Polymorphisms in human H chain V region genes from the VHIII gene family

Polymorphisms of the Ig H chain V region (VH) genes were examined with probes from the coding and flanking regions of a gene from the largest VH gene family, VHIII. The 5'-flanking probe gave the simplest pattern and revealed the largest number of polymorphic fragments. Analysis of unrelated individuals and of families identified five polymorphic loci. Two alleles were detected for each of two of the loci, whereas a polymorphic band was scored as present or absent for the other three loci. The polymorphic fragments segregated in the expected Mendelian fashion and parental haplotypes could be assigned in all cases. Comparison of the patterns obtained with the flanking and coding region probes suggests that the human VHIII gene family is highly polymorphic and may contain several hundred V genes. This method, as well as the polymorphism detected, can be used to investigate the organization and germ-line variation of H chain V genes and their inheritance in normal individuals and in individuals with immunologic disorders.     

Regulation of Histamine Release and Synthesis in the Brain by Muscarinic Receptors

The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with L-[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+-induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1-receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with Ki values of 1-6 X 10(-8) M. 11-[(2-[(Diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116), considered a preferential M2-receptor antagonist, reversed the carbachol effect with a mean Ki of approximately 2 X 10(-7) M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+-induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H- labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1-receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF-DX 116.     

Characterization of Robertsonian translocations by using fluorescence in situ hybridization.

Fluorescence in situ hybridization with five biotin-labeled probes (three alphoid probes, a probe specific for beta-satellite sequences in all acrocentric chromosomes, and an rDNA probe) was used to characterize 30 different Robertsonian translocations, including three t(13;13); one t(15;15), four t(21;21), three t(13;14), two t(13;15), two (13;21), two t(13;22), one t(14;15), eight t(14;21), two t(14;22), and two t(21;22). Of 8 de novo homologous translocations, only one t(13;13) chromosome was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. The three monocentric nonhomologous translocations included both of the t(13;21) and one t(21;22). Two of 26 translocations studied using the beta-satellite probe showed a positive signal, while rDNA was undetectable in 10 cases studied. These results indicate that most homologous Robertsonian translocations appear monocentric, while the bulk of nonhomologous translocations show two alphoid signals. A majority of the breakpoints localized using this analysis seem to be distal to the centromere and just proximal to the beta-satellite and nuclear-organizing regions.     

Structure-activity relationships on hyperglycemia by representatives of the adipokinetic/hyperglycemic hormone family in Blaberus discoidalis cockroaches

Summary Several members of the adipokinetic/hyperglycemic neurohormone family from several different invertebrate species have been prepared by solid-phase peptide synthesis and assayed by a modified in vivo hyperglycemic bioassay in Blaberus discoidalis cockroaches. The hypertrehalosemic hormone (HrTH) is the endogenous hypertrehalosemic factor for B. discoidalis and was the most potent peptide in the assay. The more divergent the sequence of a family member from Blaberus HrTH, the less potent was the bioanalog. Manduca adipokinetic hormone is the most divergent peptide of the family and was totally inactive in the bioassay. Locusta adipokinetic hormone I had reduced maximum activity in the assay, which suggests that Ser⁵ is an important residue for the transduction of the hyperglycemic response. The direct relation between bioanalog similarity to Blaberus HrTH sequence and potency suggests that the hormone and target cell receptor for HrTH have evolved to maintain an optimal fit.Abbreviations AKH adipokinetic hormone - HrGH hyperglycemic hormone - HrTH hypertrehalosemic hormone - RPCH red pigmentconcentrating hormone - CAH cardioacceleratory hormone. Hormone abbreviations are according to the convention of Gäde and Raina (1989) except that the genus names are not abbreviated     

Glycerol accumulation while recycling thin stillage in corn fermentations to ethanol

Summary By succesive recycling of the thin stillage in mashing and fermenting fresh corn, the glycerol content in each fermentation increased by about 0.4% and accumulated to a high of 2.1% in the beer of the fifth recycle. Glycerol concentration declined after the fifth recycle. The original fermentation contained 0.8% glycerol.Presented in part at the Society for Industrial Microbiology Annual Meeting, August 7–12, 1988, Chicago, IL.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Microbial oxidation of ebastine

