Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Kombinationsformen der Arteriitis

Zusammenfassung Ausgehend von den UntersuchungenRössles zum Formenkreis der rheumatischen Gewebsveränderungen, wird an Hand von 4 eigenen Fällen die Problematik der morphologischen Differentialdiagnose zwischen rheumatischer Arteriitis, Periarteriitis (Panangiitis) nodosa und Thrombangitis obliterans unter Berücksichtigung der Literatur aufgezeigt. Mit der morphologischen Definition der verwendeten Begriffe fibrinöse Entzündung und proliferierende und granulierende Angiitis wird versucht, die drei Formen der Gefäßwandentzündung morphologisch abzugrenzen. Die überschneidungen der morphologischen Symptome und die Kombinationsformen werden aus Beschreibung und Abbildung einschlägiger veröffentlichter Fälle aufgezeigt. Aus dem Nachweis dieses Schrifttumsvergleichs und dem der Differentialdiagnose der 4 eigenen Fälle wird gefolgert, daß bei solchen nicht-klassischen Gefäßentzündungen die Annahme von Kombinationsformen richtiger ist als die immer stärkere Erweiterung des sog. rheumatischen Formenkreises.

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Summary With reference to the investigations ofRössle On the Classification of the Rheumatic Tissue Changes, the problems that are associated with the differential diagnosis of rheumatic arteritis, periarteritis (panangiitis) nodosa, and thromboangiitis obliterans are discussed, exemplified by the report of four cases, and by a review of the literature. By applying the morphological criteria of fibrinous inflammation, proliferative and granulomatous angiitis, an attempt is made histologically to differentiate these three forms of inflammation of the vascular wall. The overlapping of the morphological criteria and the combined forms that mav occur are illustrated in the four cases reported. As shown by comparing the cases in the literature, and as exemplified by the differential diagnosis of the four cases, the inference is made, that with such types of non-classical vascular inflammation, it is better to refer to combined forms than to the more diffuse so-called rheumatic classification.

     

Bioactive 5-Reduced 16α,17α-Cyclohexanoprogesterone Derivatives Weakly Interact with Proteins of the Soluble Uterine Fraction

We studied competitive activities of 16,17-cyclohexano-5- and 5-dihydroprogesterone in replacing ₃H-progesterone and ₃H-16,17-cyclohexano-6-methylprogesterone from protein complexes. Direct binding of ₃H-5-reduced derivatives with proteins of soluble fractions from rat and rabbit uteri was also assayed. Cd values for 5-reduced derivatives were in the micro- or submicromolar range. The data suggest that biological effects of these analogues are not mediated via soluble uterine receptors.     

Immunohistochemical localization of αl-antitrypsin and αl-antichymotrypsin in salivary pleomorphic adenomas

Summary Immunohistochemical identification of l-antitrypsin (l-AT) and l-antichymotrypsin (l-ACh) in pleomorphic adenomas of salivary glands is reported in order to compare their distribution profiles with those of lysozyme and lactoferrin, already described elsewhere.Normal salivary glands indicated positive l-AT staining in ductal segments and had no l-ACh in any glandular cell. Pleomorphic adenomas displayed moderate positivity to l-AT staining in duct-like, tubular and glandular epithelia which was particularly intense in luminal cells. The limited number of tumour cells which showed duct-like structures with a single cellular layer arrangement, displayed the highest staining to l-ACh. Strongly l-AT positive tumour cells located on the inner side of luminal cavities were also markedly positive to l-ACh. Spindle shaped tumour cells existed outside tubular and ductal structures and were negative to l-AT and l-ACh.Distribution of l-AT in salivary glands was similar to that of lysozyme as is usual in ductal segments or their transformed cells, and occurrence of l-ACh localization rather resembled that of lactoferrin, with occurrence in acinar compartments and changed epithelia within acini.The biological role of a specific immunohistochemical distribution of l-AT and l-ACh in pleomorphic adenomas may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases.     

