Search

To investigate the association of calmodulin (CaM) with microtubules (MTs) in the mitotic apparatus (MA), the distributions of CaM and tubulin were examined in cells in which the normal spindle organization had been altered. A fluorescent CaM conjugate with tetramethylrhodamine isothiocyanate (CaM-TRITC) and a dichlorotriazinyl aminofluorescein conjugate with tubulin (tubulin-DTAF) were injected into cells that had been treated with the MT inhibitor nocodazole. With moderate nocodazole concentration (0.3 ??g/ml, 37??C, 4 h) in live cells, CaM-TRITC and tubulin-DTAF concentrated identically on or near the centrosomes and kinetochores. In serial sections of these cells, small MT segments were observed by transmission electron microscopy (TEM) in the regions where fluorescent protein had concentrated. When a higher drug concentration was used (3.0 ??g/ml, 37??C, 4 h), no regions of CaM-TRITC or tubulin-DTAF localization were observed, and no MTs were observed when serial sections were examined by TEM. However, following release from the high-concentration nocodazole block, CaM-TRITC colocalized with newly formed MTs at the kinetochores and centrosomes. Later in the recovery period, when chromosome-to-pole fibers had formed, CaM association with kinetochores diminished, ultimately attaining its normal pole-proximal association with kinetochore MTs in cells that progressed through mitosis. We interpret these observations as supporting the hypothesis that in the MA, CaM attains a physical association with kinetochore MTs and suggest that CaM-associated MTs may be inherently more stable.

Samples of human plantar and palmar skin were excised and incubated in 20 mM EDTA after which the epidermis was gently separated from the dermis with the plane of separation occurring in the lamina lucida. Scanning electron microscopic examination of the dermal component revealed the classically described series of regularly spaced grooves and papillae that characterize the epidermal-dermal junction in thick skin. Primary dermal grooves exhibited evenly spaced tunnels that were originally occupied by sweat gland ducts. The basement membrane (basal lamina) in the primary grooves was relatively smooth but did exhibit a flattened, reticulated pattern at high magnifications. The basement membrane of secondary dermal grooves and papillae was in the form of numerous, elevated microridges off of which septae arose at roughly right angles. The surface appearance of the basement membrane in these areas was that of a honeycomb owing to the numerous compartments and recesses formed by the ridges and septae. Degradation of the basement membrane by trypsin demonstrated that the foundation for the highly folded and compartmentalized basement membrane was composed of dermal collagen fibrils, 60–70 nm in diameter, that were arranged in a series of variably sized, interconnected collagen bundles or walls. Epidermal basal cells extended cytoplasmic (foot) processes into two or more compartments, formed by the ridges and septae, which considerably amplified the basement membrane surface available for epidermal attachment. Scanning electron microscopic studies of the epidermal-dermal junction confirm the variable surface character of this interface previously reported by others using sectioned material. This regional variation in surface architecture apparently distinguishes between areas in which epidermal basal cells are specialized for attachment (papillae, secondary dermal grooves) and regions occupied by slow cycling epidermal stem cells from which mitotically active keratinocytes

The objective of this study was to determine how long myogenic cells can survive in the central ischemic zone of early free muscle grafts in the rat. The study was conducted on free grafts of a large (rectus femoris) and a small (extensor digitorum lon-gus) muscle. At times ranging from zero hr to five days post-grafting, the central zones were isolated, minced, and implanted under the back skin of mice. After five days the minces were removed and examined histologically for the presence of rat myotubes, which should form only in minces that contain viable myogenic cells. The results show that myogenic cells survive two to four hr in the ischemic centers of the large rectus femoris grafts; after longer post-grafting intervals, rat myotubes did not arise in central zone minces. In grafts of small muscles, myotubes consistently appeared in central zone minces. Since the formerly ischemic central areas of rectus muscle grafts are ultimately replaced by regenerating muscle fibers, we conclude that these regenerating muscle fibers are derived from precursor cells located outside of the ischemic zone.

Cone photoreceptors in the zebrafish retina are arranged in a crystalline lattice, with each spectral subtype at a specific position in the array: rod photoreceptors are inserted around the cones. Patterning events and developmental mechanisms that lead to the formation of the cone mosaic are not known. To begin investigating this issue, we examined the initial stages of opsin expression in zebrafish embryos by in situ hybridization with goldfish opsin cRNA probes to determine how and when the cone mosaic pattern arises. We found both differences and similarities in the spatiotemporal patterns of rod and cone development, which suggest the following: (1) Expression of opsin message (including rod opsin, blue and red cone opsins) was initiated at 50–52 hours postfertilization by a few photoreceptors which were consistently found in a ventral patch of retina located nasal to the choroid fissure. (2) The cone mosaic pattern was generated by a crystallization-like process initiated in the precocial ventral patch and secondarily in nasal retina, which then swept like a wave into dorsotemporal retina. (3) The pattern of differentiation of rods in the ventronasal patch differed substantially from that in the remainder of the retina, suggesting that these precocial rods might differ from typical rods. (4) Developmental maturation of rods in zebrafish, as reflected by expression of opsin, may be accelerated compared to cones, which are thought to become postmitotic before rods. These data are consistent with a model in which lateral inductive interactions among differentiating photoreceptors lead to patterning of the array. © 1995 Wiley-Liss, Inc.

Our radioimmunoassay data reveal that ovariectomy for either one or two months resulted in a significant decrease in the GnRH content of the mouse basal hypothalamus, while in mice ovariectomized for two weeks the GnRH levels did not differ significantly from controls. Parallel immunocytochemical analyses gave similar results. Tissue sections from specific regions of the median eminence in intact and ovariectomized mice were immunolabeled for GnRH with the peroxidase-antiperoxidase procedure of Sternberger. Differences in area and average unit density of the reaction product were measured with a Quantiment 720 image analyzer. At two weeks after ovariectomy, a majority of matched sections from intact and ovariectomized animals, taken in each sampled region of the median eminence, showed no visually apparent difference in GnRH immunoreactivity. Quantitative image analysis of these preparations revealed no statistically significant difference in either the area of neural tissue covered by deposit or the average unit density of this material in any given region. In contrast, in mice ovariectomized for either one or two months, immunolabeled GnRH was decreased throughout the cephalo-caudal extent of the median eminence. In each region, a reduction in reaction deposit was observed over most of the area in which GnRH is localized, especially in the medial aspects of this distribution. Image analysis of these preparations revealed a significant reduction of both specimen area immunolabeled for GnRH and the average unit density of reaction product in each region of the median eminence. While the decrease in density was uniform throughout the median eminence, the greatest reduction in area covered by deposit occurred in the infundibular region. The demonstration of a decrease in basal hypothalamic GnRH in the chronically ovariectomized mouse suggests that GnRH neurosecretion is increased in response to the absence of the ovaries and supports the premise that tonic secretion o

In this study, we have compared spatial and temporal aspects of development of new rods and cones in the adult goldfish by using a combination of bromodeoxyuridine immunocytochemistry and opsin in situ hybridization to determine the intervals between terminal mitosis (cell "birth") and expression of opsin mRNA for each photoreceptor cell type. The goldfish opsins include rod opsin and four different cone opsins: red, green, blue, and ultraviolet. In a cohort of photoreceptors born at the same time, rods expressed opsin mRNA within 3 days of cell birth, while expression of cone opsin mRNA required at least 7 days. This temporal discrepancy in differentiation, coupled with a discordance in the site of cell genesis of rods and cones, allowed opsin expression to commence in both cell types in approximately the same retinal location. Commitment to the generic cone phenotype occurred within approximately 6 days throughout the cone cohort, as indicated by expression of interphotoreceptor retinoid-binding protein (IRBP) mRNA, but expression of a specific spectral phenotype was delayed until rods differentiated nearby. Onset of expression of cone opsin mRNA followed a phenotype-specific sequence: red, then green, then blue, and finally ultraviolet; in situ hybridization with two opsin probes confirmed that individual photoreceptors expressed only one type of opsin as they differentiated. This stepwise process of cone differentiation is consistent with the hypothesis that cell-cell interactions among developing photoreceptors may coordinate selection of specific photoreceptor phenotypes. J. Comp. Neurol. 382:272-284, 1997. © 1997 Wiley-Liss, Inc.

Cell adhesion molecules in the cadherin family have been implicated in histogenesis and maintenance of cellular structure and function in several organs. Zebrafish have emerged as an important new developmental model, but only three zebrafish cadherin molecules have been identified to date (N-cadherin, paraxial protocadherin, and VN-cadherin). We began a systematic study to identify other zebrafish cadherins by screening zebrafish cDNA libraries using an antibody raised to the cytoplasmic domain of mouse E-cadherin. Here, we report a partial cDNA with extensive sequence homology to R-cadherin. Spatial and temporal expression of this putative zebrafish R-cadherin was examined in embryos and adults by Northern analysis, RNase protection, and in situ hybridization. R-cadherin message increased during embryogenesis up to 80 hours postfertilization (hpf) and persisted in adults. In the embryonic brain, R-cadherin was first expressed in groups of cells in the diencephalon and pretectum. In adult zebrafish brain, R-cadherin continued to be expressed in several specific regions including primary visual targets. In the retina, R-cadherin was first detected at about 33 hours postfertilization in the retinal ganglion cell layer and the inner part of the inner nuclear layer. Expression levels were highest during periods of axon outgrowth and synaptogenesis. Retrograde labeling of the optic nerve with 1,12 dioctadecyl-3,3,32 32 tetramethylindocarbocyanine perchlorate (DiI) followed by in situ hybridization confirmed that a subset of retinal ganglion cells in the embryo expressed R-cadherin message. In the adult, R-cadherin expression continued in a subpopulation of retinal ganglion cells. These results suggest that R-cadherin-mediated adhesion plays a role in development and maintenance of neuronal connections in zebrafish visual system. J. Comp. Neurol. 410:303-319, 1999. © 1999 Wiley-Liss, Inc.

The primary purpose of the present study was to determine whether a rhodopsin-like gene, which has been postulated to represent the green cone pigment in several species, is in fact expressed in cone photoreceptors instead of rods. The expression patterns of rod opsin and blue and red cone opsins were also examined in both goldfish and zebrafish retinas using colorimetric in situ hybridization. The results demonstrate that the rhodopsin-like gene is expressed in green cones, as predicted. A subset of small cones that do not hybridize with these cRNA probes are tentatively identified as ultraviolet receptors. The results also demonstrate that opsin message in cones is restricted to the perinuclear region, whereas in rods, it is both perinuclear and adjacent to the ellipsoid.

Daptomycin is a lipopeptide antibiotic with strong bactericidal effects against Gram-positive bacteria and minor side effects on skeletal muscles. The type and magnitude of the early effect of daptomycin on skeletal muscles of rats was quantified by histopathology, examination of contractile properties, Evans Blue Dye uptake, and effect on the patch repair process. A single dose of daptomycin of up to 200 mg/kg had no effect on muscle fibers. A dose of 150 mg/kg of daptomycin, twice per day for 3 days, produced a small number of myofibers (???0.22%) with loss of plasma membrane integrity and/or infiltration by neutrophils and/or macrophages. Multiple doses of daptomycin are required for a quantifiable effect on skeletal muscles of rats. Some fibers were Evans Blue Dye???positive but were not yet infiltrated by neutrophils. This suggests that the sarcolemma may be the primary target for the observed effects. Muscle Nerve, 2010

Cytomegaloviruses undergo the classical morphologic stages of replication in hedgehog submandibular gland duct cells. A profound cytomegaly is associated with the infection. These features indicate that the hedgehog may be an excellent model in which to study the biology of cytomegalovirus-salivary gland interactions.