The microbial oxidation of ebastine to carebastine was investigated. Among the 15 micro-organisms examined, only theCunninghamella strains showed the desired biotransformation.Cunninghamella blakesleeana oxidised the substrate within 7 days, via the intermediates alcohol and aldehyde, mainly to carebastine, the corresponding carboxylic acid. Optimisation of the culture conditions increased the yield from initially 10% up to a reproducible 40%. For the synthesis of carebastine a substrate concentration of 200 mg/l, a starting pH of 5.0 and the addition of 1% poly(vinyl alcohol) is favourable. The results achieved in experiments with shaking flasks are transferable to the fermentation scale and yielded 270 mg carebastine in a 3-l fermentation of 600 mg ebastine. The progress of the reaction was detected by TLC and HPLC, the products were identified by mass spectrometry and NMR.     

The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor.

The ubiquitin-dependent protein degradation system has recently been implicated in downregulation of signal transducing receptors. Growth hormone receptor (GHR) cDNA was transfected into Chinese hamster ovary cells, which exhibit a temperature-sensitive defect in ubiquitin conjugation (CHO-ts20), as well as into wild-type cells (CHO-E36). Upon binding of growth hormone (GH), two GHR polypeptides dimerize and initiate signal transduction. In CHO-E36 and in CHO-ts20 at the permissive temperature the GHR was ubiquitinated and degraded in a GH-dependent fashion. However, at the non-permissive temperature in CHO-ts20 cells, neither GH-dependent uptake nor degradation of the GHR was observed, while in CHO-E36 cells both GHR uptake and degradation were accelerated. Incubation of CHO-E36 cells with inhibitors of endosomal/lysosomal function (NH4Cl, bafilomycin A1) markedly reduced ligand-induced GHR degradation. Our results indicate that a functional ubiquitin conjugating system is required for GH-induced endocytosis and that degradation of both the exoplasmic and cytoplasmic portions of the GHR occurs within the endosomal/lysosomal compartment.     

Evidence for structural heterogeneity from molecular cytogenetic analysis of dicentric Robertsonian translocations.

Most Robertsonian translocations are dicentric, suggesting that the location of chromosomal breaks leading to their formation occur in the acrocentric short arm. Previous cytogenetic and molecular cytogenetic studies have shown that few Robertsonian translocations retain ribosomal genes or beta-satellite DNA. Breakpoints in satellite III DNA, specifically between two chromosome 14-specific subfamilies, pTRS-47 and pTRS-63, have been indicated for most of the dicentric 14q21q and 13q14q translocations that have been studied. We have analyzed the structure of 36 dicentric translocations, using several repetitive DNA probes that localize to the acrocentric short arm. The majority of the translocations retained satellite III DNA, while others proved variable in structure. Of 10 14q21q translocations analyzed, satellite III DNA was undetected in 1; 6 retained one satellite III DNA subfamily, pTRS-47; and 3 appeared to contain two 14-specific satellite III DNA sub-families, pTRS-47 and pTRS-63. In 10/11 translocations involving chromosome 15, the presence of satellite III DNA was observed. Our results show that various regions of the acrocentric short arm, and, particularly, satellite III DNA sequences, are involved in the formation of Robertsonian translocations.     

Analysis of centromeric activity in Robertsonian translocations: implications for a functional acrocentric hierarchy

Approximately 90% of human Robertsonian translocations occur between nonhomologous acrocentric chromosomes, producing dicentric elements which are stable in meiosis and mitosis, implying that one centromere is functionally inactivated or suppressed. To determine if this suppression is random, centromeric activity in 48 human dicentric Robertsonian translocations was assigned by assessment of the primary constrictions using dual color fluorescence in situ hybridzation (FISH). Preferential activity/constriction of one centromere was observed in all except three different rearrangements. The activity is meiotically stable since intrafamilial consistency of a preferentially active centromere existed in members of six families. These results support evidence for nonrandom centromeric activity in humans and, more importantly, suggest a functional hierarchy in Robertsonian translocations with the chromosome 14 centromere most often active and the chromosome 15 centromere least often active.