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Ultrastructure of the salivary glands in the midtongue of the common vampire bat, Desmodus rotundus.

BACKGROUND: All examined mammals have at least two sets of lingual salivary glands: von Ebner's glands and Weber's glands. A third set, the glands of Blandin and Nuhn, is present in the tongues of some but not all mammals. Vampire bats, Desmodus rotundus, are unusual in that they possess another set of lingual glands, these being in the midtongue region. METHODS: The anterior half of the tongue was extirpated from several adult vampire bats, dissected, and tissue blocks derived from the midregions of the body of the tongue prepared for transmission electron microscopy by conventional means. RESULTS: The midlingual glands are in the form of long, tubular secretory endpieces that are succeeded by ducts of simple morphology. In general, the secretory portions consist of two cell types, which may be intermingled in the same tubule or may form tubules that consist wholly of one cell type or the other. Seromucous cells usually have one or several rough endoplasmic reticulum cisternae that are hugely distended by a homogeneously dense material. Their granules have a bizonal substructure: one or several dense bands are embedded in a lighter matrix. Mucous cells are rather typical in structure, but their secretory product is different from run-of-the-mill mucous droplets. These droplets vary in density from cell to cell. In some cells, these droplets have a relatively light matrix; in other cells, the droplet is unusually dense, consisting mainly of a dark, structureless matrix with marginal lenticular lacunae of low density in which some short, irregular filaments are scattered. A rare finding is the presence of ciliated cells intermingled with secretory endpiece cells. The cilia are of conventional morphology. Secretory tubules are succeeded by ducts that resemble intercalated ducts; the epithelium of these ducts gradually increases in height to form a kind of excretory duct, without the intervention of striated ducts. As the ducts approach the lingual surface, the epithelium changes to stratified squamous. CONCLUSIONS: Saliva produced by the midlingual glands may be an aid in the reciprocal grooming behavior of vampire bats. Based on their morphology, the excurrent ducts may not modify the initial saliva elaborated by these glands and might act simply as pipelines by which the saliva reaches the mouth.

Animals↗

Calculations of the CD spectrum of bovine pancreatic ribonuclease.

CD spectra of bovine pancreatic ribonuclease A (RNase A) and its subtilisin-modified from (RNase S) have been calculated, based upon high-resolution structures from x-ray diffraction. All known transitions in the peptide and side-chain groups, especially the aromatic and disulfide groups, have been included. Calculations have been performed with both the matrix method and with first-order perturbation theory. A newly developed method for treating the electrostatic interactions among transition charge densities and between static charge distributions and transition charge densities is used. The effects of local electrostatic fields upon the group transition energies are included for all transitions. Rotational strengths generated by the matrix method were combined with Gaussian band shapes to generate theoretical CD spectra. The calculated spectra reproduce the signs and approximate magnitudes of the near-uv CD bands of both RNase A and S. Agreement is most satisfactory for the negative 275 nm band, dominated by tyrosine contributions. In agreement with two previous studies by other workers, coupling between Tyr 73 and Tyr 115 is the single most important factor in this band. The positive band observed near 240 nm is dominated by disulfide contributions, according to our results. The far-uv CD spectrum is poorly reproduced by the calculations. The observed 208 nm band, characteristic of alpha-helices, is absent from the calculated spectrum, probably because the helices in RNase are short. A strong positive couplet centered near 190 nm is predicted but not observed. Possible reasons for these incorrect predictions of the current theoretical model in the far-uv are discussed.

Animals↗

The lack of a structured blood-brain barrier in the onychophoran Peripatus acacioi.

Onychophorans are 'living fossils' frequently purported to have evolved from the same ancestor as the arthropods and annelids. In the CNS of Peripatus acacioi, beneath an outer acellular neural lamella, glial cells ensheath the cerebral ganglion and the nerve cords. These glial cells are, however, attenuated and rather few in number and, although they interdigitate with one another, they seem to lack intercellular junctions. Exogenous tracers penetrate between them and into the underlying neuropile, suggesting that there is no structural blood-brain barrier. Throughout the nervous tissue, extracellular spaces occur which contain banded collagen fibrils embedded in a matrix material. Thin glial cell processes, characterized by dense filaments, surround these regions and frequently form hemi-desmosomes with the extracellular matrix. The peripheral nerve cell bodies have a range of diameters; some have the characteristics of neurosecretory neurons. Granules in such neurons are produced by the Golgi saccules and associated fenestrated membranes which also possess many coated vesicles. Comparable granules are also found in axonal tracts, but no distinct peripheral neurohaemal areas have been found. Lysosomes are common in the nerve cell bodies and are frequently in the form of multivesicular bodies or large phagocytic vacuoles. Beneath the outer nerve cells lie many tracheae, arranged as a ring around the central neuropile which consists of glial processes, extracellular matrix, axons and nerve terminals. These nerve terminals occur throughout the central neuropile and are characterized by dense pyramidal presynaptic specializations and postsynaptic subsurface cisternae. The nervous system of Peripatus is relatively simple in its organization, in the lack of glial intercellular junctions and in the ready accessibility of substances from the external milieu.