The medial nucleus of the amygdala is important for the neural control of reproductive behavior in the adult male Syrian hamster. Two types of signals are essential for this behavior, chemosensory stimuli and gonadal steroids; these signals appear to be received in different parts of the medial nucleus. The anterior region receives input from olfactory and vomeronasal systems, both of which are required for this behavior, whereas the posterior region receives gonadal hormone inputs. Behavioral studies have also suggested a functional differentiation of these two areas; electrolytic lesions of the anterior, but not the posterior, part eliminates normal sexual behavior. In this study, the efferent projections of the anterior and posterior divisions of the medial nucleus of the amygdala in the Syrian hamster were analyzed throughout the forebrain after injections of the anterograde neuronal tracer, Phaseolus vulgaris -leucoagglutinin. Neurons of the anterior, but not the posterior, medial nucleus, were found to project to numerous olfactory bulb projection areas and to the ventral striatopallidal complex. Within areas of the chemosensory circuitry that control reproductive behavior, the anterior region of the medial nucleus projects to the intermediate part of the posterior bed nucleus of the stria terminalis and the lateral part of the medial preoptic area, whereas the posterior region of the medial nucleus projects to the medial parts of these areas. Differences in targets were also observed in the ventromedial nucleus of the hypothalamus where the anterior region projects to the core while the posterior part projects to the shell of this nucleus. Furthermore, reciprocal projections between the anterior and posterior regions of the medial nucleus were observed. Taken together, these studies support the hypothesis that the anterior and posterior regions of the medial amygdaloid nucleus provide substantially different contributions to the control of reproductive behavi

The neuroanatomical distribution of the prodynorphin precursor molecule in the forebrain of the male Syrian hamster ( Mesocricetus auratus ) has been studied with a novel antiserum directed against the C-terminus of the leumorphin [dynorphin B (1–29)] peptide product. C-peptide staining in sections from colchicine-treated hamsters is compared to staining in sections from untreated animals. In addition, the pattern of C-peptide immunostaining in hamster brain is compared to that in the rat brain. Finally, the C-peptide immunolabeling patterns in hamsters and rats are compared to those obtained with antisera to dynorphin A (1–17) and dynorphin B (1–13). Areas of heaviest prodynorphin immunoreactivity in the hamster include the hippocampal formation, lateral septum, bed nucleus of the stria terminalis, medial preoptic area, medial and central amygdaloid nuclei, ventral pallidum, substantia nigra, and numerous hypothalamic nuclei. Although this C-peptide staining pattern is similar to dynorphin staining reported previously in the rat, several species differences are apparent. Whereas moderate dentate gyrus granule cell staining and no CA4 cell staining have been reported in the rat hippocampal formation, intense immunostaining in the dentate gyrus and CA4 cell labeling are observed in the hamster. In addition, the medial preoptic area, bed nucleus of the stria terminalis, and medial nucleus of the amygdala stain lightly for prodynorphin-containing fibers and cells in the rat, compared to heavy cell and fiber staining in the hamster in all three of these regions. In the rat there is no differential staining between tissues processed with the C-peptide, dynorphin A, and dynorphin B antisera, but numerous areas of the hamster brain show striking differences. In most hamster brain areas containing prodynorphin peptides, the C-peptide antiserum immunolabels more cells and fibers than the dynorphin B antiserum, which in turn labels more cells and fibers than dynorphin A antis

This study demonstrated that distal branches of the anterior cerebral artery (ACA) are joined by interarterial anastomoses to rami of the middle cerebral artery (MCA) in the normal Wistar rat. Arteries of 36- and 56-day-old animals were dilated with papaverine and injected with Vultex. Vultex arrived at corresponding ACA and MCA collaterals simultaneously as determined by microscopy through a skull window and photography. There were about 29 ACAMCA junctions per hemisphere. Junction density was nearly constant along the frontal-occipital axis. The anastomoses were most numerous between 2 and 3 mm lateral to the midline and were less than 120 Μm in internal diameter. No significant difference was found between total numbers of junctions for right versus left hemispheres or between age groups. The most evident collateral pattern was characterized by two ACA end rami joining two MCA end branches to form a closed, diamond-shaped collateral unit. Considerations were given to alternate routes of blood flow into the MCA tissue field. We conclude abundant dorsal anastomoses exist in 36- and 56-day-old rats and are the prime potential source for ACA collateral supply to the MCA tissue field.

The relationships of mesenchymal cells to the basal lamina underlying regions of the palatal-shelf epithelium that are known to increase in cell density during shelf reorientation are quantitatively different from those of cells underlying neighboring regions that do not increase in cell density. Chlorcyclizine-induced alterations of the extracellular matrix were used to investigate the possible contribution of extracellular matrix to these differences. Chlorcyclizine causes hyaluronate and the chondroitin sulfates to be degraded into pieces with smaller molecular weights and lower charge densities, with little or no effect on their synthesis, and also results in cleft palate. Pregnant CD-1 mice were gavaged with chlorcylizine on days 10.5, 11.5, and 12.5 of gestation, and the fetuses were harvested on day 13.5. Some palatal shelves were excised immediately and fixed for electron microscopy; other heads were partially dissected and incubated for 4 hr prior to fixation. In normal heads differences in mesenchymal cell configurations are detectable after 4 hr in vitro . Electron micrographs were taken of the epithelial-mesenchymal interface in nasal and oral regions that increased in epithelial cell density and in nasal and oral regions which did not. Several variables of mesenchymal cell configuration were measured in a 500-nm-wide zone delimited on photographic prints. Chlorcyclizine-induced glycosaminoglycan alterations resulted in quantifiable, region-specific differences in mesenchymal cell relationships to the basal lamina and in the ultrastructural appearance of the zone immediately subjacent to the basal lamina. These results suggest that the epithelial-mesenchymal interface and sublaminar zone of the nasal and oral regions as well as their active and inactive segments may be constitutively different.

