Immunocytochemical study of seminal γ-glutamyltransferase using monoclonal antibodies

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal -glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal -glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal -glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal -glutamyltransferase but recognize different epitopes.     

Developmental expression of transforming growth factor-α and epidermal growth factor receptor proteins in the human pancreas and digestive tract

This study was designed to localize transforming growth factor alpha (TGF-) and epidermal growth factor receptor (EGFR) expression in the developing human gastrointestinal tract and pancreas. Immunohistochemical techniques using specific antibodies against human TGF- and EGFR were performed on digestive tissues of fetuses from 9 to 10 to 24 weeks of gestation, children and adults. In fetuses, TGF- and EGFR proteins were expressed in all epithelial tissues studied with a good correlation and from an age as early as 9 to 10 weeks of gestation, except for TGF- in the esophagus. The strongest TGF- immunostaining was noted in the stomach and the proximal colon. Unexpectedly, immunoreactive gut endocrine cells were observed with the two antibodies used. Relatively numerous in fetuses, they decreased in number with age and were rare in adults particularly along the colon. Enteroglucagon-secreting cells were shown to express TGF- while some gastrin, somatostatin and pancreatic glucagon cells were immunostained with EGFR antibodies. The presence of TGF- and of its recetor in digestive tract epithelium and pancreatic tissues early in fetal life suggests a functional role for TGF- during the developmental process of the digestive system. We demonstrate that TGF- is also produced by endocrine cells and might have an additional mode of action other than paracrine, at least during fetal life.     

Basement membrane components of bronchial epithelium in humans suffering from chronic nonspecific lung diseases

Collagen IV and laminin are important constituents of the basement membrane (BM). By use of immunocytochemistry we examined the occurrence and distribution of these two components in the BM beneath normal, mucoid and metaplastic epithelium of large bronchi in 22 adults suffering from chronic nonspecific lung diseases. Both collagen IV and laminin were expressed as a thin and continuous layer beneath the epithelium in most tissue specimens with normal epithelium. In a few specimens the layer showed interruptions with a patchy distribution of the immunoreactivity. Three patterns of distribution of BM components were found under the metaplastic epithelium. Total absence of immunoreactive collagen IV and laminin was the most common variant. Weak and scarce staining for both proteins in the BM characterized the second pattern. The third variant showed strong collagen IV immunoreactivity but lack of laminin. The BM beneath the mucoid epithelium was characterized by irregular distribution of collagen IV and laminin. We suggest that the occurrence and distributional pattern of the BM components are related to the type of overlying epithelium and connected with an altered synthesis of these components.     

Maxi K+ channels on human vas deferens epithelial cells

The vas deferens forms part of the male reproductive tract and extends from the cauda epididymis to the prostate. Using the patch clamp technique, we have identified a Ca²⁺-activated, voltage-dependent, maxi K⁺ channel on the apical membrane of epithelial cells cultured from human fetal vas deferens. The channel had a conductance of 250 pS in symmetrical 140 mm K⁺ solutions, and was highly selective for K⁺ over Na⁺. Channel activity was increased by depolarization and by an elevation of bath (cytoplasmic) Ca²⁺ concentration, and reduced by cytoplasmic Ba²⁺ (5 mm) but not by cytoplasmic TEA (10 mm). Channel activity was also dependent on the cation bathing the cytoplasmic face of the membrane, being higher in a Na⁺-rich compared to a K⁺-rich solution. We estimated that up to 600 maxi K⁺ channels were present on the apical membrane of a vas cell, and that their density was 1–2 per ² of membrane. Activity of the channel was low on intact cells, suggesting that it does not contribute to a resting K⁺ conductance. However, fluid in the lumen of the human vas deferens has a high K⁺ concentration and we speculate that the maxi K⁺ channel could play a role in transepithelial K⁺ secretion.Funded by grants from the Cystic Fibrosis Trust and the Medical Research Council (UK). We thank Mr. David Stephenson for excellent technical assistance.     

L-Alanine absorption in human intestinal Caco-2 cells driven by the proton electrochemical gradient

In human Caco-2 intestinal epithelial layers, xxxl-alanine absorption can be energized by a proton gradient across the brush-border membrane. Acidification of the apical medium, even in Na⁺-free media, is associated with a saturable net transepithelial absorption of xxxl-alanine. xxxl-Alanine transport causes cytosolic acidification consistent with proton/amino acid symport. xxxl-Alanine transport in Na⁺-free media is rheogenic, stimulating an inward short-circuit current in voltageclamped epithelial monolayers. By measurement of rapid xxxl-alanine influx across the apical membrane, xxxl-alanine-stimulated inward short-circuit current and intracellular acidification in the same cell batch, we estimate xxxl-alanine/proton stoichiometry to be 10.62 ±0.25 (xxxsd) (short-circuit current) or 10.73 ±0.19 (intracellular acidification). From competition studies, it is likely that xxxl-proline, -aminoisobutyric acid, and -alanine, but not xxxl-valine and xxxl-serine, are substrates for protonlinked, substrate transport in the brush border of Caco-2 cells.This study was supported by the Wellcome Trust (to D.T.T. and N.L.S.) and the LINK Programme in Selective Drug Delivery and Targeting (funded by the SERC/MRC/DTI and Industry). Charlotte Ward gave excellent technical assistance.     

