Ligand-independent tyrosine phosphorylation of the receptor encoded by the c-neu oncogene.

Incubation of Swiss 3T3 murine fibroblasts at low temperatures induces phosphorylation on tyrosine of a transmembrane protein of 175 kDa. This phenomenon is time and temperature dependent and reaches a maximum after 2 hr at 4 degrees C. The 175 kDa protein phosphorylated in vivo at low temperatures can be immunoprecipitated by phosphotyrosine antibodies and displays auto-kinase activity in vitro in the presence of radiolabelled ATP. This molecule was found to react with anti-peptide antibodies directed against the product of the HER2/neu proto-oncogene only when immunoprecipitated with phosphotyrosine antibodies from cold-stimulated cells. Activation of protein kinase-C by treatment of the cells with phorbol esters, bombesin or PDGF inhibits the effect of the exposure to low temperatures. Phosphorylation of p175 is not induced by treatment of the cells with the phosphatases inhibitor sodium orthovanadate. These results suggest that, at low temperatures, the tyrosine kinase associated with the putative receptor encoded by c-neu is activated by physico-chemical modifications of the plasma membrane.     

The insulin-like growth factor 1 receptor gene is normally biallelically expressed in human juvenile tissue and tumours

We have examined Insulln-like growth factor 1 receptor (IGF1R)gene expression for evidence of Imprinting in 15 informativepatients with embryonal tumours. Blallellic expression was observedIn all but one sample of normal juvenlle kidney and liver, andIn 9/10 associated Wllms' tumours, 3/3 hepatoblastomas and 2/2adrenal tumours. A single patient with Beckwlth—WledemannSyndrome (BWS) demonstrated monoallellc expression of the maternallyderived IGF1R allele In normal kidney, associated Wllms' tumourand In peripheral blood lymphocytes. The observed blallellcexpression of the IGF1R gene in all but one patient stronglysuggests that the human gene Is not normally Imprinted.     

MAFA-L,an ITIM-containing receptor encoded by the human NK cell gene complex and expressed by basophils and NK cells

The natural killer cell gene complex on human chromosome 12p12-13 encodes several C-type lectin receptor genes expressed by NK cells and other hematopoietic cells. We have identified a novel receptor gene in this region encoding a putative type II transmembrane glycoprotein. The product is 54 % identical to the rat mast cell function-associated antigen (MAFA), which inhibits mast cell activation by IgE. The human MAFA-like receptor (MAFA-L) and the rat MAFA protein are expressed by basophils and both have an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic tail, consistent with an inhibitory role in basophil activation. Unlike rat MAFA, expression of the MAFA-L gene is not limited to mast cells and basophils. In common with other genes in the NK cell gene complex MAFA-L is also expressed by natural killer cells as well as the monocyte-like cell-line U937. Expression in NK cells is restricted to peripheral blood NK cells, decidual NK cells do not express MAFA-L. While MAFA-L and rat MAFA might have a similar role in basophils, the expression of MAFA-L in other cell types implies additional functions for this molecule. The presence of the MAFA-L gene in the human NK cell complex indicates that this locus encodes C-type lectin receptors expressed by a variety of cells important in host defense.     

Detection and characterization of the protein encoded by the v-rel oncogene

To identify the protein encoded by v-rel, the oncogene of reticuloendotheliosis virus (REV-T), antisera have been raised to three synthetic peptides derived from the translation of our previously published v-rel DNA sequence [R.M. Stephens, N.R. Rice, R.R. Hiebsch, H.R. Bose, Jr., and R.V. Gilden, Proc. Natl. Acad. Sci. USA 80, 6229-6233 (1983)]. Sera to all three peptides precipitate a 59,000 Da protein from REV-T-transformed chicken lymphoid cells. This protein is not detectable in uninfected chick embryo fibroblasts, and its observed size is in good agreement with the 56,000 Da predicted by the DNA sequence. We conclude that this protein is the v-rel product and designate it p59rel. To search for evidence of post-translational processing of this protein, cells were grown in the presence of glycosylation inhibitors. These resulted in no detectable difference in the size of p59rel. Nor was its size detectably altered during the course of a pulse-chase experiment. Growth of cells in the presence of [32P] orthophosphate, however, revealed that p59rel is a phosphoprotein. It is also closely associated with a protein kinase activity, for precipitation with one of the peptide antisera (but not the other two) resulted in strong kinase activity in the immune complex pellet. During this reaction, p59rel itself becomes phosphorylated. Kinase activity was retained in the immune complex following detergent and high salt washes, leaving open the possibility that p59rel is itself a kinase.     