We have studied the shape and size distribution of membrane-bound polysomes in somatotropes and mammotropes, which are the sources, respectively, of growth hormone and of prolactin in the rat pituitary. The observations were made in conventional electron micrographs of these cells in situ , where occasional surface or en face views of the rough endoplasmic reticulum allow the polysomes to be seen as rows of ribosomes arranged in distinctive patterns on the membranes. It is possible by this means to characterize the shape and number of ribosomes for the total population of bound polysomes in the respective cell types. The great majority of membrane-bound polysomes in these two cell types (81% in somatotropes, 78% in mammotropes) have an approximately circular shape and contain an average of 6.8 (somatotropes) or 6.5 (mammotropes) ribosomes, which is an appropriate size for translation of the polypeptide hormones produced by these cells. About 17% of the membrane-bound polysomes in somatotropes and 20% in mammotropes have a spiral shape, resembling somewhat the letter “G,” and contain about eight to nine ribosomes in both cell types. The preponderance of circular polysomes on the rough endoplasmic reticulum of somatotropes and mammotropes suggests the possibility that ribosomes (or the 40S ribosomal subunit) may recycle on the polysome after the translation of growth hormone or of prolactin.

The myelin-deficient Shiverer (Shi/Shi) mutant mouse may be a useful model in assessing the dependence of brain (Na + +K +)-ATPase concentration and composition on myelin membrane formation. Brain microsomal membranes from age-matched control (+/+) and Shiverer (Shi/Shi) mice were fractionated by differential centrifugation and sucrose gradient sedimentation. No reduction in (Na + +K +)-ATPase specific activity was measured in whole homogenates, high-and low-speed fractions or gradient fractions from brains of Shi/Shi mice as compared to those of +/+ mice. In addition, sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with antisera specific for mouse brain (Na + +K +)-ATPase revealed no significant difference in catalytic subunit composition between fractions of +/+ and Shi/Shi brains. The similar results obtained for both +/+ and myelin-deficient Shi/Shi mice suggest that myelin contributes little to total brain (Na + +K +)-ATPase.

The carbonic anhydrase (CA)- like protein, CA VIII, lacks the typical carbon dioxide hydrase activity of the CA isozymes. However, the high degree of amino acid sequence similarity between the products of the mouse and the human CA VIII genes suggests an important biological function. We have attempted to investigate the function of this gene in mammalian development by conducting an in situ hybridization study on sagittal sections of mouse embryos at gestation days of 9.5--16.5 using a 35S-labelled riboprobe. Results indicate that this gene (called Car8 in mice) is expressed as early as day 9.5 in a variety of organs including liver, branchial arches, neuroepithelium and developing myocardium. Between days 10.5 and 12.5, it showed a widespread distribution of mRNA expression that became more restricted as development progressed. The level of expression of Car8 mRNA was relatively high in the brain, liver, lung, heart, gut, thymus and epithelium covering the head and the oronasal cavity

T lymphocytes, synovial macrophages, and synovial fibroblasts are the three most abundant cell populations in rheumatoid arthritis synovial tissue, and each is believed to play an important role in the pathogenesis of joint inflammation and destruction. While interactions between T cells and macrophages and between macrophages and fibroblasts have been carefully studied, less attention has been paid to potential interactions between T lymphocytes and synovial fibroblasts. In this review we consider available data which suggests that cell???cell contact between T lymphocytes and synovial fibroblasts may lead to activation of each cell type. This interaction is likely to be significant in the pathophysiology of rheumatoid arthritis.

Controlled and localized release of drugs in the intestine can be achieved by enteric coating. The design of enteric-coated tablets has so far remained empirical, in part because of the lack of a quantitative description of the drug release kinetics. In this paper, a mathematical model is presented that describes the dissolution of the polymer coating and release kinetics of weakly acidic drugs from enteric-coated tablets in buffered media. This model can also be used to predict the time of onset of core disintegration. The model assumes that the release rate is limited by diffusion, and furthermore, all the reactions are considered as reversible and instantaneous. Dissolution and reaction are assumed to take place in the polymer layer and a hypothetical stagnant liquid film adjacent to the polymer layer (the classical film theory approach). The dissolution of the enteric coating is found to depend on the intrinsic solubilities and p K a 's of the drug and polymer and the medium properties. The release rate of the drug is found to depend on the intrinsic solubilities and p K a 's of drug and polymer, the medium properties, i.e., pH and buffer capacity, and a mass transfer coefficient. Explicit relationships between the release rates and all these factors are derived. Successful prediction of experimental data indicates that the model provides an adequate description of release from enteric coated tablets. Limitations of the model and its potential application to the design of appropriate in vitro testing conditions and to the formulation of enteric coated tablets are also discussed.

In the course of making antibodies against various yeast (S. cerevisiae) proteins, we have noted that it is common to observe reactivity of rabbit sera with a number of extraneous bands on Western transfers of yeast proteins. The pattern of reactive bands can change within a period of weeks, even when the rabbit has not been injected with antigen. A simple method of affinity purification, using antigen bound to nitrocellulose, is employed to remove the reactivity with these extraneous bands from immune sera. The importance of affinity purification is demonstrated by our attempts to immunolocalize a 55 kd yeast protein (p55). Immune serum stains yeast cells to give a striking pattern of spots and blotches not seen with preimmune serum. However, affinity purification of anti-p55 antibody shows that this pattern is not due to staining by anti-p55 antibody; rather the pattern is due to staining left in the serum depleted of anti-p55 antibody.

The innervation pattern of skeletal muscles was studied in the normal and regenerating tail of Notophthalmus viridescens . Silver staining for nerve endings and histochemical localization of acetylcholinesterase (AChE) were used for light microscopy. In normal musculature, AChE positive reactions were localized at the ends of the muscle fibers where they are anchored on connective tissue septa by myotendinous junctions. At this level, silver staining shows nerve terminals forming endplates. During regeneration, positive reactions for AChE appear de novo as dense plates localized at the ends of the newly formed myotubes. The mechanisms involved in the localization of AChE on this surface seem to operate before previous local contacts by nerve terminals. From the ultrastructural data and immunohistochemical results with anti-laminin antibody, these observations suggest that regenerating muscle fibers determine a region of post-synaptic specialization in close relation with the organization of myotendinous regions and basement membrane formation. Nerve-muscle contacts appear at these levels at stage IV (15-20 days after amputation) in the stump and in the rostral part of the regenerate (transition zone). These nerve terminals are provided by the disorganized peripheral nervous system of the injured segment. In the regenerate a similar pattern of AChE reaction can be seen in every myotube, differentiating according to a rostro-caudal gradient. Innervation at the ends of the muscle fibers is in spatiotemporal relation with the exits of the ventral roots from the regenerating nerve cord as the regenerate continues to grow in length.

