Developmental changes in inhibin-alpha gene expression in the mouse testis

Inhibin is a gonadal hormone which is composed of an alpha-subunit and one of two related beta-subunits (betaA, betaB). Inhibin is important for pituitary FSH regulation, normal follicle development and maintenance of the estrous cycle in the female, whereas the role of inhibin in the male is less clear. Thus, we examined the expression of the inhibin-alpha gene in testis during sexual maturation in male mice, to try to gain insight into its functions in the male. Male mice of the ICR strain attained fertility at 6 weeks of age, and histological analysis revealed that a functional testis was formed, with seminiferous tubules which contain mature sperm and with an abundant population of Leydig cells. Parallel with this sexual maturation, inhibin-alpha subunit protein synthesis increased, whereas synthesis of the activin betaA and activin betaB followed with a delayed time course. Inhibin-alpha mRNA also increased during this critical period, and this corresponded to a change in the methylation status of the inhibin-alpha gene. Taken together, our data reveal that activation of inhibin-alpha gene during testis development correlated with the histological maturation of the testis and the acquisition of fertility in male mice.     

<Emphasis Type="Italic">Flavobacterium koreense</Emphasis> sp. nov., <Emphasis Type="Italic">Flavobacterium chungnamense</Emphasis> sp. nov., and <Emphasis Type="Italic">Flavobacterium cheonanense</Emphasis> sp. nov., isolated from a freshwater reservoir

Taxonomic studies were performed on three strains isolated from Cheonho reservoir in Cheonan, Korea. The isolates were Gram-negative, aerobic, rod-shaped, non-motile, catalase-positive, and oxidase-positive. Colonies on solid media were cream-yellow, smooth, shiny, and circular. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belong to the genus Flavobacterium. The strains shared 98.6–99.4% sequence similarity with each other and showed less than 97% similarity with members of the genus Flavobacterium with validly published names. The DNA-DNA hybridization results confirmed the separate genomic status of strains ARSA-42^T, ARSA-103^T, and ARSA-108^T. The isolates contained menaqui-none-6 as the predominant menaquinone and iso-C_15:0, iso-C_15:0 3-OH, iso-Ci_15:1 G, and iso-C_16:0 3-OH as the major fatty acids. The genomic DNA G+C content of the isolates were 31.4–33.2 mol%. According to the phenotypic and genotypic data, these organisms are classified as representative of three novel species in the genus Flavobacterium, and the name Flavobacterium koreense sp. nov. (strain ARSA-42^T =KCTC 23182^T =JCM 17066^T =KACC 14969^T), Flavobacterium chungnamense sp. nov. (strain ARSA-103^T =KCTC 23183^T =JCM 17068^T =KACC 14971^T), and Flavobacterium cheonanense sp. nov. (strain ARSA-108^T =KCTC 23184^T =JCM 17069^T =KACC 14972) are proposed.     

Consistent manufacturing and quality control of a highly complex recombinant polyclonal antibody product for human therapeutic use

The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well-characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost-efficient cell banking and single-batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled-up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic-, protein-, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition.     

The etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus HERV-Fc1

We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.     

Neuropeptide profiling of the bovine hypothalamus: thermal stabilization is an effective tool in inhibiting post-mortem degradation

The hypothalamus is the central regulatory region of the brain that links the nervous system to the endocrine system via the pituitary gland. It synthesizes and secretes neuropeptide hormones, which in turn act to stimulate or inhibit the secretion of pituitary hormones. We have undertaken a detailed MS investigation of the peptides present in the bovine hypothalamus by adapting a novel heat stabilization methodology, which improved peptide discovery to direct our studies into the molecular mechanisms involved in bovine reproduction. The untreated samples contained large numbers of protein degradation products that interfered with the analysis of the neuropeptides. In the thermally stabilized samples, we were able to identify many more neuropeptides that are known to be expressed in the bovine hypothalamus. Furthermore, we have characterized a range of post-translational modifications that indicate the presence of processed intact mature neuropeptides in the stabilized tissue samples, whereas we detected many trimmed or truncated peptides resulting from post-mortem degradation in the untreated tissue samples. Altogether, using an optimized workflow, we were able to identify 140 candidate neuropeptides. We also nominate six new candidate neuropeptides derived from proSAAS, secretogranin-2 and proTRH.     

Development of single-stranded DNA aptamers for specific Bisphenol a detection

The development of reagents with high affinity and specificity to small molecules is crucial for the high-throughput detection of chemical compounds, such as toxicants or pollutants. Aptamers are short and single-stranded (ss) oligonucleotides able to recognize target molecules with high affinity. Here, we report the selection of ssDNA aptamers that bind to Bisphenol A (BPA), an environmental hormone. Using SELEX process, we isolated high affinity aptamers to BPA from a 10(15) random library of 60 mer ssDNAs. The selected aptamers bound specifically to BPA, but not to structurally similar molecules, such as Bisphenol B with one methyl group difference, or 4,4'-Bisphenol with 2 methyl groups difference. Using these aptamers, we developed an aptamer-based sol-gel biochip and detected BPA dissolved in water. This novel BPA aptamer-based detection can be further applied to the universal and high-specificity detection of small molecules.     

Pig transgenesis by Sleeping Beauty DNA transposition

Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.     

Impacts of introduced common wasps (Vespula vulgaris) on experimentally placed mealworms in a New Zealand beech forest

An introduced social wasp Vespula vulgaris may compete with native birds for honeydew and invertebrates in New Zealand forests. Experimentally hidden mealworms (Tenebrio molitor) persisted longer at two sites following wasp poisoning that at two sites where wasps were not poisoned. Mealworms persisted longer in the morning than in the afternoon within all study sites. An unusually low mealworm removal rate during a morning trial before wasp poisoning heavily influences the results of this experiment but we have no ecological reason to ignore it. Wasps may therefore be having a heavy impact on invertebrate abundance on very short time scales (within a day following dawn emergence). They may also remove cached food items that would otherwise be retrieved by the South Island robin (Petroica australis australis) during cold or dark feeding conditions.     

Protease-Activated Receptor 2 Activation Inhibits N-Type Ca2+ Currents in Rat Peripheral Sympathetic Neurons

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type Ca²⁺ currents (I_Ca-N) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated Ca²⁺ currents (I_Ca), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on I_Ca. This PAR-2-induced inhibition was almost completely prevented by ω-CgTx, a potent N-type Ca²⁺ channel blocker, suggesting the involvement of N-type Ca²⁺ channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited I_Ca–N in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ω-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type Ca²⁺ channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type Ca²⁺ channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals.