Identification of a nuclear localization signal in activin/inhibin betaA subunit; intranuclear betaA in rat spermatogenic cells

Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin betaA subunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of beta-galactosidase fusion proteins that the betaA subunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear betaA was demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX-XII. In some pachytene spermatocytes, the pattern of betaA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, betaA immunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of betaA before spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear betaA species with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin betaA subunit present in activin dimers. These results suggest that activin/inhibin betaA may elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed betaA sequence acting directly within the nucleus. According to our immunohistochemical data, betaA may play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis.     

Polarized endocytosis and transcytosis in the hypothalamic tanycytes of the rat

Four types of tanycytes can be distinguished in the rat hypothalamus: ₁ and ₂ tanycytes establish an anatomical link between the ventricular cerebrospinal fluid (CSF) and the arcuate nucleus, whereas ₁ and ₂ tanycytes establish a link between CSF and portal blood. Endocytosis and transcytosis in these cells have been investigated by (1) immunocytochemistry with antibodies against molecular markers of the endocytotic and transcytotic pathways; (2) the administration of wheat germ agglutinin (WGA) into the ventricular or subarachnoidal CSF and following its internalisation by and its routing through tanycytes. The four populations of tanycytes show marked differences concerning the expression and subcellular location of proteins involved in endocytosis and transcytosis, such as clathrin, caveolin-1, Rab4 and ARF6. Thus, _1,2 tanycytes express caveolin-1 at the ventricular cell pole and at their terminals contacting the portal capillaries, whereas _1,2 tanycytes do not, suggesting that caveolae-dependant endocytosis does not occur in the latter and that, in _1,2 tanycytes, it may occur at both cell poles. In _1,2 tanycytes, clathrin is only expressed at the ventricular cell pole indicating that clathrin-dependant endocytosis operates for compounds present in the ventricular CSF and not for those exposed to the terminals. This agrees with the property of _1,2 tanycytes of internalising WGA through the ventricular cell pole but not through the terminals. The subcellular distribution in _1,2 tanycytes of WGA and of the proteins clathrin and Rab4 indicates that part of the internalised WGA follows the degradative pathway and part is sorted to a transcytotic pathway and that the transcytotic and the secretory pathways might intersect. Financial support was provided by grants 01/1050, from FIS, Spain (to J.L.B.) and 1030265, from FONDECYT, Chile (to E.M.R.)     

Selectins and monocyte chemotactic peptide as the markers of atherosclerosis activity

The role of adhesive selectin molecules in the process of atherogenesis is an open question. These molecules are known as markers of atherosclerosis activity, however, only some biological mechanisms are known up to now. In this study we examined the levels of soluble forms of E-, P-selectin and monocyte chemotactic protein (MCP-1) in the process of extracorporeal cholesterol elimination by LDL-apheresis. We measured the levels of sE-, sP-selectin and MCP-1 in the plasma before and after LDL-apheresis and in the washout solution from immunoabsorption columns Lipopak. Eighty measurements were performed repeatedly in 6 patients with severe familial hypercholesterolemia (FH) on long-term LDL-apheresis treatment. Before the procedure P-selectin levels were 204+/-179 ng/ml, E-selectin 32.1+/-33.7 ng/ml, MCP-1 323.8+/-121 pg/l, whereas after the procedure we found P-selectin levels 131.6+/-34 ng/ml, E-selectin 33.1+/-51 ng/ml, and MCP-1 200.4+/-15 pg/l. Levels of P-selectin were increased in the blood of patients with FH in spite of long-term intensive extracorporeal LDL-elimination, documenting thus the activity of atherosclerosis. The levels of P-selectin and MCP-1 decreased significantly after the hypolidemic procedure and could be used as another marker showing the effectivity of the extracorporeal LDL-cholesterol elimination (immediately after the procedure), and, after further verification, may serve as a marker for controlling the therapy efficacy.     