The bovine gene for the nuclear-encoded heart/muscle isoform of cytochrome c oxidase subunit VIa (COX6A1) was isolated from a library of bovine genomic DNA in lambda EMBL3 and sequenced. The gene spans 760 bp and comprises three exons and two small introns. Exon 1 encodes a 193 bp 5' untranslated region, a 12 amino acid presequence, and the first 12 amino acids of the mature COX VIa protein. Exon 2 encodes amino acids 13 to 58, and exon 3 amino acids 59 to 85 plus the 35 bp 3' untranslated region. Exons 2 and 3 are separated by a small intron of only 96 bp. All exon-intron boundaries matched the consensus splice junction sequences. COX6A1 transcripts are present in RNA from bovine heart but not brain. Primer extension and ribonuclease protection assays were used to map the 5' ends of COX6A1 transcripts in heart; both methods identified several clusters of transcription initiation sites, indicating that COX6A1 mRNA is heterogeneous at the 5' end. The proximal 5' flanking region is AT-rich and contains potential basal promoter elements, such as TATA and CCAAT boxes, associated with tissue-specific genes. A single consensus binding site for the muscle-specific transcription factor, MyoD1, was also located within this AT-rich region. The distal promoter region contained a perfect AP4 site plus potential binding sites for enhancer elements (NRF-1, Mt1, Mt3, and Mt4) proposed to regulate expression of genes for mitochondrial proteins.

Tyrosine phosphorylation plays a central role in the control of neuronal cell development and function. Yet, few neuronal protein tyrosine phosphatases (PTPs) have been identified. We examined rat olfactory neuroepithelium for expression of novel PTPs potentially important in neuronal development and regeneration. Using the polymerase chain reaction with degenerate DNA oligomers directed to the conserved tyrosine phosphatase domain, we identified 6 novel tyrosine phosphatases. One of these, PTP NE-3, is a receptor-type PTP expressed selectively in both rat brain and olfactory neuroepithelium. In the olfactory neuroepithelium, PTP NE-3 expression is restricted to neurons and describes a novel pattern of expression with a high level in the immature neurons and a lower level in mature olfactory sensory neurons.

To confirm previous immunocytochemical findings in colchicine-treated Syrian hamsters, in situ hybridization was used to investigate the distribution of TH mRNA-containing cells in the medial amygdaloid nucleus (Me) and the thalamic reticular nucleus (Rt) of untreated hamsters. TH mRNA-producing neurons were observed in anterior and posterior Me and throughout Rt, similar to the distribution of TH-immunostained cells in these areas of animals receiving colchicine. These data confirm that TH is normally produced in amygdaloid and thalamic cell groups which lie outside the classical catecholamine systems.

The 90-kDa heat shock protein (hsp90) is present in cells at high levels in the cytoplasm and is composed of two separate gene products, hsp86 and hsp84. Rabbit polyclonal antibodies to the murine N-terminal sequences of the 86- and 84-kDa heat shock proteins were isolated from serum by peptide affinity chromatography, Antibodies against each form of hsp90 are capable of immunoprecipitating hsp90. Each antibody preparation is specific against either hsp86 or hsp84 when tested on a protein blot of Hepa 1c1c7 cytosol. The overall ratio of hsp84/hsp86 in Hepa 1 cytosol was estimated to be 2 to 1. Each antibody preparation was used to immunoprecipitate hsp84 or hsp86 from Hepa 1 cytosol to test whether hsp86/84 exists as a homo- and/or heterodimer. After electrophoresis, silver staining revealed that anti-hsp86 antibodies immunoprecipitated both hsp86 and hsp84. This result would suggest that hsp86 forms heterodimers with hsp84. In contrast, the anti-hsp84 antibodies immunoprecipitated almost entirely hsp84, suggesting that hsp84 exists largely as homodimers. Both anti-hsp86 and hsp84 antibodies were able to immunoprecipitate the 2-azido-3-[125I]iodo-7, 8-dibromodibenzo-p-dioxin-labeled Ah receptor from Hepa 1 cytosol, indicating that these antibodies are able to bind to hsp90 when it is complexed with other proteins. Both antibody preparations recognize hsp90 in mouse, rat, and human cell lines. Immunofluorescence and confocal microscopy were performed using both antibody preparations, and the results indicated that both hsp86 and hsp84 were located in the cytoplasm and nucleus of Hepa 1 cells. Hsp86 was found to localize unevenly in the cytoplasm, while hsp84 was found evenly dispersed throughout the cytoplasm. Hsp86 also appeared to be localized to a greater degree than hsp84 in the vicinity of the nuclear envelope.

The medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), and medial amygdaloid nucleus (Me) are essential for male sexual behavior in the Syrian hamster. These nuclei received chemosensory stimuli and gonadal steroid signals, both of which are required for mating behavior. The objective of this study was to compare the distribution of androgen- and estrogen-concentrating neurons in MPOA, BNST, and Me in the adult male hamster using steroid autoradiography for estradiol (E2), testosterone (T) and dihydrotestosterone (DHT). Adult males (n = 4 per group) received two i.p. injections of tritiated steroid 4-7 days after castration. Six-[mu]m frozen sections through the brain were mounted onto emulsion-coated slides, and exposed for 11-16 months. In MPOA, BNST, and Me, neurons were more abundant and heavily labelled after [3H]E2 treatment than after either [3H]T or [3H]DHT. Tritiated estradiol- and DHT-labeled cells were found throughout the rostrocaudal extent of Me, with a high concentration in posterodorsal Me. Tritiated testosterone treatment labelled cells largely within posterodorsal Me. In MPOA, the majority of E2-, T-, and DHT-labelled neurons were in the medial preoptic nucleus (MPN) and the preoptic continuation of the posteromedial bed nucleus of the stria terminalis (BNSTpm). Few T-labelled cells were present outside these subdivisions. In the BNST, E2- and DHT-labelled neurons were present in all subdivisions, whereas T labelling was confined to the antero- and posteromedial subdivisions of BNST. These results suggest that the distribution of androgen- and estrogen receptor-containing neurons overlap considerably in nuclei which transmit chemosensory signals in the control of mating behavior.

