Intracellular pathway and kinetics of protein secretion in the coagulating gland of the mouse

The coagulating gland of male rodents is part of the prostatic complex. Various mechanisms of secretion have been postulated, in part because organelles commonly involved in the secretory process possess unusual features, such as extreme distension of the rough endoplasmic reticulum. In the present study, the pathway, kinetics, and mode of secretion in the coagulating gland of the mouse were studied by electron microscope autoradiography at intervals between 5 min and 8 h after administration of 3H-threonine. The percentage of grains associated with the rough endoplasmic reticulum was initially high and generally decreased throughout the experiment, while a pronounced rise in the proportion of grains associated with the Golgi apparatus and secretory granules was observed 6 h after injection of precursor. In addition, there was a smaller elevation in the percentage of grains over the Golgi apparatus and secretory granules between 1 and 4 h, and radioactive material first reached the lumen of the gland 4 h after injection of the precursor. Although the general pathway of intracellular transport of secretory protein resembles that in other cells, the results indicate that there are several unusual aspects to the secretory process in the coagulating gland. First, the rate of transport was markedly slower than in most other exocrine gland cells, since the bulk of the labeled protein did not reach the Golgi apparatus and secretory granules until 6 h after administration of precursor. This reflected prolonged retention of secretory products in the endoplasmic reticulum. Second, in addition to the major bolus of labeled material that traversed the cells at about 6 h, a smaller wave of radioactivity appeared to pass through the Golgi apparatus and secretory granules and reach the lumen earlier, within the first few hours after the injection. Finally, the primary mode of secretion in the coagulating gland appears to be merocrine because the secretory granules contained much labeled protein.     

Generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6

Spontaneous pathologic arterial calcifications in childhood can occur in generalized arterial calcification of infancy (GACI) or in pseudoxanthoma elasticum (PXE). GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. However, the genetic basis in subsets of both disease phenotypes remains elusive. We hypothesized that GACI and PXE are in a closely related spectrum of disease. We used a standardized questionnaire to retrospectively evaluate the phenotype of 92 probands with a clinical history of GACI. We obtained the ENPP1 genotype by conventional sequencing. In those patients with less than two disease-causing ENPP1 mutations, we sequenced ABCC6. We observed that three GACI patients who carried biallelic ENPP1 mutations developed typical signs of PXE between 5 and 8 years of age; these signs included angioid streaks and pseudoxanthomatous skin lesions. In 28 patients, no disease-causing ENPP1 mutation was found. In 14 of these patients, we detected pathogenic ABCC6 mutations (biallelic mutations in eight patients, monoallelic mutations in six patients). Thus, ABCC6 mutations account for a significant subset of GACI patients, and ENPP1 mutations can also be associated with PXE lesions in school-aged children. Based on the considerable overlap of genotype and phenotype of GACI and PXE, both entities appear to reflect two ends of a clinical spectrum of ectopic calcification and other organ pathologies, rather than two distinct disorders. ABCC6 and ENPP1 mutations might lead to alterations of the same physiological pathways in tissues beyond the artery.     

The diversity of O-linked glycans expressed during Drosophila melanogaster development reflects stage- and tissue-specific requirements for cell signaling

Appropriate glycoprotein O-glycosylation is essential for normal development and tissue function in multicellular organisms. To comprehensively assess the developmental and functional impact of altered O-glycosylation, we have extensively analyzed the non-glycosaminoglycan, O-linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by beta-elimination, we detected novel as well as previously reported O-glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Galbeta1-3GalNAc) is the predominant glycan in the total profile. HexNAcitol, hexitol, xylosylated hexitol, and branching extension of core 1 with HexNAc (to generate core 2 glycans) were also evident following release and reduction. After Galbeta1-3GalNAc, the next most prevalent glycans were a mixture of novel, isobaric, linear, and branched forms of a glucuronyl core 1 disaccharide. Other less prevalent structures were also extended with HexA, including an O-fucose glycan. Although the expected disaccharide product of the Fringe glycosyltransferase, (GlcNAcbeta1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAcbeta1-3(GlcAbeta1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcA-extended form of the Fringe product was enriched in the dorsal portion of the wing imaginal disc. Furthermore, loss of Fringe activity reduced the prevalence of the O-Fuc trisaccharide. Therefore, O-Fuc glycans necessary for the modulation of important signaling events in Drosophila are, as in vertebrates, substrates for extension beyond the addition of a single HexNAc.     

Autonomy and Reproductive Rights of Married Ikwerre Women in Rivers State,Nigeria

A woman’s lack of or limited reproductive autonomy could lead to adverse health effects, feeling of being inferior, and above all being unable to adequately care for her children. Little is known about the reproductive autonomy of married Ikwerre women of Rivers State, Nigeria. This study demonstrates how Ikwerre women understand the terms autonomy and reproductive rights and what affects the exercise of these rights. An exploratory research design was employed for this study. A semi-structured interview schedule was used to conduct thirty-four in-depth interviews and six focus group discussions with purposively sampled educated, semi-educated, and uneducated Ikwerre women in monogamous or polygynous marriages. The collected data was analysed qualitatively with MAXQDA 11 using open and axial coding. The interviews and focus group responses reveal a low level of awareness of autonomy and reproductive rights amongst the Ikwerre women in Nigeria. While some educated women were aware of their reproductive rights, cultural practices were reported to limit the exercise of these rights. Participants reported that Ikwerre culture is a patriarchal one where married women are expected to submit and obey their husbands in all matters; and a good married woman according to Ikwerre standard is one who complies with this culture. Women’s refusal of sexual advances from their husbands is described as not being acceptable in this culture; and hence rape in marriage is not recognized in Ikwerre culture. Education and awareness creation on the importance of women’s reproductive autonomy could improve their reproductive rights and autonomy in marital settings. Overcoming the patriarchal aspects of Ikwerre culture—for example, the greater value placed on male children than female children and treating women as incompetent individuals—is necessary to promote gender equality as well as help improve women’s reproductive autonomy.     

Biochemical Characterization of the Chlamydomonas reinhardtii α-1,4 Glucanotransferase Supports a Direct Function in Amylopectin Biosynthesis

Plant α-1,4 glucanotransferases (disproportionating enzymes, or D-enzymes) transfer glucan chains among oligosaccharides with the concomitant release of glucose (Glc). Analysis of Chlamydomonas reinhardtii sta11-1 mutants revealed a correlation between a D-enzyme deficiency and specific alterations in amylopectin structure and starch biosynthesis, thereby suggesting previously unknown biosynthetic functions. This study characterized the biochemical activities of the α-1,4 glucanotransferase that is deficient in sta11-1 mutants. The enzyme exhibited the glucan transfer and Glc production activities that define D-enzymes. D-enzyme also transferred glucans among the outer chains of amylopectin (using the polysaccharide chains as both donor and acceptor) and from malto-oligosaccharides into the outer chains of either amylopectin or glycogen. In contrast to transfer among oligosaccharides, which occurs readily with maltotriose, transfer into polysaccharide required longer donor molecules. All three enzymatic activities, evolution of Glc from oligosaccharides, glucan transfer from oligosaccharides into polysaccharides, and transfer among polysaccharide outer chains, were evident in a single 62-kD band. Absence of all three activities co-segregated with the sta11-1 mutation, which is known to cause abnormal accumulation of oligosaccharides at the expense of starch. To explain these data we propose that D-enzymes function directly in building the amylopectin structure.     

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.