A reevaluation of the amino acid sequence of human follitropin beta-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin -subunit sequence (hFSH), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSH with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.     

A unique DR-related B cell differentiation antigen

The Ia or class II molecules in both mouse and man are the basis for the genetic control of the immune response. In addition to DR, other class II antigens have been described in man. We describe a new human Ia antigen K19, recognized by three monoclonal antibodies (HK-9, HK-19, and HK-20). This antigen has the general biochemical characteristics of an Ia antigen but is different from a DR antigen. Further, this antigen is found only on mature B lymphocytes and not on monocytes and activated T cells. Thus, this antigen may represent a new Ia-like molecule that is preferentially expressed on mature B cells.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. III. A single VKII gene and one of several JK genes are joined by an invariant arginine to form the most common L chain V region

To characterize the L chain V region repertoire of IgG anti-Haemophilus influenzae type b capsular polysaccharide (Hib-PS) antibodies, clonal antibodies were purified from immune serum and internal amino acid sequences of VKII anti-Hib-PS L chains obtained. We examined VKII L chains because it is the most common VL family expressed in the anti-Hib-PS response. Comparison of VKII amino acid sequences, including the entire CDR2 and CDR3 regions, of five anti-Hib-PS clonal antibodies from four unrelated individuals revealed complete identity with the exception of a single CDR3 residue from one antibody. When the sequence of these antibodies was compared with known VKII genes and myeloma proteins, it was found to be identical to the human VKII gene, A2, whose genomic sequence is presented here. In addition, all five of the VKII anti-Hib-PS antibodies examined contain an arginine inserted at the V-J junction. Finally, in contrast to the extraordinary homology of the VKII-encoded residues, there is variability in the JK gene utilization by these antibodies. These results demonstrate that the most common L chain V region in IgG anti-Hib-PS antibodies is the product of a single germ-line gene. The invariant arginine insertion suggests that this residue has an important role in Ag binding.     

25OHD, a circulating vitamin D metabolite in fish.

Serum and hepatic 25-hydroxyvitamin D (25OHD), and serum calcium, phosphate, 25OHD₃ binding capacity and binding affinity were measured in male and female trout. Both serum and hepatic 25OHD levels are decreased in female trout with elevations in protein bound calcium and phosphate. Whereas the apparent dissociation constant (Kd) for serum binding of 25OHD₃ of 1.0–2.0 × 10^?9M is similar in males and females, the 25OHD₃ binding capacity of hypercalcemic spawning trout (1.39 × 10^?7M) is significantly less than that of male fish (1.88 × 10^?7M). At circulating serum concentrations of 25OHD which average 9.5 × 10^?9M only 5–7% of trout serum 25OHD binding sites are occupied.     

Tbc1d15–17 regulates synaptic development at the Drosophila neuromuscular junction

Members of the Tre-2/Bub2/Cdc16 (TBC) family of proteins are believed to function as GTPase-activating proteins (GAPs) for Rab GTPases, which play pivotal roles in intracellular membrane trafficking. Although membrane trafficking is fundamental to neuronal morphogenesis and function, the roles of TBC-family Rab GAPs have been poorly characterized in the nervous system. In this paper, we provide genetic evidence that Tbc1d15–17, the Drosophila homolog of mammalian Rab7-GAP TBC1d15, is required for normal presynaptic growth and postsynaptic organization at the neuromuscular junction (NMJ). A loss-of-function mutation in Tbc1d15–17 or its presynaptic knockdown leads to an increase in synaptic bouton number and NMJ length. Tbc1d15–17 mutants are also defective in the distribution of the postsynaptic scaffold Discs-large (Dlg) and in the level of the postsynaptic glutamate subunit GluRIIA. These postsynaptic phenotypes are recapitulated by postsynaptic knockdown of Tbc1d15–17. We also show that presynaptic overexpression of a constitutively active Rab7 mutant in a wild-type background causes a synaptic overgrowth phenotype resembling that of Tbc1d15–17 mutants, while a dominant-negative form of Rab7 has the opposite effect. Together, our findings establish a novel role for Tbc1d15–17 and its potential substrate Rab7 in regulating synaptic development.     

Development of SSR markers by next-generation sequencing of Korean landraces of chamoe (Cucumis melo var. makuwa)

The oriental melon (Cucumis melo var. makuwa), called ‘chamoe’ in Korean, is a popular fruit crop cultivated mainly in Asia and a high-market value crop in Korea. To provide molecular breeding resources for chamoe, we developed and characterized genomic SSR markers from the preliminary Illumina read assemblies of Gotgam chamoe (one of the major landraces; KM) and SW3 (the breeding parent). Mononucleotide motifs were the most abundant type of markers, followed by di-, tri-, tetra-, and pentanucleotide motifs. The most abundant dinucleotide was AT, followed by AG and AC, and AAT was the most abundant trinucleotide motif in both assemblies. Following our SSR-marker development strategy, we designed a total of 370 primer sets. Of these, 236 primer sets were tested, exhibiting 93 % polymorphism between KM and SW3. Those polymorphic SSRs were successfully amplified in the netted and Kirkagac melons, which respectively exhibited 81 and 76 % polymorphism relative to KM, and 32 and 38 % polymorphism relative to SW3. Seven selected SSR markers with a total of 17 alleles (2–3 alleles per locus) were used to distinguish between KM, SW3, and four chamoe cultivars. Our results represent the first attempt to provide genomic resources for Korean landraces for the purposes of chamoe breeding, as well as to discover a set of SSR markers capable of discriminating chamoe varieties from Korea and the rest of Asia, which possess little genetic diversity. This study establishes a highly efficient strategy for developing SSR markers from preliminary Illumina assemblies of AT-rich genomes.     

Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes ≫ heart > cerebrum ≥ ileum > stomach = liver = jejunum ≥ epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.     

Quorum sensing: an emerging link between temperature and membrane biofouling in membrane bioreactors

Lab-scale membrane bioreactors (MBRs) were investigated at 12, 18, and 25?°C to identify the correlation between quorum sensing (QS) and biofouling at different temperatures. The lower the reactor temperature, the more severe the membrane biofouling measured in terms of the transmembrane pressure (TMP) during filtration. More extracellular polymeric substances (EPSs) that cause biofouling were produced at 18?°C than at 25?°C, particularly polysaccharides, closely associated with QS via the production of N-acyl homoserine lactone (AHL). However, at 12?°C, AHL production decreased, but the release of EPSs due to deflocculation increased the soluble EPS concentration. To confirm the temperature effect related to QS, bacteria producing AHL were isolated from MBR sludge and identified as Aeromonas sp., Leclercia sp., and Enterobacter sp. through a 16S rDNA sequencing analysis. Batch assays at 18 and 25?°C showed that there was a positive correlation between QS through AHL and biofilm formation in that temperature range.     

Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans

A method to rapidly generate gene replacement constructs by fusion PCR is described for Aspergillus nidulans. The utility of the approach is demonstrated by green fluorescent protein (GFP) tagging of A. nidulans ndc80 to visualize centromeres through the cell cycle. The methodology makes possible large-scale GFP tagging, promoter swapping, and deletion analysis of A. nidulans.