CAP is a component of the S. cerevisiae adenylyl cyclase complex. The N-terminal domain is required for cellular RAS responsiveness. Loss of the C-terminal domain is associated with morphological and nutritional defects. Here we report that cap cells bud randomly and are defective in actin distribution. The morphological and nutritional defects associated with loss of the CAP C-terminal domain are suppressed by over-expression of PFY, the gene encoding profilin, an actin- and polyphosphoinositide-binding protein. The phenotype of cells lacking PFY resembles that of cells lacking the CAP C-terminal domain. Study of mutated yeast profilins and profilins from Acanthamoeba suggests that the ability of profilin to suppress cap cells is dependent upon a property other than, or in addition to, its ability to bind actin. This property may be its ability to bind polyphosphoinositides. We propose that CAP and profilin provide a link between growth signals and remodeling of the cellular cytoskeleton.

To assess whether calmodulin (CaM) could have a role in the behavioral sensitization induced by repeated intermittent amphetamine, CaM content was determined in several brain areas from rats repeatedly administered saline or amphetamine. Rats were treated with amphetamine using an escalating dose paradigm and withdrawn for either 4 weeks (withdrawn group) or 30 min (non-withdrawn group). CaM content was measured in cytosol and 100,000 x g membrane fractions from striatum, limbic forebrain, medial prefrontal cortex, hippocampus and cerebellum. In the withdrawn group, CaM was significantly increased in striatal membranes and cytosol and in the mesolimbic membranes from amphetamine-treated rats. There were no changes in CaM in the medial prefrontal cortex, hippocampus or cerebellum. In the non-withdrawn group, there was no significant change in CaM in striatal or mesolimbic fractions but CaM was significantly decreased in cytosol of the medial prefrontal cortex and hippocampus as compared to saline controls. This decrease could be related to the tolerance that has developed to the amphetamine after the repeated treatments. In the withdrawn group, challenge with a low dose of amphetamine (1 mg/kg) elicited a translocation of CaM from membranes to cytosol in the striatum and limbic forebrain of rats repeatedly treated with amphetamine, but not in saline-treated rats. Our findings that the change in CaM occurs in striatum and limbic forebrain, requires time after treatment to develop and exhibits persistence after withdrawal correlate with known characteristics of behavioral sensitization to amphetamine. These results suggest that CaM could contribute to neurochemical events underlying behavioral sensitization to amphetamine.

Iontophoretic injections of the fluorescent retrograde tract tracer, Fluoro-gold, into the entopeduncular nucleus of the rat (homologous to the internal segment of the primate globus pallidus) resulted in a substantial number of retrogradely labeled neurons in the ipsilateral globus pallidus (homologous to the external segment of the primate globus pallidus). In experiments confirming this projection, iontophoretic injections of the anterograde tract tracer, Phaseolus vulgaris-leucoagglutinin, in the globus pallidus resulted in dense fiber and terminal labeling in the ipsilateral entopeduncular nucleus. This projection is topographically organized in rostral-caudal, medial-lateral and dorsal-ventral orientations.

The role of Muller glial cells in the process of degeneration and regeneration of the goldfish retina is poorly understood. One potential role is phagocytosis of neuronal debris in degenerating retinas. We investigated the phagocytic capacity of Muller glial cells of the goldfish retina both in vitro and in vivo. Muller glial cells from primary or first passage cultures were incubated with latex beads to assess their phagocytic ability, and acridine orange staining was used to identify phagolysosomes in living Muller glial cells. These experiments showed that Muller glial cells are phagocytic in culture. Cell identity was verified with an antibody raised against glial fibrillary acidic protein (GFAP). For the in vivo experiments fluorescent latex beads alone or in combination with the metabolic poison ouabain were injected into the posterior chamber. At various intervals (4 days to 8 weeks) after injection the retinas were prepared for immunocytochemistry. Polyclonal anti-GFAP and NN-1, a monoclonal antibody which recognizes macrophages and microglia within the goldfish retina, were used to identify the phagocytic cell. When the beads were injected into the eye, they were phagocytosed by macrophages/microglia cells but not by Muller cells.

Expression of adult-type nicotinic acetylcholine receptors at the neuromuscular junction is thought to result from selective induction of their genes in endplate-associated nuclei due to local neurotrophic control. However, denervation studies indicate that endplate-specific expression can be maintained in the absence of the nerve. We investigated the role played by the basal lamina in this expression by assaying for the adult-type-specific [var epsilon] RNA in noninnervated regenerating muscle. We found that this RNA is locally expressed beneath the old endplates after 10 days of regeneration. At earlier times [var epsilon] RNA is also found in areas other than the endplate region. These results indicate that in adult muscle the basal lamina contains all the components necessary to direct nicotinic acetylcholine receptor gene expression to the endplate.

Prostacyclin release from endothelial cells in culture appears increased by the presence of plasma, but occurrence of a similar phenomenon in intact vessels has not been established. In the present investigation release of 6-keto-PGF1[alpha], the stable breakdown product of prostacyclin, was quantitated from canine veins perfused ex vivo for 15 minute periods, using three different perfusates: 1) Hank's balanced salt solution (HBSS), 2) 20% platelet poor plasma (PPP) derived from heparinized blood, in HBSS, and 3) 20% PPP in HBSS with added arachidonic acid (AA). No differences in initial 6-keto-PGF1[alpha] release existed among the three perfusates. However, PPP and PPP + AA solutions were associated with lesser declines in release, than occurred with HBSS alone, these differences being statistically significant after 60 min of perfusion (p<0.05). When PPP derived from heparinized and citrated blood rather than from only heparinized blood was used, there was a significantly lower release of prostacyclin (p<0.05). The latter may be due to citrate binding of calcium. These data indicate that autologous plasma does not alter initial prostacyclin release from freshly harvested canine veins, but that it weakens the decline in release usually following prolonged periods of perfusion.

