Influence of genotypes at SAA1 and SAA2 loci on the development and the length of latent period of secondary AA-amyloidosis in patients with rheumatoid arthritis

To examine whether polymorphism at the SAA loci is associated with the development of amyloid protein A (AA)-amyloidosis, we determined the genotypes at the SAA1 and SAA2 loci in 43 AA-amyloidosis patients (amyloidosis population) and 77 patients with rheumatoid arthritis (RA) who had been ill for less than 5 years (early RA population). We also compared the frequencies of the genotypes at the SAA1 locus among 90 Korean, 95 Taiwanese, and 103 Japanese healthy subjects. The frequencies of the gamma/gamma genotype and gamma alleles at the SAA1 locus were significantly higher in the amyloidosis population than in the early RA population (34.9% versus 7.8%, and 58.1% versus 33.8%, chi2 test P=0.0001). The frequencies of the gamma allele at the SAA1 locus in Koreans, Taiwanese, and Japanese were 41.6%, 35.6%, and 37.4%, respectively. The length of the latent period of AA-amyloidosis was significantly longer in the patients with smaller numbers of the gamma allele at the SAA1 locus (Spearman's correlation coefficient: -0.42, P<0.05). On the other hand, the mean C-reactive protein (CRP) level during 2 years prior to the diagnosis of AA-amyloidosis was significantly higher in the patients with larger numbers of the gamma allele at the SAA1 locus (Spearman's correlation coefficient: 0.34, P<0.05). No significant association was found between amyloidosis and polymorphism at the SAA2 locus. We postulate that the allele SAA1gamma renders an RA patient susceptible to amyloidosis, possibly by affecting the severity of inflammation in RA.     

High-efficient expression of porcine IL-2 with recombinant baculovirus infected silkworm,Bombyx mori

Biologically active porcine Interleukin-2(poIL-2) was produced fromin vitro andin vivo baculovirus expression systems, namely theAutographa californica nuclear polyhedrosis virus (AcNPV)-cell culture system and the Hybrid nuclear polyhedrosis virus (HyNPV)— silkworm larva system. The concentration of the recombinant poIL-2(rpoIL-2) in the larvae hemolymph was 1 to 3 mg/mL, which was about 7 to 20 times those of the cell culture systems. The level of this expression efficiency is equal to that with transgenic livestock, secretion products in milk.     

Synthesis of N-labeled peptidyl AMP

This paper deals with the synthesis of a new type of N-labeled peptidyl AMP, which would be used as a good substrate for analysis of the peptidyl transfer reaction on ribosome and for co-crystallization with ribosome. 4-(Dimethylamino)azobenzene-4'-sulfonyl (Dabsyl) was selected as the labeling group. (N-Dabsylglycyl)-L-leucyl AMP was synthesized from glycyl-L-leucine via a three-step procedure.     

Production and characterization of the recombinant human mu-opioid receptor from transgenic silkworms

The production of useful quantities of G protein-coupled receptors is a major problem not only for screening of various drug compounds but also in performing structural biology studies. To solve this problem, we investigated the possibility of using transgenic silkworms for the production of these receptors. Using the human mu-opioid receptor gene, we constructed three transgenic silkworm strains that produced mu-opioid receptors. The silkworms expressed significant amounts of the receptor in the fat body and silk gland. The product was evaluated using a saturation ligand-binding assay. The expressed receptor exhibited ligand affinity similar to that of an authentic sample, and the yield from the transgenic silkworm was comparable to that obtained using an Sf9-baculovirus expression system. As the mass rearing of transgenic silkworms has already been established, the silkworms can be adapted for production of large quantities of receptors.     

Expression of protein engineered NADP<Superscript>+</Superscript>-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD⁺-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP⁺. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP⁺-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP⁺-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.     