Prognostic value of apoptosis in breast cancer (pT1-pT2). A TUNEL, p53, bcl-2, bag-1 and Bax immunohistochemical study

Apoptosis or programmed cell death produces cells breaking into several fragments of nuclei, cytoplasm or both nuclei and cytoplasm, known as apoptotic bodies which can be visualized in haematoxylin-eosin staining. Some genes (promoters and suppressors) control this process and certain mutations may induce the expression of abnormal proteins, which can be detected by immunohistochemical staining. Apoptosis can be detected by the TUNEL method either identifying apoptotic bodies or cells at the initial stages of the fragmentation process. We have studied 186 cases of infiltrating ductal breast carcinoma, stages pT1-pT2, and analysed the prognostic significance of tumour recurrence and overall survival of apoptotic index (AI) through univariate and multivariate analysis. We have also studied the immunohistochemical protein expression of apoptosis promoter and suppressors gene (p53, nuclear expression; bcl-2 and Bax, cytoplasm expression; BAG-1, nuclear and cytoplasm expression). The results indicate prognostic significance of p53 and bcl-2 related to patient death and bcl-2 and tumour size to tumour recurrence, bcl-2 acting as a protector factor (apoptotic suppressor) in both situations. On the other hand, we have not found useful prognostic information of AI either to tumour recurrence or overall survival in univariate or multivariate studies. In this study, Bax expression does not provide a new prognostic role in breast carcinoma, although it contrasts to the bcl-2 action and accelerates death.     

Mapping of QTL for Body Conformation and Behavior in Cattle

Genome scans for quantitative trait loci (QTL) in farm animals have concentrated on primary production and health traits, and information on QTL for other important traits is rare. We performed a whole genome scan in a granddaughter design to detect QTL affecting body conformation and behavior in dairy cattle. The analysis included 16 paternal half-sib families of the Holstein breed with 872 sons and 264 genetic markers. The markers were distributed across all 29 autosomes and the pseudoautosomal region of the sex chromosomes with average intervals of 13.9 cM and covering an estimated 3155.5 cM. All families were analyzed jointly for 22 traits using multimarker regression and significance thresholds determined empirically by permutation. QTL that exceeded the experiment-wise significance threshold (5% level) were detected on chromosome 6 for foot angle, teat placement, and udder depth, and on chromosome 29 for temperament. QTL approaching experiment-wise significance (10% level) were located on chromosome 6 for general quality of feet and legs and general quality of udder, on chromosome 13 for teat length, on chromosome 23 for general quality of feet and legs, and on chromosome 29 for milking speed. An additional 51 QTL significant at the 5% chromosome-wise level were distributed over 21 chromosomes. This study provides the first evidence for QTL involved in behavior of dairy cattle and identifies QTL for udder conformation on chromosome 6 that could form the basis of recently reported QTL for clinical mastitis.     

Association of human DEAD box protein DDX1 with a cleavage stimulation factor involved in 3'-end processing of pre-MRNA

DEAD box proteins are putative RNA helicases that function in all aspects of RNA metabolism, including translation, ribosome biogenesis, and pre-mRNA splicing. Because many processes involving RNA metabolism are spatially organized within the cell, we examined the subcellular distribution of a human DEAD box protein, DDX1, to identify possible biological functions. Immunofluorescence labeling of DDX1 demonstrated that in addition to widespread punctate nucleoplasmic labeling, DDX1 is found in discrete nuclear foci approximately 0.5 microm in diameter. Costaining with anti-Sm and anti-promyelocytic leukemia (PML) antibodies indicates that DDX1 foci are frequently located next to Cajal (coiled) bodies and less frequently, to PML bodies. Most importantly, costaining with anti-CstF-64 antibody indicates that DDX1 foci colocalize with cleavage bodies. By microscopic fluorescence resonance energy transfer, we show that labeled DDX1 resides within a F?rster distance of 10 nm of labeled CstF-64 protein in both the nucleoplasm and within cleavage bodies. Coimmunoprecipitation analysis indicates that a proportion of CstF-64 protein resides in the same complex as DDX1. These studies are the first to identify a DEAD box protein associating with factors involved in 3'-end cleavage and polyadenylation of pre-mRNAs.