The rate of synthesis of secretory proteins increases significantly in rat parotid glands after stimulated discharge of stored proteins. How any difference in the amount of secretory protein discharge affects the rate of subsequent protein synthesis, and whether this post-secretory synthesis is regulated at the level of messenger RNA, was now examined. One group of rats was stimulated to secrete 97% of stored secretory proteins by an intraperitoneal injection of isoproterenol. The other group received a much smaller dose to induce the discharge of about 40% of the proteins. Despite this difference in secretion, the subsequent rates of total protein synthesis, as well as of amylase, were increased to about the same extent. The amylase messenger RNA (mRNA) was identified and quantified by hybridization with a 32P-labelled amylase complementary DNA (cDNA) probe. The amylase mRNA in stimulated and unstimulated rats was of the same molecular size (Northern blot analyses). The amount of amylase mRNA, determined by dot blot analyses, were also increased in stimulated rats, although this increase was not as great as that in the rate of amylase protein synthesis. The implications of this discrepancy concern the possibility that the mechanism of regulation of secretory protein synthesis in parotid glands is at the translational level.

To determine the extent of colocalization of substance P (SP) and prodynorphin peptides within neurons of the medial nucleus of the amygdala (AMe), medial bed nucleus of the stria terminalis (BNSTm) and medial preoptic area (MPOA), we incubated colchicine-treated Syrian hamster brain tissue in an antiserum mixture containing rat anti-SP antibody combined with 1 of 3 rabbit antibodies against prodynorphin peptides: anti-dynorphin A(1-17), anti-dynorphin B(1-13) or anti-C-peptide. This was followed by incubation in a secondary antiserum mixture containing fluorescein-labelled anti-rabbit and rhodamine-labelled anti-rat antibodies. Sections were viewed with an epifluorescence microscope using blue light excitation for fluorescein and green light excitation for rhodamine. Colocalization of SP and prodynorphin labelling was observed in neurons of the caudal parts of AMe, BNSTm and MPOA, areas which are essential for male mating behavior. The colocalization was most extensive in the dorsolateral part of the caudal MPOA, the caudodorsal part of the BNSTm, and in the posterodorsal subdivision of AMe. Although all 3 dynorphin peptides coexisted with SP in these areas, dynorphin B did so less than C-peptide, and dynorphin A less than dynorphin B.

Electrolytic lesions were made in the medial preoptic area (MPOA) and bed nucleus of the stria terminalis (BNST) to evaluate their participation in the neural regulation of copulatory and chemoinvestigatory behaviors in male hamsters. Damage to either the MPOA or the BNST caused severe deficits in copulatory performance in a subset of the animals in each group. In the MPOA group all males displaying severe deficits had lesions which included a small central region of the caudal MPOA. In the BNST group, animals with severe copulatory deficits all had large lesions which covered most of both the medial and lateral parts of the nucleus. In contrast, MPOA and BNST lesions differentially affected chemoinvestigatory behaviors. MPOA lesions did not affect any of the males' anogenital investigation rates or attraction to female odors, even though some of these hamsters had stopped mating completely. Males with BNST lesions, on the other hand, all displayed significant reductions in their chemoinvestigatory responding even though the majority of them continued to mate normally. We suggest that the MPOA and BNST may in part regulate male sexual behavior by differentially responding to `attractant' and `mounting' substances within female hamster vaginal secretion.

Dividing cells and their progeny in retinae of young goldfish were labeled with [3H]thymidine, and selected cells were reconstructed from serial sections processed for electron microscopic autoradiography. Our goals were to characterize the cells that were identified as rod precursors in previous light microscopic autoradiographical studies and to determine their origin and fate. (In fish the population of rods increases several-fold postembryonically by proliferation of rod precursor cells scattered across the retina.) Over 200 labeled cells taken from 11 retinas were examined, and 20 of these were reconstructed in their entirety. Some retinas were examined at short intervals (1 to 48 hr) after [3H]thymidine injection in order to study mitotically active cells, and others were examined after longer intervals (9 or 14 days) to discover the nature of the progeny of labeled dividing cells. Previous evidence from thymidine studies in larval goldfish suggested that proliferating cells destined to produce rods appear first in the inner nuclear layer and later in the outer nuclear layer, where they continue to divide and generate new rods (P.R. Johns, (1982) J. Neurosci. 2, 179). The present results provide morphological evidence in support of the suggestion that rod precursors migrate from inner to outer nuclear layer and, furthermore, show that the precursors are closely associated with, and perhaps guided by, the radial processes of Muller glial cells. Examination of EM autoradiographs of labeled cells at 9 and 14 days after a pulse label with thymidine confirms that the differentiated progeny of dividing precursor cells are exclusively rods. To our knowledge, rod precursors are the first example of a neuronal germinal cell in the vertebrate central nervous system that under normal conditions produces only one type of neuron.

This relationship between the function of the muscles of mastication and craniofacial form was investigated in young adult monkeys by increasing the functional length of the elevator muscles of the mandible non-invasively by a bite-opening splint cemented to the maxillary dentition. The major adaptations to increased vertical dimension were (1) marked superior and some anterior displacement of the maxillary complex, (2) dental intrusion and (3) rotation of the mandible. These alterations produced a shortening of the lengthened masseter muscle, i.e. a reduction in the amount of muscle stretch brought about by the appliance. Migration of the masseteric insertion along the ramus did not contribute significantly to the pattern of adaptation. The role of the masseter muscle in craniofacial adaptations to altered vertical dimension was determined by detaching and re-attaching the insertion of the masseter muscle in one group of experimental animals. The myotomized monkeys experienced significantly less anterior displacement of the maxilla than the non-myotomized monkeys, indicating that the surgery may have lessened some of the anteriorly-directed distracting forces of the lengthened masseter. Masseter myotomy alone was not sufficient to eliminate the vertically-directed distracting forces of the remainder of the mandibular elevator muscles brought about by increasing the vertical dimension of the lower face.