Animals↗

Structural correlation between collagen VI microfibrils and collagen VI banded aggregates.

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.

Aged↗

Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells.

A secreted cysteine protease (CP) fraction from Trichomonas vaginalis is shown here to induce apoptosis in human vaginal epithelial cells (HVEC) and is analyzed by mass spectrometry. The trichomonad parasite T. vaginalis causes one of the most common non-viral sexually transmitted infection in humans, trichomoniasis. The parasite as well as a secreted cysteine protease (CP) fraction, isolated by affinity chromatography followed by Bio-Gel P-60 column chromatography, are shown to induce HVEC apoptosis, as demonstrated by the Cell Death Detection ELISA(PLUS) assay and annexin V-fluorescein isothiocyanate flow cytometry analyses. Initiation of apoptosis is correlated with protease activity because the specific CP inhibitor E-64 inhibits both activities. SDS-PAGE analysis of the CP fraction reveals triplet bands around 30 kDa, and matrix-assisted laser desorption ionization time-of-flight MS indicates two closely associated peaks of molecular mass 23.6 and 23.8 kDa. Mass spectral peptide sequencing of the proteolytically digested CPs results in matches to previously reported cDNA clones, CP2, CP3, and CP4 (Mallinson, D. J., Lockwood, B. C., Coombs, G. H., and North, M. J. (1994) Microbiology 140, 2725-2735), as well as another sequence with high homology to CP4 (www.tigr.org). These last two species are the most abundant components of the CP fraction. The present results, suggesting that CP-induced programmed cell death may be involved in the pathogenesis of T. vaginalis infection in vivo, may have important implications for therapeutic intervention.

Amino Acid Sequence↗

An extended QBASIC program for the normalization and computation of whole-cell protein profiles and the application to clinically important Candida species.

The computer program described enables a rapid calculation of relative molecular masses of proteins from different yeasts and microbial pathogens, by interpolation from a molecular weight calibration curve that comprises stepwise linear regression between the protein bands produced by the internal standards. A similarity matrix can then be produced, taking into account variations between calculated molecular masses caused by small differences in bandwidths and/or positions of specific protein bands. This program has been applied to different Candida species and the similarity data obtained further analysed numerically utilizing CLUSTAN II on a SPERRY 1100 multi-processor.

Bacterial Typing Techniques↗

Coulomb energy differences in t = 1 mirror rotational bands in (50)Fe and (50)Cr.

Gamma rays from the N = Z-2 nucleus (50)Fe have been observed, establishing the rotational ground state band up to the state J(pi) = 11+ at 6.994 MeV excitation energy. The experimental Coulomb energy differences, obtained by comparison with the isobaric analog states in its mirror (50)Cr, confirm the qualitative interpretation of the backbending patterns in terms of successive alignments of proton and neutron pairs. A quantitative agreement with experiment has been achieved by exact shell model calculations, incorporating the differences in radii along the yrast bands, and properly renormalizing the Coulomb matrix elements in the pf model space.

Journal Article↗

The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins.

The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.

Acid Anhydride Hydrolases↗

Light microscopy of the exfoliation syndrome.