A molecular genetic linkage map of mouse chromosome 18, includingspm,Grl-1, Fim-2/c-fms,andMbp

Restriction endonuclease fragment length variations (RFLV) were detected in mice with DNA probes for myelin basic protein (Mbp), glucocorticoid receptor-1 (Grl-1), and Friend MuLV integration site-2 (Fim-2). RFLV of theMbp gene were found inSacI restriction patterns, RFLV of theGrl-1 gene were found inEcoRV patterns, and RFLV of theFim-2 were found inBglII patterns. A three-point backcross was carried out by the backcross mating (C57BL/KsJ-spm/spm × MOL-MIT)F₁ males × C57BL/KsJ-spm/spm; spm is an autosomal recessive gene causing sphingomyelinosis. From the results,spm, Grl-1, Fim-2, andMbp loci were mapped on chromosome 18, and the following order of genes is proposed, with distances between genes in parentheses: centromere—spm—(7.8 cM)—Grl-1—(7.8 cM)—Fim-2—(39.1 cM)—Mbp—telomere. All laboratory strains and two European subspecies (Mus mus domesticus andM. m. brevirostris) carry theGrl-1 ^a ,Fim-2 ^a , andMbp ^a alleles. In contrast, another wild subspecies from Europe (M. m. musculus) and some Asian subspecies (M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai) carry theGrl-1 ^b ,Fim-2 ^b , andMbp ^b alleles. Onlycastaneus strains carry the intermediate combination of theGrl-1 ^b ,Fim-2 ^a , andMbp ^b alleles.     

Phylogenetic analyses of Japanese species of Phyllosticta sensu stricto

Although the genus concept of Phyllosticta s. str. (teleomorph: Guignardia) as defined by van der Aa is widely accepted, the species concept is still controversial because it is often based on the morphology on host plants. In this study, the culture characteristics within Phyllosticta s.str. were examined, and the phylogenetic relationships among Japanese species of Phyllosticta s.str. and its teleomorph Guignardia were analyzed using 18S rDNA sequences. Phyllosticta s. str. formed a monophyletic clade. ITS-28S rDNA sequences extracted from fungal cultures derived from various host plants were divided into two subgroups. The first group included cultures from a wide range of host plants and were mainly derived as endophytes from a symptom-less plant. In the second group, cultures from each host plant genus formed distinct clades; these were often isolated as leaf pathogens from diverse plants. Isolates belonging to the first lineage generally grew faster on oatmeal agar. To classify species of Phyllosticta it is necessary to consider an integrated approach such as molecular phylogeny, host plant, colony growth, symptoms, and morphological characteristics of the conidiomata.     

Production of D-Alanine by Corynebacterium fascians

A strain identified as Corynebacterium fascians was found to accumulate extracellular D-alanine from glycerol. Cultural conditions for the accumulation of D-alanine were investigated and, as a result, a yield of 7 g of D-alanine per liter was obtained after a 96-h incubation in a medium containing 5% glycerol, 4% (NH(4))(2)HPO(4), and 0.3% corn steep liquor. Optical purity of D-alanine was dependent upon the concentration of corn steep liquor. At the optimal condition, almost optically pure D-alanine was formed and readily isolated (5 g/liter) from the fermentation broth. The product was not contaminated with any detectable amount of other amino acids, except for glycine which was present at a concentration of less than 1 percent.     

Emigration behavior and molting during the sea‐to‐land transition of terrestrial hermit crabs under laboratory conditions

Terrestrial hermit crabs in the family Coenobitidae (genera Coenobita and Birgus) must migrate onto land after completing a pelagic larval stage in the ocean. Better knowledge of emigration behavior would assist in the conservation and management of coenobitid populations by helping identify and protect the habitats they need to complete their life cycles. We cultured laboratory‐born individuals of five coenobitid species (Coenobita cavipes, C. purpureus, C. rugosus, C. violascens, and Birgus latro) from megalopae to early juveniles (first, second, and/or third crabs) in vessels containing seawater and a hard substrate, and analyzed their behavior and molting in conjunction with our published data for C. brevimanus. Our results confirm that the coenobitids migrated from sea to land at the megalopal stage. Megalopae and early juveniles tended to select shells based on their body size. Inland‐dwelling coenobitids, such as C. brevimanus, C. cavipes, and B. latro, had a longer duration from landing to first molt and had a prolonged first crab intermolt period compared with those of the beach‐dwelling coenobitids C. purpureus, C. rugosus, and C. violascens, probably because of the adaptive traits for migrating to inland habitats. Little burrowing behavior was observed by megalopae of B. latro, but they had a strong tendency to be cryptic under shelters. Additionally, megalopae and early juveniles of Coenobita spp. created and utilized burrows somewhat differently. Our results suggest that coenobitids require specific microhabitats for completing their early life stages in the wild. In particular, megalopae of B. latro may need structurally complex refuges to migrate from the sea.