Quantitative study of testicular angiotensin-converting enzyme on the surface of human spermatozoa

Expression of testicular angiotensin-converting enzyme on the surface of human spermatozoa was studied by means of flow cytometry with monoclonal antibodies. Expression of testicular angiotensin-converting enzyme on the cell surface depended on functional and morphological characteristics of spermatozoa. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 1, pp. 41–44, January, 2006     

Identification of the mineralocorticoid receptor in human spermatozoa

Aldosterone seems to play a role in the regulation of the electrolyte content of sperm and in the motility of spermatozoa. The aim of the study was to evaluate the presence of the mineralocorticoid receptor (MR) in human ejaculated spermatozoa. We have assayed MR on spermatozoa of freshly ejaculated sperm from healthy donors. The identification of MR was made by using immunohistochemistry and immunofluorescence analyses, while MR mRNA expression was evaluated by real-time PCR assay. The immunohistochemical and immunofluorescence analyses showed positive staining both in the midpiece and in the tail of the spermatozoa. Relative quantification of MR by using real-time PCR shows that the mRNA expression of MR in spermatozoa is lower than in mononuclear leukocytes (positive controls). Sequencing showed complete identity between the sequence obtained from spermatozoa and the human MR cDNA sequence. Further studies should be performed in order to elucidate a possible physiological role of aldosterone in regulating electrolyte concentration, and the pro-oxidant effect of excess aldosterone in this new target tissue.     

C-MET is expressed in the majority of penile squamous cell carcinomas and correlates with polysomy-7 but is not associated with MET oncogene amplification,pertinent histopathologic parameters,or with cancer-specific survival

We assessed c-MET expression and oncogene amplification in a cohort enrolling 92 surgically treated penile squamous cell carcinomas (PSCCs). A tissue microarray was constructed for c-MET immunohistochemistry (IHC) and chromogenic silver in situ hybridization (SISH). Two independent pathologists evaluated IHC by employing the breast cancer scoring rules, and scored the presence of MET oncogene amplification and/or polysomy-7. Eighty study cases (87%) showed c-MET expression. No study case had MET oncogene amplification, but 42 patients (45.7%) had polysomy-7. Polysomy-7 showed a significant positive correlation with c-MET expression (ρ = 0.323, p = 0.002). Neither c-MET expression nor polysomy-7 was associated with histopathologic parameters or with cancer-specific survival (median post-surgical follow-up 32 months). Our data suggest that the majority of PSCCs exhibit c-MET expression which is not associated with oncogene amplification, but might be attributable to polysomy-7. Further studies should investigate the expression and activation of downstream molecules functionally involved in c-MET pathway signaling, and clarify the so far unresolved role of c-MET inhibitors as potential targeted therapies in PSCCs with metastatic dissemination.     

The lipopolysaccharide co-receptor CD14 is present and functional in seminal plasma and expressed on spermatozoa

CD14 is a 54 000-molecular weight (MW) glycolipid-anchored membrane glycoprotein, expressed on myeloid cells, which functions as a member of the lipopolysaccharide (LPS) receptor complex. Soluble forms of CD14 have been reported in plasma, cerebrospinal fluid, amniotic fluid and breast milk. In plasma and breast milk, soluble CD14 has been implicated as a regulator of T- and B-cell activation and function. Expression of CD14 in the male reproductive system has not previously been investigated. We here show that soluble CD14 is present in seminal plasma at levels comparable to those in serum. Spermatozoa expressed CD14 on their membranes, as demonstrated by fluorescence microscopy and flow cytometry. Post-vasectomy, the levels of seminal plasma CD14 (spCD14) were much reduced, implying an origin distal to the point of transection of the vas deferens. Ultracentrifugation analyses demonstrated that spCD14 was not associated with lipid complexes, indicating that it lacks the glycolipid anchor. Purified spCD14 mediated activation by LPS of CD14-negative cells. These findings suggest that CD14 may play a hitherto unexplored role in immune defence and cell activation in the male reproductive tract.     

Integrins and adhesion molecules: P-selectin is expressed on the oolemma of human and hamster oocytes following sperm adhesion and is also detected on the equatorial region of acrosome-reacted human spermatozoa

Selectins are a family of adhesive molecules, involved in theinteractions between leukocytes and endothelium and in plateletadhesion. P-selectin, one of the members of this family, isstored in     

The human cationic antimicrobial protein (hCAP-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa

Innate immunity is important for the integrity of the host against potentially invasive pathogenic microorganisms in the environment. Antibiotic peptides with broad antimicrobial activity are part of the innate immune system. We investigated the presence of the cathelicidin, human cationic antimicrobial protein (hCAP-18), in the male reproductive system. We found strong expression of the hCAP-18 gene by in situ hybridization and hCAP-18 protein, as detected by immunohistochemistry, in the epithelium of the epididymis, but not in the testis. The highest expression in the epididymis was in the caudal part. Western blotting showed a doublet band, the upper part corresponding to the size of hCAP-18 in plasma and neutrophils. Using a specific enzyme-linked immunosorbent assay (ELISA), levels of 86.5 +/- 37.8 microg/ml (mean +/- standard deviation; range, 41.8 to 142.8 microg/ml; n = 10) were detected in seminal plasma from healthy donors, which is 70-fold higher than the level in blood plasma. Flow cytometry and immunocytochemistry revealed the presence of hCAP-18 on spermatozoa. ELISA measurement showed levels of 196 ng/10(6) spermatozoa, corresponding to 6.6 x 10(6) molecules of hCAP-18 per spermatozoon. Our results suggest a key role for hCAP-18 in the antibacterial integrity of the male reproductive system. The attachment of hCAP-18 to spermatozoa may implicate a role for hCAP-18 in conception.     

The human beta-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal beta-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the beta-glucan receptor (betaGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional betaGR isoforms - betaGR-A and betaGR-B - were expressed by these cell populations in peripheral blood; however, only betaGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other beta-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major beta-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.     

Leptin receptor is expressed by epidermis and skin appendages in dog

Leptin is a polypeptide secreted by adipocytes which binds to a specific receptor (Ob-R) that is expressed in various tissues. The wide distribution of the Ob-R suggests that leptin might exert diverse biological functions, not only by regulating energy metabolism and appetite, but also by acting as a mitogen in many cell types, including keratinocytes.In this study, the presence and localization of Ob-R was investigated in the skin of the dog using RT-PCR and immunohistochemical techniques. RT-PCR revealed the presence of Ob-R m-RNA in the skin specimens collected from the dorsal region of two smooth coat breed dogs. Through immunohistochemistry performed on the skin of five dogs, the expression of the receptor was observed in the basal layer of the epidermis, in the hair follicles as well as in the apocrine sweat and sebaceous glands. No staining for Ob-R was detected in the suprabasal epidermis layers. Strong positive signals were observed in many cells of the outer root sheath of hair follicles in growing and in regressive phases. The identification of Ob-R in the above targets suggests that leptin may play a role in the regulation of cyclic renewal of the epidermis and skin appendages in dog. This study represents an important contribution to understand the complex mechanisms that are involved in the skin biology in this species.     

The human EGF-TM7 family member EMR2 is a heterodimeric receptor expressed on myeloid cells

The EGF-TM7 family is a group of class B seven-span transmembrane (TM7) receptors expressed predominantly by cells of the immune system. Family members CD97, EMR1, EMR2, EMR3, and ETL are characterized by an extended extracellular region with a variable number of N-terminal epidermal growth factor (EGF)-like domains coupled to a TM7 domain by a stalk. The EGF domain region of the recently identified EMR2 differs from that of CD97 in only 6 out of 236 amino acids. Although small, this difference has been shown to alter ligand specificity. To analyze the structure and cellular distribution of EMR2, a specific monoclonal antibody (2A1) was generated. Use of 2A1 has demonstrated EMR2, like CD97, to be expressed as a heterodimeric receptor consisting of an extracellular alpha part and a TM7/cytoplasmic beta part. Analysis of EMR2 expression on primary blood leukocytes, on hematopoietic cells lines, and in situ revealed a myeloid-restricted profile. Highest expression levels were detected on the more mature CD16(+) blood monocytes, on macrophages, and on BDCA-3(+) myeloid DC, whereas little if any expression was found on granulocytes. Unlike CD97, no expression was observed on resting or activated lymphocytes. Different expression patterns and the inability of EMR2 to interact with the CD97 ligand CD55 indicate that the molecular twins EMR2 and CD97 likely have nonredundant functions.     

The role of mannose receptor on HIV-1 entry into human spermatozoa

In this opinion article we consider the possibility that human spermatozoa have receptors for human immunodeficiency virus-1 (HIV-1). It is clear that sperm cells have the potential for transmitting HIV-1, but the mechanisms responsible for spreading or the virus by this vector are not known. In contrast to the traditional HIV-1 target cells, spermatozoa do not express CD4 receptors or the CCR5/CXCR4 co-receptors. Recent evidence indicates that astrocytes, which also do not express these molecules, can be infected with HIV-1 through the mannose receptor. Furthermore, a 160-kDa sperm receptor that interacts with the HIV gp 120 has been described. Therefore, we hypothesize that the mannose receptor, of 165-175 kDa, is the receptor that HIV-1 uses to invade spermatozoa, which could lead to both vertical and horizontal transmission of HIV-1.