An autoradiographic study of uptake of exogenous glycine by vertebrate spinal cord splices in vitro

Summary Spinal cord slices from rat and goldfish were incubated with³H-glycine and³H-leucine. After fixation, the slices were examined by both light and electron microscopic autoradiography. Light microscopic autoradiograms showed, in slices incubated with³H-glycine, a high level of uptake at discretely localized sites in the ventral horn grey matter with particular concentrations around the perikarya of motor neurons. Electron microscopic autoradiograms revealed that glycine had been taken up by axon terminals containing flat synaptic vesicles. There was no uptake into terminals containing round vesicles. The spinal cord slices incubated with³H-leucine showed very low levels of radioactivity randomly scattered throughout the tissue.     

Prostate specific antigen and prostate specific acid phosphatase in adenocarcinoma of Skene's paraurethral glands and ducts

An autopsy case of adenocarcinoma of Skene's paraurethral gland co-incident with renal cell carcinoma is described. The adenocarcinoma showed distinct prostate specific antigen and prostate specific acid phosphatase pointing to the equivalence between the male prostate and Skene's paraurethral glands and ducts. Skene's gland are the homologue of the prostate in females and tumours arising from them are immunohistochemically similar to male prostate carcinoma.In the title and text the authors used the official term of Nomina Anatomica paraurethral (Skene's) glands and ducts. Nevertheless recently published data on cross-antigenicity between the male prostate and Skene's glands and the newly discovered exocrine and neuroendocrine parameters of the prostate homologue in the female, comparable with the male prostate (Zaviai 1987), support the use of the same term — the prostate — for prostatic tissue in both sexes (Zaviai 1987, Zaviai et al. 1985). The designations female prostate homologue or female prostate equivalent are a compromise between terms the female prostate and Skene's paraurethral glands.     

αN-catenin expression in the normal and regenerating chick sciatic nerve

Summary The Ca²⁺-dependent intercellular adhesion molecule cadherin is known to be linked to the cytoskeleton by the protein catenin, an association of which appears to be important for the cell-adhesion function of cadherin. Catenin consists of three subtypes-, , and . In our previous study, N-cadherin was shown to be localized on the plasmalemma of normal and regenerating chick peripheral nerve. Thus, as N-catenin is a subtype of -catenin (which is specifically associated with N-cadherin), we investigated the immunolocalization of N-catenin in normal and regenerating chick sciatic nerve. In normal nerve, unmyelinated axons exhibited either intense or weak N-catenin immunoreactivity throughout the axoplasm, whereas myelinated axons were completely immunonegative. Regenerating axons, including those derived from parent myelinated axons, showed N-catenin immunoreactivity of variable intensities in growth cones and axon shafts. Schwann cells were invariably devoid of immunoreactivity. Thus N-catenin is not necessarily bound to the surface plasmalemma, but is distributed throughout the cytoplasm, suggesting that most N-catenin molecules are dissociated from N-cadherin.     

Interferon β1a Treatment Modulates TH1 Expression in γδ + T Cells from Relapsing-Remitting Multiple Sclerosis Patients

A paradigm exists that multiple sclerosis is causally related to dysregulation of T_H1 inflammatory cytokines and T_H2 antiinflammatory cytokines. The cytokine source(s) that initiate the imbalances are unknown. In this study, , CD4, and CD8 T cell receptor-positive (TCR+) cells were isolated from the blood of 26 definitive relapsing-remitting multiple sclerosis patients prior to interferon -1a (IFN1a) therapy and following 8–10 weeks of this therapy. The bioactivities of interferon (IFN), interleukin 10 (IL10), and interleukin 12 (IL12) were determined. The concentrations of IFN, IL10, and IL12 from each cell type did not change significantly with IFN1a treatment. The IL10 secreted by TCR+ cells strongly correlated with the IL12 secreted by the same TCR+ cells, supporting the paradigm. Furthermore, IFN1a therapy decreased the TCR+ cell secretion of T_H1 cytokines after 8–10 weeks of therapy.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Endothelial cell dynamics under pulsating flows: significance of high versus low shear stress slew rates (d(tau)/dt)