Suspension culture of single adult rat flexor digitorum brevis (FDB) muscle fibres in Vitrogen, a purified collagen, on tissue culture plastic or glass with mesh ring supports is superior to culture upon other substrates including collagen-, laminin-, or Vitrogen-coated tissue culture plastic. The Vitrogen gel-fibre mixture which attaches to glass or plastic provides at least 10 times more fibres per dish than does plating fibres on other substrates. Use of Vitrogen gel permits variable plating densities and the production of adequate numbers of cultures for long-term experimental comparisons of acetylcholinesterase (AChE) and rhodamine-[alpha]-bungarotoxin (RBTX) distribution on muscle fibres. Use of 40 [mu]/ml ovotransferrin (OT) instead of chick embryo extract in the culture medium significantly improves long-term survival. Cultured fibres, with or without the addition of ventral spinal cord explants, may also be examined with electrophysiological techniques.

Male hamsters in which the stria terminalis (ST) had been interrupted either by electrolytic lesions or knife cuts, or normal control males, received iontophoretic injections of horseradish peroxidase in either the bed nucleus of the stria terminalis (BNST) or the medial preoptic-anterior hypothalamic area (MPOAH). Comparison of intact and St-lesioned brains revealed the existence of a ventral non-striatal pathway, from cells in the medial amygdaloid nucleus (M) to the preoptic portion of the BNST but not to the MPOAH. Since bilateral lesions of M completely eliminate male hamster mating behavior, but ST lesions do not, we suggest that the ventral pathway to the BNST may be an important route by which M influences male copulatory behavior.

Electron microscopic examination of the developing neural tube in 11th to 13th day rat fetuses revealed a new cell surface relationship between differentiating neuroepithelial cells. Cytoplasmic projections possessing terminal dilations were observed extending from neuroepithelial cells through cytoplasmic furrows into large coated pits at the surface of adjacent cells. This cell-to-cell relationship provides a mechanism for the internalization of surface molecules and possibly even cytoplasmic constituents. Communication between donor and recipient cells mediated in this way suggests a route for the sharing of macromolecules, including cytoplasmic fragments, which could function as regulators in embryonic development and differentiation.

Calmodulin has been labeled with rhodamine isothiocyanate (CaM-RITC) and used as a probe for the location of calmodulin in vivo. CaM-RITC retains its capacity to regulate the activity of brain phosphodiesterase in a Ca2+-dependent manner in vitro, indicating that the labeled protein is still active. After injection into living mammalian cells CaM-RITC incorporates rapidly into the mitotic spindle; the details of its localization there mimic closely the distribution of Calmodulin seen by immunofluorescence. In interphase cells the CaM-RITC is excluded from the nucleus, but shows no region of specific concentration within the cytoplasm. Neither a 2-fold increase in cellular CaM nor the injection of anti CaM has any observable effect on the progress of mitosis.

Exposure to 2-methoxyethanol (ME) or its major metabolite, methoxyacetic acid (MA), results in spermatocyte depletion and testicular atrophy in experimental animals. The site of spermatogenesis is within the seminiferous tubule. Sertoli cells support spermatogenesis, synthesizing and secreting proteins, and metabolic substrates for utilization by differentiating germ cells in the seminiferous tubule lumen. One of these substrates, lactate, is preferentially metabolized by spermatocytes. Therefore, because germ cells are dependent upon the metabolic products of Sertoli cells, the effect of ME and MA on production of lactate and protein synthesis was measured in cultured rat Sertoli cells. Cell cultures were incubated with ME or MA at 0, 3, or 10 m for up to 12 hr. No significant difference was seen in total protein synthesis as measured by [3H]leucine incorporation. ME and MA had no apparent effect on cell viability. However, lactate concentrations and rates of lactate accumulation were significantly decreased by MA, but not ME, at both 3 and 10 m following incubation for 6, 9, and 12 hr. The results suggest that inhibition of Sertoli cell lactate production resulting from ME or MA exposure could account for the inhibitory action of these compounds on spermatogenesis.

Daylength regulates reproduction in golden hamsters through a mechanism which involves the pineal indoleamine, melatonin. Retinal input to the suprachiasmatic nucleus of the hypothalamus (SCN) and sympathetic innervation of the pineal are critical to the inhibition of reproduction by short photoperiods. Since the hypothalamic paraventricular nucleus (PVN) receives extensive input from the SCN in the rat, and may influence autonomic function via its brainstem and spinal cord projections, we studied the role of this nucleus in photoperiodically induced gonadal regression in the hamster. Bilateral electrolytic destruction of either the paraventricular nucleus (PVN) or suprachiasmatic nucleus (SCN) of the hypothalamus completely blocked testicular regression induced by either blinding or exposure to short days (10L:14D). Lesions in the retrochiasmatic hypothalamus (RCA) which may have interrupted the pathway of previously identified efferents from the SCN to the PVN were also effective in preventing short day-induced gonadal regression. Pineal melatonin content was measured in intact and lesioned hamsters sacrificed 3-5 h before lights on, at the time of the expected nocturnal peak. While SCN and RCA lesions significantly reduced pineal melatonin content, PVN lesions were still more effective in this regard. We conclude that the hamster's neuroendocrine response to photoperiod is mediated by neural pathways which include retinohypothalamic input to the SCN and efferents from this nucleus to the PVN which travel dorsocaudally through the retrochiasmatic area of the hypothalamus. Effectiveness of lesions restricted to the PVN suggests that direct projections from the PVN to spinal autonomic centers convey photoperiodic information which regulates pineal, and hence gonadal, function.