The exfoliation syndrome affects all structures of the ocular anterior segment, as well as the conjunctiva and occasionally, nonocular structures. The exfoliative material has been shown by a series of light microscopic and gross anatomic studies to be only loosely adherent to the anterior lens capsule, zonules and anterior vitreous face, and firmly adherent to the equatorial lens capsule and posterior epithelium of the iris and the nonpigmented ciliary epithelium. Electron microscopy demonstrates that, in these latter regions, exfoliation material consisting of characteristic, cross-banded fibrils embedded in an amorphous matrix, is present both within the epithelial cells and associated with a disorganized, reduplicated basement membrane. These findings suggest that the material arises from the epithelium of the lens, iris and ciliary body, possibly the result of an underlying metabolic disorder. From these areas, the material enters the aqueous humor and later deposits on the anterior lens capsule, zonules, vitreous face, anterior surface of the iris, and trabecular meshwork. Histochemical studies demonstrate the presence of glycosaminoglycans, which may comprise the interfibrillar portion of the exfoliative material. Other studies demonstrate histochemical similarities between exfoliative material and zonules and are supported by recent work suggesting that the exfoliative fibrils are related to the microfibrillar portion of elastin. Although some reports suggest similarities between exfoliative material and amyloid, a majority of histochemical studies do not support this possibility.

Amyloid↗

Increased Tyr phosphorylation of ZO-1 during modification of tight junctions between glomerular foot processes.

The slit diaphragms between the glomerular epithelial foot processes represent a variant of the tight junction that are rapidly replaced by typical tight junctions after perfusion with protamine sulfate (PS). To investigate the mechanism of signaling involved, tyrosine phosphorylation of glomerular proteins was analyzed in newborn, PS-treated, and control rats using antiphosphotyrosine immunoglobulin G. In glomeruli of normal adults, phosphotyrosine (Ptyr) staining was confined largely to mesangial cells by immunofluorescence, whereas in newborn and PS-treated rats, the Ptyr signal was dramatically increased in the glomerular epithelium. By immunogold labeling, it was found that newly phosphorylated proteins were concentrated along the newly formed tight junctions (cell-cell junctions) and the basal membrane of the foot processes (cell-matrix junctions). By immunoblotting, several prominent bands were detected with anti-Ptyr in glomerular lysates of controls; in PS-treated rats, additional bands were detected at 225, 180, and 100 kDa. The 225-kDa protein was identified as ZO-1 by immunoprecipitation with anti-ZO-1 followed by immunoblotting with anti-Ptyr. These findings indicate that ZO-1 is one of the targets for tyrosine phosphorylation after PS treatment. They indicate that phosphorylation of tight junction and other proteins occurs during the formation of tight junctions in glomeruli under circumstances where there are rapid changes in epithelial cell shape.

Animals↗

The development of medial geniculate body in man: changes in the cholinesterase (CHE) activity during fetal and perinatal life.

In the present study cholinesterase histochemical (ChE) techniques have been used to demonstrate differentiation and maturation of the medial geniculate body (MGB) in the human fetuses (ranging between 10.5 to 28 weeks) and infants (premature and three postnatal months). In the youngest specimens examined (10.5 to 15 weeks) moderate ChE activity was found in the neuropil of MGB. In the next developmental stage ChE activity is significantly increased with stronger reactivity in the peripheral part of MGB. In the fetuses between 22 to 26 weeks of gestation peak ChE reactivity was observed in MGB. Reactivity is strong and homogeneous. In the premature infant (28 weeks and older) ChE reactivity becomes inhomogeneous and irregular with appearance of ChE positive bands in the moderately stained MGB matrix. In addition, strongly ChE reactive cell bodies can be discerned. In 3-month-old infant brain we have found significant decrease of ChE reactivity in the neuropil of the MGB. In conclusion we can say that MGB shows very strong, transient ChE reactivity during second half of gestation. Since a great majority of ChE reactivity disappears during later postnatal development, it is very likely that strong fetal activity is related to the histogenetic events and maturation of MGB.

Cholinesterases↗

Immunocytochemical localization of copper,zinc superoxide dismutase in peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons.

In previous works using cell fractionation methods we demonstrated the presence of a Cu,Zn-containing superoxide dismutase in peroxisomes from watermelon cotyledons. In this work, this intracellular localization was evaluated by using western blot and EM immunocytochemical analysis with a polyclonal antibody against peroxisomal Cu,Zn-SOD II from watermelon cotyledons. In crude extracts from 6-day old cotyledons, analysis by western blot showed two cross-reactivity bands which belonged to the isozymes Cu,Zn-SOD I and Cu,Zn-SOD II. In peroxisomes purified by sucrose density-gradient centrifugation only one cross-reactivity band was found in the peroxisomal matrix which corresponded to the isozyme Cu,Zn-SOD II. When SOD activity was assayed in purified peroxisomes two isozymes were detected, Cu,Zn-SOD II in the matrix, and a Mn-SOD in the membrane fraction which was removed by sodium carbonate washing. EM immunocytochemistry of Cu,Zn-SOD on sections of 6-day old cotyledons, showed that gold label was mainly localized over plastids and also in peroxisomes and the cytosol, whereas mitochondria did not label for Cu,Zn-SOD.

