developing FMN from animals 5 to 25 days of postnatal age (PN). The specific nuclei were identified using methylene blue stained 150 μm thick vibratome cut sections. The FMN and HMN were then dissected out for processing, guaranteeing that visualization at the EM level would be confined to areas specific to the nuclei of interest.

Initial analysis of the FMN indicates a marked increase of detectable synapses between 5 and 15PN.

Sperm cells in flowering plants are non-motile and are passive participants in their movement to the female reproductive cells. It is believed that actomyosin interaction may play a key role for sperm cell transmission in the pollen tube as well as in the embryo sac. However, indirect evidence has shown that the surface of sperm cells lacks amounts of myosin sufficient to support movement. Immunofluorescence microscopy was used in this study to further assess the presence of myosin on the surface of sperm cells of Plumbago Zeylanica.

Sperm cells of Plumbago Zeylanica were isolated according to published methods. Isolated sperm cells were blocked 20 min with 1% bovine serum albumin and 2% normal goat serum in phosphate buffered saline (PBS) (pH 7.3, in 15% sucrose), incubated 2 hrs in anti-myosin antibody (M-7648, Sigma Chemical Co., St. Louis, MO) diluted 1:10 with blocking solution, washed three times (5 min) with blocking solution and incubated 1 hr in 1:30 FITC-conjugated anti-rabbit IgG as secondary antibody (EY Labs, Inc., San Mateo, CA) in blocking solution. Samples were rinsed in PBS and mounted in an anti-fading solution with 1:1 PBS:glycerol with 3% n-propyl gallate.

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca2+ concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis suggests a role for CaR in local regulation of cellular functions. Although extracellular Ca2+ regulates the differentiation of keratinocytes, the role of CaR in the epidermis is not established. In this work using knockout mice lacking full length CaR, we sought to determine the role of CaR in epidermal differentiation.

Dorsal skin of Casr−/− knockout mice lacking full length CaR, and Casr+/+ (wild type) control mice, aged 4 to 7 days after birth was fixed in 4% formaldehyde in PBS, and in 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M cacodylate buffer. The samples were embedded in paraffin (for immunohistochemistry and for in situ hybridization) or in Spurr’s or LR White resins. Digoxigenin labeled antisense and sense RNA probes for loricrin and filaggrin were used for in situ hybridization.

Decorin is the predominant proteoglycan isolated from bone of several animal species. Bone matrix decorin appears to bind transforming growth factor β (TGF-β) and enhances its bioactivity. TGF-β is stored in bone matrix in abundant amounts and modulates the synthesis of bone matrix proteins by osteoblasts. Thus it appears to play a role in regulation of bone formation during the bone remodeling process. The effect of TGF-β on decorin expression in bone cells has been evaluated in murine osteoblastic cells, but the results are divergent depending on the experimental conditions and cell types used. The present study investigated the effect of TGF-βl on the expression of decorin mRNA in two clonal rat osteoblastic cell lines with different stages of differentiation, ROS-C26 (C26) and ROS-C20 (C20); C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes; C20 is a more differentiated osteoblastic cell line.

Decorin and biglycan are the predominant proteoglycans (PGs) isolated from bone of several animal species. Previous light microscope (LM) immunohistochemical studies of fetal human bone demonstrated that osteoid and osteoblasts reacted with both biglycan and decorin antibodies, but min-eralized matrix did not; however, the precise distribution of immunostaining was not examined at the electron microscope (EM) level. The present study examines LM and EM immunolocalization of decorin and biglycan in osteoblasts and early bone matrix of developing mandible of fetal rats, using polyclonal antibodies (LF113, LF106) directed against synthetic peptides corresponding to nonho-mologous regions of the two core proteins.

Fetuses were collected at embryonic day 15-18 from pregnant Wistar rats. After aldehyde fixation, developing jaws with and without osmium post-fixation were dehydrated, and embedded in paraffin, Spurr's resin, or LR gold resin for morphological and immunohistochemical observations. Sections cut from paraffin- or LR gold resin-embedded specimens were immunostained with LF113 specific for rat decorin or LF106 specific for rat biglycan, which were kindly provided by Dr. L.W. Fisher, National Institute of Dental Research, NIH, Maryland.

Gonomery, the separate grouping of maternal and paternal chromosomes during the first few mitoses following fertilization (Fig. 1) and chromatin diminution, the fragmentation and elimination of whole chromosome regions during specific stages of early cleavage in all primordial cells of the soma, occur in several species of copepods, ascarid nematodes, ciliated protozoa, Japanese hagfish 9 and a few other invertebrates. Because of the marked loss of DNA from all presumptive somatic cells by selective chromatin diminution during the 4th cleavage division in M. edax, it was of interest to determine DNA levels in chromosomes during gonomery in anaphases preceding diminution (Fig. 1). It was also important to estimate the DNA content of sperm as an index of the relative contribution by the male genome at fertilization in this species.

Mature males and egg-carrying females were fixed in 3:1 methanol/acetic acid for 3-5 mins, swollen in 45% acetic acid for 2-3 mins, squashed, frozen in liquid N2 for coverslip removal, thawed in 2 changes of absolute ETOH and air dried.

The nature and distribution of large hyaluronate-binding proteoglycans (PGs) in early bone formation bf developing rat mandible were biochemically and immunohistochemically investigated with a monoclonal antibody (MAb) 5D5, which is derived from bovine sciera and specifically recognizes the core protein of the large PGs such as versican, neurocan and brevican, but not that of aggrecan.