Shear stress modulates endothelial cell (EC) remodeling via realignment and elongation. We provide the first evidence that the upstroke slopes of pulsatile flow, defined as shear stress slew rates (positive / affect significantly the rates at which ECs remodel. We designed a novel flow system to isolate various shear stress slew rates by precisely controlling the frequency, amplitude, and time-averaged shear stress _ave of pulsatile flow. Bovine aortic endothelial cell (BAEC) monolayers were exposed to three conditions: (1) pulsatile flow (1 Hz) at high slew rate (293 dyn/cm² s), (2) pulsatile flow (1 Hz) at low slew rate (71 dyn/cm² s), and (3) steady laminar flow at /t=0. All of the three conditions were operated at _ave=50{dyn/cm}². BAEC elongation and alignment were measured over 17 h. We were able to demonstrate the effects of shear stress slew rates /t on EC remodeling at a fixed spatial shear stress gradient (/x). We found that pulsatile flow significantly increased the rates at which EC elongated and realigned, compared to steady flow at /t=0. Furthermore, EC remodeling was faster in response to high than to low slew rates at a given tau _ave © 2002 Biomedical Engineering Society. PAC2002: 8719Tt, 8719Rr     

Rhinovirus detection using probes from the 5′ and 3′ end of the genome

Summary This study investigated the abilities of cDNA probes from the 5 and 3 ends of the genome of human rhinoviruses (HRV-) 14, 9, and 1B to detect RNA from 59 rhinovirus serotypes. The results show that probes from the 5 end of the genomes of HRV-14, 9, and 1B detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. In contrast, all the 3 end probes were specific for the homologous virus. However, along HRV-9 probe detected a large number of serotypes.It was concluded that such cDNA probes would not detect all serotypes with equal efficiency. Synthetic oligonucleotides corresponding to short but highly conserved regions in the 5 non coding region may overcome this problem.     

Distribution of renal integrin receptors and their ligands in congenital nephrotic syndrome of the finnish type

The aim of this study was to examine the distribution of 1 and v integrins (Ints) and some of their ligands in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF) and in controls using indirect immunofluorescence with monoclonal antibodies. The mesangial reactivity of Int 1 and Int 1 subunits was more variable and an increased glomerular reactivity with Int 3 and Int-6 antibodies was found in CNF kidneys than in controls. Int 2 subunit was either completely missing from or found in significantly lesser amounts in CNF kidney glomeruli. The immunoreactivity for Int v was more variable, fainter and also more granular in CNF samples than in control kidneys. The glomerular reactivity for Int 5 was more diffuse and weaker, and in sclerotic Bowman's capsules more intense in CNF kidneys than in controls. Immunoreactivity for Int 6 was restricted and was comparable in extent in CNF and control kidneys. Of the extracellular matrix components studied, the expression of EDAFn, EDBFn, OncFn, Ln 2 chain, Ln 1 chain and tenascin was increased. This is also seen in several glomerular diseases with inflammation and sclerosis. Immunoreactivity for vitronectin was decreased. Several differences were found in the intensity or location of the immunostaining for the 1 and v Ints and their ligands in CNF kidneys compared with controls, which have not been found in any other proteinuric disease. Disturbed Int expression pattern in CNF may specifically reflect the disturbance of glomerular function caused by the primary defect in this disease.     

Postsynaptic and extrasynaptic localization of κ-opioid receptor in selected brain areas of young rat and chick using an anti-receptor monoclonal antibody

Summary -opioid receptors were visualized by light and electron microscopical immunohistochemistry in young rat and chick brains, using a monoclonal antibody KA8 (IgG1, ) raised against a -opioid receptor preparation from frog brain, which recognizes selectively the -type receptor with preference for the -2 subtype. The most pronounced -opioid receptor-like immunoreactivity was observed in the hypothalamic nuclei of the rat brain and in the chick optic tectum, in regions where the functional significance of -opioid receptors is well documented. Both neurons and glia were stained, the former on both somata and dendrites. At the ultrastructural level, the receptor-like immunoreactivity was similar in both species. Immunoprecipitate decorated the inner surface of the plasma membrane of glial cells, neuronal somata and dendrites, in a discontinuous arrangement. In the cytoplasm, labelling was associated with ribosomes, polyribosomes and rough endoplasrnic reticulum membranes but not with Golgi cisternae. In the neuropil, the immunoprecipitate was observed along the dendritic microtubules and was also associated with postsynaptic sites. Nuclei and axons were devoid of label and immunoreactivity was never visible presynaptically. Our findings indicate that the antibody used in the present study marks various forms of the -opioid receptor protein including those synthesised in ribosomes, transported along dendritic microtubules and incorporated into postsynaptic and non-synaptic membranes. The antibody also recognizes glial opioid receptors. The observed subcellular distribution appears to be conserved in phylogenetically distant species.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