Conservation of human Y chromosome sequences among male great apes: Implications for the evolution of Y chromosomes

Nine newly described single-copy and lowcopy-number genomic DNA sequences isolated from a flow-sorted human Y chromosome library were mapped to regions of the human Y chromosome and were hybridized to Southern blots of male and female great ape genomic DNAs (Gorilla gorilla, Pan troglodytes, Pongo pygmaeus). Eight of the nine sequences mapped to the euchromatic Y long arm (Yq) in humans, and the ninth mapped to the short arm or pericentromeric region. All nine of the newly identified sequences and two additional human Yq sequences hybridized to restriction fragments in male but not female genomic DNA from the great apes, indicating Y chromosome localization. Seven of these 11 human Yq sequences hybridized to similarly-sized restriction endonuclease fragments in all the great ape species analyzed. The five human sequences that mapped to the most distal subregion of Yq (deletion of which region is associated with spermatogenic failure in humans) were hybridized to Southern blots generated by pulsed-field gel electrophoresis. These sequences define a region of approximately 1 Mb on human Yq in which HpaII tiny fragment (HTF) islands appear to be absent. The conservation of these human Yq sequences on great ape Y chromosomes indicates a greater stability in this region of the Y than has been previously described for most anonymous human Y chromosomal sequences. The stability of these sequences on great ape Y chromosomes seems remarkable given that this region of the Y does not undergo meiotic recombination and the sequences do not appear to encode genes for which positive selection might occur. Correspondence to: B. Steele Allen     

Fenfluramine Releases Serotonin from Human Brain Nerve Endings by a Dual Mechanism

Abstract: Fenfluramine is the most widely used anorexigenic drug in humans. In animal experiments d -fenfluramine has been shown to act as a potent releaser of brain serotonin [5-hydroxytryptamine (5-HT)]. Here we have investigated the effects of d -fenfluramine on the release of [³H]5-HT from isolated nerve endings of human neocortex. The drug elicited release of unmetabolized [³H]5-HT, and this effect was concentration dependent. However, the mechanism of release seems to differ profoundly depending on the concentrations of d -fenfluramine used. At 5 µ M , the release of [³H]5-HT was blocked by the 5-HT transporter inhibitor fluoxetine and was Ca²⁺ independent and insensitive to the human autoreceptor 5-HT_1D agonist sumatriptan. The release of [³H]5-HT elicited by 0.5 µ M d -fenfluramine was similarly blocked by fluoxetine, but it was strongly Ca²⁺ dependent and sensitive to sumatriptan. It is suggested that, at relatively high concentrations, d -fenfluramine largely diffuses into serotonergic terminals and causes release of 5-HT through the 5-HT carrier working in the inside-outside direction; at relatively low concentrations d -fenfluramine enters the terminals through the 5-HT transporter but elicits release of 5-HT by an exocytotic-like mechanism.     

Amino Acid Residues 226–240 of τ Which Encompass the First Lys-Ser-Pro Site of τ, Are Partially Phosphorylated in Alzheimer Paired Helical Filament-τ

Abstract: A synthetic peptide corresponding to residues 226–240 (E9 peptide) of human τ, which contains an Lys-Ser-Pro motif, was used to raise a polyclonal antibody. The antibody, E9, was 10-fold less reactive with phospho-E9 peptide than with native E9 peptide. E9 antibody was used to study the extent of phosphorylation in a modified form of τ (PHF-τ) that is found in Alzheimer's disease brain and is incorporated into paired helical filaments (PHFs). E9 immunolabeled Alzheimer's disease neurofibrillary tangles and abnormal neurites in brain sections intensely, with increased immunoreactivity detected after pretreatment of sections with phosphatase. On immunoblots and ELISA, E9 reacted with PHF-τ and recombinant human τ but not with the high and middle molecular weight neurofilament proteins. Phosphatase treatment of PHF-τ improved the E9 immunoreactivity by 30–50%. Dephosphorylated high but not middle molecular weight neurofilament protein became reactive with E9. These results indicate that <50% of the PHF-T is phosphorylated in the subregion corresponding to residues 226–240 of τ and suggest that the phosphorylation of this region may not be essential for PHF formation.