The first sign of mandibular bone formation is evident in the embryonic day 15 (Day 15). At the site of initiation of bone formation, a center of ossification was seen in the close proximity to Meckel’s cartilage. At the light microscopic level, these centers contained fine strips of the intercellular matrix surrounded by osteoblasts (Fig. 1). At the EM level, these matrices contained very fine fibrillar material, numerous, osteoblastic cell processes, and vesicular structures, but did not show any typical mineralization pattern. In Day 16, clusters of needle-like crystals or bone nodules, which varied considerably in size and shape, were seen in the intercellular matrix among osteoblasts (Fig. 2).

Recently, Seiffert using light microscope (LM) immunohistochemical methods, has localized vitronectin (Vn) in the mineralized bone matrix of mature mouse long bone after demineralization with 10 % nitric acid, indicating that Vn is a specific component of bone tissue. This raises the possibility that Vn is involved in regulation of bone metabolism. The present study extends previous studies to the electron microscope (EM) level and utilizes biochemical methods to determine the distribution and nature of Vn in early bone formation of developing rat mandible with rabbit antimurine Vn IgG (antiVn).

Developing jaws of fetuses were collected at embryonic day 15-18 from pregnant Wistar rats. After al-dehyde fixation, specimens with and without osmium post-fixation were dehydrated, and embedded in paraffin, Spurr's resin, or LR gold resin for morphological and immunohistochemical observations. Sections cut from paraffin- or LR gold resin-embedded specimens were immunostained with antiVn, which was kindly provided by Dr. D. Seiffert, The Scripps Research Institute, La Jolla, California.

The field of molecular cytogenetics is presently hampered by the need for allele- and homologue-specific hybridization probes. Vast stretches of the human genome display a considerable amount of polymorphic variation between individuals. Alpha satellite DNA is a tandemly repeated sequence located at the centromeres of all primate chromosomes and is a rich source of polymorphisms. These DNA variants are phenotypically silent; they are also stable and heritable in Mendelian fashion. We have shown that we can use this genetic diversity to create homologue-specific probes using fluorescence in situ hybridization (FISH) However unlike most classical heteromorphisms, which are detected by banding techniques, our approach does not depend upon detection of gross alterations in chromosome structure, but on the basis of sequence composition. We have constructed highly-specific oligonucleotide probes that are the first reported FISH probes to discriminate between the cytogenetically indistinguishable chromosomes of normal individuals.

These sequence-specific probes have been used to follow the transmission of a single chromosome 17 through three generation families, similar to a typical polymorphic marker.

Current molecular biological approaches were developed primarily for characterization of single genes, not entire genomes, and, as such, are not ideally suited to analysis of complex traits and population-based molecular genetics. Despite rapid progress in the human genome project effort, there is little doubt that radically new conceptual approaches are needed before routine whole genome-based analyses can be undertaken by both basic research and clinical laboratories.

Physical mapping of genomes, using restriction endonucleases, has played a major role in the identification and characterizing various loci, for example, by aiding clone contig formation and by characterizing genetic lesions. Restriction maps provide precise genomic distances, unlike ordered sequence-based landmarks such as Sequence Tagged Sites (STSs), that are essential for optimizing the efficiency of sequencing efforts, and for determining the spatial relationships of specific loci. When compared to tedious hybridization-based fingerprinting approaches, ordered restriction maps offer relatively unambiguous clone characterization that is useful in contig formation, establishment of minimal tiling paths for sequencing, and preliminary characterization of sequence lesions.

Oligonucleotide probes that can be cyclized by ligation (“padlock probes”) provide a very high degree of recognition specificity. Nilsson et al. have demonstrated the used of padlock probes for the cytological localization of alphoid repeats in chromosome 12. We have been extending the use of padlock probes to the detection of single copy sequences, and with this in mind have explored the amplification of DNA circles.

We designed a primer complementary to the arbitrary backbone (non-probing) sequence of a 92-base closed circular probe oligonucleotide and investigated the kinetics of rolling circle replication. Using the highly processive, strand-displacing DNA polymerase of phage ∅29 (kindly provided by Dr. Margarita Salas, CSIC, Madrid, Spain) we demonstrated that several hundred tandem copies of the circular oligonucleotide are generated in a few minutes of incubation at 32°C. Because the amplified DNA remains hybridized to the circle in a rolling circle reaction, this method of amplification offers unique advantages for in situ gene detection since the amplified DNA can not diffuse away from the site of synthesis.

In recent years, Tyramide Signal Amplification (TSA) has gained acclaim as a very sensitive detection method for immunocytochemsitry and fluorescence in situ hybridization (FISH). To maximally exploit the great signal generation capacity of TSA in mRNA-FISH, minimizing signals emanating from non-specifically bound nucleic acid probe becomes of prime importance, because a specificity check of the signals observed in the cytoplasm is virtually impossible. We reasoned that utilization of synthetic oligonucleotides (ONTs) in stead of commonly used cDNAs or cRNAs would diminish non-specific probe binding and that direct Horse Radish Peroxidase (HRP) labelling of ONTs and TSA would enable their in situ detection.

This approach was first tested in metaphase DNA-FISH using chromosome-specific repeats as targets. Using bifunctional crosslinking chemistry and HPLC, 5’-hexylamino oligonucleotides for chromosome specific simple satellite and alphoid sequences were conjugated to HRP and purified. Following 15 - 20 min of situ hybridization of a single HRP-ONT probe to metaphase chromosomes and a direct flurochrome-tyramide detection step, such repeat targets could be visualized with high specificity and excellent signal-to noise ratio.