Blotting, Western↗

Experimental intraocular fibrovascular proliferation through sclerotomy wound.

In the process of closing scleral wounds caused by various conditions, incarceration of conjunctiva, Tenon's capsule, or vitreous in the wound can occur unexpectedly. We created such conditions experimentally in order to discover their intraocular complications. The experimental materials consisted of 12 albino rabbits (24 eyes) divided into two groups (Groups I & II). Vitrectomy was performed in the Group I rabbits (12 eyes) but not in the Group II rabbits (12 eyes). Flaps of conjunctiva and Tenon's capsule were made and inserted into the vitreous cavity through the sclerotomy site, which was soon closed. Fundal examination of the rabbits was carried out using an indirect ophthalmoscope at intervals after the procedure; first at 3 days, then at 1, 3, and 6 weeks, and then at 3 months and 6 months, respectively. Enucleation of the rabbits' eyes 4 from two different rabbits at each of these intervals was carried out, and the extracted eyes were examined under a light microscope at each interval. The results are summarized as follows: 1. All rabbit eyes studied showed intraocular fibrovascular proliferation. 2. The extent of tissue proliferation, which was proportional to the amount of vitreous hemorrhage, was greater in Group II than in Group I. 3. The proliferated tissue developed to "band" by three weeks postexperiment, after which it gradually regressed. 4. The fibrovascular band was made of fibroblasts, stromal matrix, and capillaries.

Animals↗

Adrenoreceptor blockade in angiotensin-induced hypertension: effect on rat coronary arteries and myocardium.

Adrenoreceptor blockade has been used to separate the actions of elevated blood pressure, angiotensin II, and catecholamines on the coronary vasculature and myocardium of rats. Twenty-two male Wistar-Kyoto rats received phentolamine (an alpha-receptor blocker, 10 mg/kg body weight) and/or propranolol (a beta-receptor blocker, 1 mg/kg body weight) followed by an infusion for 2 hours of angiotensin amide (1.7 micrograms/min/kg) or saline. Sections of left ventricle were examined by light and electron microscopy. Blood pressure was elevated only in animals receiving angiotensin II with or without propranolol. Epicardial arteries were devoid of lesions in all animals. Small intramural arteries and arterioles in the hypertensive animals exhibited vasoconstriction, endothelial cell vacuolization with bleb formation, and medial smooth muscle cell fragmentation and necrosis. Foci of irreversible ischemic or anoxic myocardial injury consisting of contraction zones and bands and translocated mitochondria with granular matrix densities were seen in angiotensin-infused animals. Similar but less severe myocardial changes were found in the animals pretreated with propranolol. Vascular lesions were also seen in animals receiving phentolamine, propranolol, and angiotensin II; but myocardial alterations consisted solely of areas with contraction zones. Vascular but not myocardial lesions were observed in animals that received angiotensin II and phentolamine. It is concluded that angiotensin II can produce vascular injury in the absence of elevated systemic blood pressure or catecholamine effects. In contrast, irreversible myocardial injury seems to depend upon the increased pressure and/or coronary artery vasoconstriction associated with angiotensin administration.

Adrenergic alpha-Antagonists↗

Morphologic, microscopic, and immunohistochemical investigations into the function of the primate TMJ disc.

The distribution and arrangement of extracellular matrix proteins were examined in the primate temporomandibular joint disc and posterior attachment using a combination of light microscopic, immunohistochemical, and biochemical techniques. The band areas of the disc contain a complex collagenous (type I) fiber network consisting of a mediolaterally directed fiber bundle system that interlaces or becomes continuous with an anteroposteriorly directed collagenous fiber array that runs through the intermediate zone. Thin, branching, elastic fibers are a significant component of the disc and are generally oriented parallel to the collagenous fiber network. Interfibrillar spaces in band areas contain numerous chondrocytes encased within a matrix that is rich in a high molecular weight, predominantly chondroitin-sulfate proteoglycan and type II collagen. The intermediate zone appears tendinous in its construction and is composed of anteroposteriorly oriented elastic and collagenous fibers, scattered chondrocytes, and reduced amounts of chondroitin-sulfate proteoglycan and type II collagen. The posterior attachment is composed of fibrocytes, larger caliber elastic fibers, loosely organized type I collagenous fibers, and low molecular weight dermatan-sulfate proteoglycan. These results indicate that the primate temporomandibular joint disc is a microheterogenous tissue with distinct regional specializations.