The tubular vacuolation process in amphibian skeletal muscle

The exposure of amphibian muscle to osmotic shock through the introduction and subsequent withdrawal of extracellular glycerol causes vacuolation in the transverse tubules. Such manoeuvres can also electrically isolate the transverse tubules from the surface (detubulation), particularly if followed by exposures to high extracellular [Ca2+] and/or gradual cooling. This study explored factors influencing vacuolation in Rana temporaria sartorius muscle. Vacuole formation was detected using phase contrast microscopy and through the trapping or otherwise of lissamine rhodamine dye fluorescence within such vacuoles. The preparations were also examined using electron microscopy, for penetration into the transverse tubules and tubular vacuoles of extracellular horseradish peroxidase introduced following the osmotic procedures. These comparisons distinguished for the first time two types of vacuole, open and closed, whose lumina were respectively continuous with or detached from the remaining extracellular space. The vacuoles formed close to and between the Z-lines, but subsequently elongated along the longitudinal axis of the muscle fibres. This suggested an involvement of tubular membrane material; the latter appeared particularly concentrated around such Z-lines in the electron-micrograph stereopairs of thick longitudinal sections. Open vacuoles formed following osmotic shock produced by extracellular glycerol withdrawal from a glycerol-loaded fibre at a stage when one would expect a net water entry to the intracellular space. This suggests that vacuole formation requires active fluid transport into the tubular lumina in response to fibre swelling. Closed vacuoles only formed when the muscle was subsequently exposed to high extracellular [Ca2+] and/or gradual cooling following the initial osmotic shock. Their densities were similar to those shown by open vacuoles in preparations not so treated, suggesting that both vacuole types resulted from a single process initiated by glycerol withdrawal. However, vacuole closure took place well after formation of open vacuoles, over 25 min after glycerol withdrawal. Its time course closely paralleled the development of detubulation reported recently. It was irreversible, in contrast to the reversibility of open vacuole formation. These findings identify electrophysiological detubulation of striated muscle with closure of initially open vacuoles. The reversible formation of open vacuoles is compatible with some normal membrane responses to some physiological stresses such as fatigue, whereas irreversible formation of closed vacuoles might only be expected in pathological situations as in dystrophic muscle.This revised version was published online in September 2005 with corrections to the Cover Date.     

A structural model for the genome of echovirus 22

Summary We have proposed previously that the structural model for the echovirus 22 genome is a single-stranded RNA molecule that has folded back upon itself to form a stable hairpin at the 5-terminus. The vRNA of echovirus 22 has been characterized further by digestion with selective ribonucleases, electrophoresis in composite gels, hydrodynamic studies in density gradients of Cs₂SO₄ and sucrose, thermal denaturation and 3-terminal ribonucleotide analysis. Based on these observations, the genome of echovirus 22 is a single-stranded RNA molecule having a region of secondary structure located at the 5-terminus that may be characterized as a snapback hairpin with hydrogen-bonded base-pairing. In addition, a VPg-like protein is attached (presumably to the 5-end of the RNA) and the 3-terminus contains a polyadenylic acid tract [poly(A)].     

Influence of adoptively transferred thioglycollate-elicited peritoneal macrophages on metastasis formation in mice with depressed or stimulated NK activity

