Absence of polymorphism between HLA-B27 genomic exon sequences isolated from normal donors and ankylosing spondylitis patients

Ninety percent of individuals with ankylosing spondylitis (AS) express HLA-B27. To determine if HLA-B27 coding sequences from normal vs AS individuals show differences that might relate to the etiology of the disease, the gene coding for this allele was cloned from three different partial genomic libraries. These libraries were made with DNA from three different cell lines expressing HLA-B27: MRWC (HLA-B27, 14), obtained from an AS patient; KCA (HLA-B27, w44), obtained from a known normal individual; and MVL (HLA-B27, 27), a homozygous consanguineous cell line of unknown origin. To increase the number of clones coding for the HLA-B locus, partial libraries were made using a complete Eco RI digestion of genomic DNA in the lambda vector 607. The libraries were screened with two probes; one probe hybridizes to all HLA-A, B, C class I genes, and the other to a small subpopulation of class I genes, including the B locus. DNA from clones hybridizing with both probes was transfected into murine L cells. Cell surface expression of HLA-B27 on murine L cells was detected with a polymorphic monoclonal antibody (ME1) specific for HLA-B27, 7, 22. DNA from those clones positive for HLA-B27 by transfection was subcloned into the Xba I site of M13mp18 and the DNA sequence for exons 2 through 4 (encoding domains alpha 1, alpha 2, and alpha 3) was determined by the dideoxy technique by using synthetic oligonucleotide primers or the M13 primer. The resulting sequences show no difference between HLA-B27 alpha 1, alpha 2, alpha 3 domains from a known AS patient and a known normal individual.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Permanent suppression of phase separation cataract in calf lens using amine modification agents

Low temperature induced opacification (cold cataract) of the nucleus of young mammalian lenses is associated with a phase separation of proteins in the lens cell cytoplasm. Calf lenses were treated with a variety of imido-esters and N-hydroxysuccinimide-esters, which react specifically with amino groups. Many potent inhibitors of phase separation cataract were identified which lower the opacification temperature by 6 degrees C or more. Lenses generally remain clear, colorless and soft. Furthermore, suppression of the cold cataract temperature is permanent upon removal of excess reagent.     

Evolution of the HLA-DR1 gene family. Structural and functional analysis of the new allele "DR-BON".

The molecules of the MHC are highly polymorphic and involve in Ag presentation; their striking genetic polymorphism allows probable interactions with a large variety of antigenic fragments when these are presented to the TCR. It is therefore of interest to explore the extent of this polymorphism and the mechanisms of its generation. We have studied the class II HLA-DR blank allele DR-BON that has been previously defined by MLR, restriction fragment length polymorphism, and two-dimensional gel electrophoresis. A cDNA library was constructed from a DR-BON homozygous typing cell and cDNA corresponding to DR alpha- and DR beta-chains were sequenced. By comparison with other known alpha- and beta-chain sequences it is shown that the alpha-chain is invariant and the beta-chain differs from DR1 by only six nucleotides, clustered in the third variable region with three amino acid changes at position 67, 70, and 71. The short DNA stretch of sequence encoding the 67-71 region is also present in other DR alleles: DR4/Dw10, DRw13, and some DRw11 specificities. Therefore we propose that a gene conversion-like event has occurred between the DRB1 *0101 (DR1/Dw1) gene and one of these three DRB1 genes. Extensive typing has been performed with a DR-BON-specific 17-mer oligonucleotide. Cross-hybridization with other genes than the ones expected from DNA sequence comparison was not observed. A selected panel of DR-BON reactive T cell clones shows three patterns of reactivity. Some clones are strictly DR-BON specific; some cross-reacted with DRw13 and a few cross-reacted with other haplotypes. The role of different epitopes of the third variable region of HLA-DR beta chain in allo-reaction is discussed.     

Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation

The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat?, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat+-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat? information. The transformants behave in crosses as do the reference mat+ or mat? strains, thus indicating that the transgenic mat+ and mat? are fully functional even when they have integrated at ectopic sites.     

Organophosphate and Pyrethroid Hydrolase Activities of Mutant Esterases from the Cotton Bollworm Helicoverpa armigera

Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes’ active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4–6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.     

Association between pygmy right whales (Caperea marginata) and areas of high marine productivity off Australia and New Zealand

Abstract

Opportunistic sightings and strandings of Caperea marginata (n=196) from the vicinity of Australia and New Zealand (1884 to early 2007) were used to relate geographic and temporal patterns to oceanographic and broad-scale climatic variability. Records were not uniformly distributed along the coast and more (69%) were from Australia than New Zealand. Seven coastal whale ‘hotspots’ were identified which accounted for 61% of records with locality data. Half of the hotspot records were from southeast (37) and northwest (20) Tasmania—others each had 9–15 events. Upwelling and/or high zooplankton abundance has been documented near all whale hotspots. Records of C. marginata occurred in all months, with 75% in spring and summer. Inter-annual variability showed broad agreement between increased whale records (usually in spring/summer) and strongly positive ‘Niño 3.4’ during 1980–1995 but not thereafter. Coastal upwelling and productivity increase during climatic phenomena such as El Niño and are likely to be quickly beneficial to plankton-feeding whales such as C. marginata.     

Characterization of an insulin receptor-related receptor in Biomphalaria glabrata embryonic cells

Tyrosine kinase receptors play a key role in the communication of cells with their environment. Growth hormone receptors, such as insulin receptors, are involved in the regulation of cell growth, differentiation and metabolism in multicellular organisms. Insulin-related peptides and members of the insulin receptor subfamily have been described in a wide variety of invertebrates, including freshwater molluscs. In this paper, we describe the metabolic effect of insulin on a mollusc cell line (Bge) derived from embryos of the snail Biomphalaria glabrata. Using a PCR strategy, we have cloned from Bge cells a cDNA encoding a protein (BgIR) homologous to, and exhibiting all of the typical features of insulin receptors. Northern blot analysis confirmed the expression of BgIR in B. glabrata snails and suggested its wide distribution in the snail body. Bge cells have been shown to provide the environmental conditions necessary for the in vitro development of the sporocysts of Schistosoma mansoni, a trematode parasite that uses B. glabrata as an intermediate host. The possible implication of BgIR in the activating and proliferating processes observed in Bge cells during their coculture with S. mansoni larvae is discussed.