Animals↗

Culture and characterization of equine terminal arch endothelial cells and hoof keratinocytes.

OBJECTIVES: To develop methods to isolate, culture, and characterize equine hoof endothelial cells (EC) and keratinocytes. SAMPLE POPULATION: Cells harvested from the forelimbs of 8 horses. PROCEDURE: EC were obtained via catheters placed in the palmar digital arteries of the disarticulated lower portion of the forelimbs from 4 horses that had been heparinized prior to euthanasia. Phosphate-buffered saline solution was used to remove and discard RBC from blood vessels, and collagenase was used to loosen and flush EC from the vasculature. Hoof keratinocytes were obtained from 4 recently euthanatized horses by use of dispase/trypsin dissociation of the coronary band epidermis. Use of an extracellular matrix gel as a culture flask attachment factor was important to the success of hoof keratinocyte cultures. RESULTS: EC from the palmar digital arteries were successfully cultured and characterized by in vitro morphology, uptake of a fluorescence-labeled acetylated-low density lipoprotein, and lack of expression of von Willebrand factor and smooth muscle actin. Hoof keratinocytes were characterized by morphology in culture and expression of keratin proteins, as determined by immunochemical reaction. Keratinocyte cultures were also positive for vimentin expression. CONCLUSIONS: Culture techniques to isolate and characterize hoof cells should aid investigators in their study of equine hoof pathobiologic features, especially as it relates to laminitis.

Animals↗

Estrogen receptors in the nuclear matrix: direct demonstration using monoclonal antireceptor antibody.

Estradiol-binding sites, as assayed by exchange with radiolabeled steroid, become associated with the nuclear matrix of estrogen-responsive tissues after treatment with estrogen in vivo. Using monoclonal estrogen receptor antibodies, we have now obtained direct evidence that these matrix-associated estradiol-binding sites are estrogen receptor proteins similar to those found in the cytosol before estrogen treatment. Proteins of the liver nuclear matrix from untreated or ethinyl estradiol-treated female rats were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and probed with the monoclonal estrogen receptor antibody H222Sp gamma. A single prominent immunoreactive 67,000 mol wt band, indicating the presence of estrogen receptors, was found in the liver nuclear matrix of estrogen-treated animals. This band was detectable, but of much lower intensity, in the liver nuclear matrix of untreated animals. Liver cytosol estrogen receptor from untreated rats also migrated as a 67,000 mol wt band. These immunoreactivity data corroborated data obtained by [3H]estradiol-binding assays. Scatchard analysis of specific high affinity [3H]estradiol-binding sites showed high levels of these sites in the liver nuclear matrix of estrogen-treated rats and low levels in untreated rats. Therefore, both direct and indirect methods of receptor identification demonstrate the specific association of estrogen receptors with the nuclear matrix after estrogen treatment in vivo.

Animals↗

The microanatomy of the distal arrector pili: possible role for alpha1beta1 and alpha5beta1 integrins in mediating cell-cell adhesion and anchorage to the extracellular matrix.

The arrector pili (AP) muscle is a small band of smooth muscle that attaches proximally to the bulge area of the pilosebaceous apparatus in the reticular dermis and extends up toward the epidermis. The distal anatomy of the AP and the anchorage mechanism allowing hair erection have not been previously described. Integrins are likely candidates mediating this attachment. Immunohistochemical techniques were used to determine the distribution of the following integrins: alpha1, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 as well as fibronectin. Frozen human scalp tissue was sectioned in traditional planes, obliquely and horizontally to visualize microanatomy in three dimensions. Histological examination revealed that the distal portions of smooth muscle fibers splay extensively between collagen bundles of the upper dermis. Integrin subunits alpha1, alpha5 and beta1 were expressed by the AP muscle. Analysis of the relative density of immunoreactivity in digitized sections revealed increased alpha5 subunit expression at the extracellular matrix (ECM)-muscle interface. These data suggest that anchorage of the AP muscle to the ECM is via alpha5beta1 integrin and alpha1beta1 integrin functions in muscle cell-cell adhesion. Extensive splaying of smooth muscle fibers may allow increased surface area contact between the ECM and smooth muscle cells expressing peripherally situated alpha5 integrin.

Aged↗