The effect of thioglycollate-elicited macrophages (TG-M) on natural killer (NK)-cell activity and metastases formation in mice was investigated. Intravenously (i.v.) inoculated TG-M inhibited spleen NK activity of normal mice and abrogated polyinosinic: polycytidylic (poly IC) induced augmentation of NK cell function. TG-M also inhibited the clearance of i.v.-injected radiolabeled B16 melanoma cells from the lungs of normal or poly IC stimulated mice. Formation of experimental B16 melanoma metastases was dramatically increased in mice pretreated with TG-M. Administration of TG-M increased metatasis formation to a greater extent than anti-asialo GM₁ serum, while anti-asGM₁ serum was more efficient than TG-M in depressing spleen NK cell activity. When mice with low NK reactivity (beige mice or mice treated with anti-asialo GM₁ serum) were inoculated with TG-M, there was a substantial additive augmenting effect on metastasis formation in the lungs. Treatment with poly IC elevated NK-cell activity and had profound antimetastatic effects in normal but not in TG-M pretreated mice. The metastasis augmenting effect of TG-M was fully expressed in poly IC-treated mice as well as in athymic nude mice. Inoculation of proteose peptone-elicited macrophages (PM), unlike TG-M, did not depress NK activity or augment metastasis formation in normal or poly IC-treated mice. However, since the inhibition of NK activity in TG-M-treated mice was relatively weak, and a substantial additional increase in metastases was observed in NK-depressed mice after transfusion of TG-M, it seems unlikely that the TG-M-induced inhibition of NK reactivity is entirely responsible for the augmented formation of metastases. Further studies revealed that i.v. inoculation of TG-M, but not PM, induced intravascular inflammatory reactions, and damage to endothelial cells and basement membrane of the lung vasculature. These reactions may contribute to increased tumor cell extravasation and metastasis formation in mice pretreated with TG-M.     

Denervated chicken breast muscle displays discoordinate regulation and differential patterns of expression of αf and β tropomyosin genes

Summary The expression of the fast (f) and tropomyosin (TM) genes has been analysed with muscle-specific and common cDNA probes after unilateral nerve section of the pectoralis major muscle (PM) in 4-week-old chickens. The following were observed in denervated muscles. (1) The TM mRNA, which was repressed during development, reaccumulates in a biphasic curve with the increase in the TM protein lagging behind the changes in its mRNA. Accordingly, no TM is seen in products translated in vitro from total and polyA⁺ RNA obtained 1 week after denervation. No such translation block is seen with RNA obtained from control or muscles denervated for 6 weeks. (2) No changes in the fTM mRNA and corresponding protein are observed. (3) RNA processing of the two genes is not changed. (4) In the contralateral muscles, transitory increases in f and TM mRNAs are observed while the corresponding proteins remain unchanged. Our data suggest that muscle fibres display early and long-term responses to the loss of neural input which might result from a combination of changes produced by regenerative processes and reprogramming of existing fibres. Moreover, in contrast to normal development, no reciprocal changes of f and TM expression are seen in denervated muscles.     

Binding of α-actinin to F-actin or to tropomyosin F-actin is a function of both α-actinin concentration and gel structure

Summary We have studied by electron microscopy as well as by measurements of low shear viscosity, rigidity and binding, the effect of-actinin on the gel formed at 37° C with F-actin and with tropomyosin-decorated F-actin. Contrary to previous reports in the literature,-actinin at nanomolar concentrations is an efficient actin gelling protein, even at 37° C, provided that the concentration of actin (or of tropomyosin-decorated F-actin) is low (1.2–2.4 m). The binding of-actinin to F-actin, as a function of actin concentration, is anomalous. The amount of bound-actinin increases when actin concentration increases from 0 to 1.2 m but does not change significantly when actin concentration is further increased up to 48 m. A similar result is obtained with tropomyosin-decorated F-actin.These observations can be explained by an hypothesis that binding is a function of the-actinin — F-actin association constant as well as of the rigidity of the gel. When the concentration of actin increases, the rigidity of the gel also increases and more work is required to bring two actin filaments to the reaction distance with-actinin and, consequently, a larger-actinin concentration is required to attain the same ratio of bound-actinin to actin monomers in the filaments.     

TRP channels: a TR(I)P through a world of multifunctional cation channels

The transient receptor potential (TRP) family of ion channels comprises more than 50 cation-permeable channels expressed from yeast to man. On the basis of structural homology, the TRP family can be subdivided in to seven main subfamilies: the TRPC (Canonical) group, the TRPV (Vanilloid) group, the TRPM (Melastatin) group, the TRPP (Polycystin), the TRPML (Mucolipin), the TRPA (Ankyrin) and the TRPN (NOMP) family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of cell types, tissues and species. This paper briefly reviews the TRP superfamily and the basic properties of its many members as a readers guide in this Special Issue. Hopefully, a better understanding of TRP channel physiology will provide important insight into the relationship between TRP channel dysfunction and human diseases.