Taq I-generated HLA-DQ αpolymorphism in Japanese patients with narcolepsy

Taq I-generated HLA-DQrestriction fragment length polymorphism was examined in Japanese patients with narcolepsy. All patients were DR2 positive and shared a 6.0 kb fragment, although this fragment was found only in 54 % of the healthy DR2-positive Japanese. This finding added the DQ gene to the list of candidates for the possible narcolepsy-susceptibility gene. In contrast, there was no complete association between narcolepsy and DXrestriction fragment length polymorphism. These findings suggest that a narcolepsy-susceptibility gene is located closer to the DQ locus than to the DX locus.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

An L1210 cell line (JT-1), which can grow in medium supplemented with 1 nM folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37 degrees C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23 +/- 0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37 degrees C remained as unmetabolized folic acid. Binding was also rapid at 0 degrees C but uptake at the plateau was only one-half the value obtained at 37 degrees C. Half-maximal saturation of the binding component (KD) occurred at a folate concentration of 0.065 nM at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (KD = 2.0 nM). 5-Methyltetrahydrofolate was also bound by this component (Ki = 13 nM at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (Ki = 45 nM) and methotrexate (Ki = 325 nM). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500 nM caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and      

Intra- and interspecific variation in body size ofProtohermes (Megaloptera: Corydalidae)

The life history of three populations ofProtohermes grandis and two populations ofProtohermes immaculatus (Megaloptera: Corydalidae) was compared. In general, the larvae lived in stream riffles for 2 years and the adults appeared in summer. Adult body size differed between these closely related species and also between the populations ofP. grandis. Dwarfism occurred inP. immaculatus, a species that is endemic to the small, isolated island, Amami Island. The population ofP. grandis on Yaku Island, located between Amami Island and the mainland Kyushu, had an intermediate body size between that ofP. immaculatus and the mainland population ofP. grandis. Despite being an insular population,P. grandis on Tsushima Island had a similar body size to mainlandP. grandis. In these populations with large adults, some larvae lived in the streams for 3 years. The size distribution of benthic animals, which are the prey available toProtohermes larvae, differed between the streams studied. The density of large prey was lowest on Amami Island, intermediate on Yaku Island, and highest on the mainland and Tsushima Island. Different size distributions of available prey may be caused by the differences of benthic fauna; most of Ecdyonuridae and Ephemerellidae (large mayflies) and Perlidae (large stoneflies) were not found on Amami and Yaku Islands. Thus, there is a tendency to dwarfism in the populations ofProtobermes inhabiting streams where the density of large prey is low.     

Cloning, Nucleotide Sequences and Differential Expression of the nifH and nifH-Like (frxC) Genes from the Filamentous Nitrogen-Fixing Cyanobacterium Plectonema boryanum

The frxC gene, found in liverwort chloroplast DNA, encodes aprotein of unknown function. The deduced amino acid sequenceof the protein shows significant homology to that of ni-trogenaseFe-protein encoded by the nifH gene. We have cloned the frxCand nifH genes from the nitrogen-fixing cyanobacterium Plectonemaboryanum, using frxC- and nifH-specific probes, and have determinedtheir nucleotide sequences. The amino acid sequence deducedfrom the frxC gene of P. boryanum exhibits 83% homology to thatof the protein encoded by the/rxCgene from liverwort, whereasit exhibits only 34% homology to that encoded by the nifH genefrom the same organism, namely, P. boryanum. Northern blot analysisshowed that the frxC gene was transcribed more actively undernitrogenase-repressed conditions than under nitrogenase-inducedconditions, suggesting that the FrxC protein has a functiondistinct from nitrogen fixation. These results, together withthe phylogenetic relationship between the nifH and frxC genes,indicate that the frxC and nifH genes are derived from a commonancestral gene but have evolved independently to encode proteinswith different functions. (Received April 27, 1991; Accepted August 12, 1991)     

Sequence of three members and expression of a new major subfamily of glutelin genes from rice

Three members have been isolated of an additional glutelin gene subfamily, named subfamily B, consisting of about five members per haploid rice genome. Restriction fragment length polymorphism analysis showed major differences between Japonica and Indica lines, indicating the divergence of the subfamily since the split between the two varieties. While corresponding exons of the subfamily B showed 80 to 88% nucleotide sequence homology, those exons were only 60–65% homologous to those of the glutelin A subfamily [15, 19, 24], distinguishing them from the subfamily A. Intron position and derived polypeptide structure, in addition to the nucleotide sequence, confirm the subfamily B members as glutelins. Analysis of RNA from seeds of different stages of development showed that the subfamily B members were expressed at the same time as those of subfamily A, demonstrating coordinated regulation of the two subfamilies.     

On the hoplonemertean Sagaminemertes nagaiensis (Iwata, 1957), with consideration of its systematics

Summary The polystiliferous hoplonemertean Sagaminemertes nagaiensis (Iwata, 1957) has been redescribed.A distinct pre-cerebral region is wanting. The rhynchocoel opens directly at the tip of the head, whilst the mouth opens seperately below the ventral commissure of the brain which is situated at the anterior end of the head. The dorsal ganglia are well developed and extend posteriorly far behind the ventral ganglia. The cerebral sense organs are situated behind the brain and possess posterior ciliated canals. Cephalic glands are poorly represented, but ocelli are well developed. The sickle-shaped basis of the proboscis bears about 20 central stylets. The caecal appendages of the rhynchocoel are arranged pseudometamerically throughout the body. The foregut is straight, consisting of mouth, oesophagus, stomach and pylorus. Intestinal caeca and lateral diverticula are present. The cephalic lacunae have no anterior anastomosis. A cerebral lacuna is present, there is a rhynchocoel vessel, but transverse vessels are absent. The protonephridia extend throughout the foregut region. Mature spermatogonia are well developed.A systematic discussion of Sagaminemertes nagaiensis suggests that it is taxonomically close to Siboganemertes weberi in the archireptantic family Siboganemertidae.     

Effect of substitution of glycine for arginine at position 146 of the A1 subunit on biological activity of Escherichia coli heat-labile enterotoxin.

The ADP-ribosyltransferase activity of polypeptide A1 of cholera toxin and that of Escherichia coli heat-labile enterotoxin (LT) are primarily responsible for the toxic activities of these toxins. Since the amino acid sequences of the two A1 polypeptides are very similar, their functional mechanisms are considered to be the same. Arg-146 of polypeptide A1 is thought to be involved in the active site, because this amino acid of cholera toxin has been identified as the site of self-ADP-ribosylation. However, the exact role of Arg-146 and the significance of self-ADP-ribosylation in toxicity remain unclear. We substituted Arg-146 of polypeptide A1 of LT with Gly by oligonucleotide-directed mutagenesis and examined the biological property of the resultant mutant LT. The substitution changed the mobility of subunit A on sodium dodecyl sulfate-polyacrylamide gel but did not reduce the vascular permeability activity of LT. This result indicates that Arg-146 is not absolutely required for toxic activity and that LT can express its toxic activity without self-ADP-ribosylation at Arg-146.