Functional differentiation in the leucine-rich repeat domains of closely related plant virus-resistance proteins that recognize common avr proteins

The N' gene of Nicotiana sylvestris and L genes of Capsicum plants confer the resistance response accompanying the hypersensitive response (HR) elicited by tobamovirus coat proteins (CP) but with different viral specificities. Here, we report the identification of the N' gene. We amplified and cloned an N' candidate using polymerase chain reaction primers designed from L gene sequences. The N' candidate gene was a single 4143 base pairs fragment encoding a coiled-coil nucleotide-binding leucine-rich repeat (LRR)-type resistance protein of 1,380 amino acids. The candidate gene induced the HR in response to the coexpression of tobamovirus CP with the identical specificity as reported for N'. Analysis of N'-containing and tobamovirus-susceptible N. tabacum accessions supported the hypothesis that the candidate is the N' gene itself. Chimera analysis between N' and L(3) revealed that their LRR domains determine the spectrum of their tobamovirus CP recognition. Deletion and mutation analyses of N' and L(3) revealed that the conserved sequences in their C-terminal regions have important roles but contribute differentially to the recognition of common avirulence proteins. The results collectively suggest that Nicotiana N' and Capsicum L genes, which most likely evolved from a common ancestor, differentiated in their recognition specificity through changes in the structural requirements for LRR function.     

Studies on Erythorbic Acid Production by Fermentation

Screening was carried out for erythorbic acid (EA)-producing strains from about 5,000 newly isolated fungi and bacteria. Penicillium notatum FY 115 was screened out as most powerful EA producer. Only Penicillium, but no other genera, was obtained as EA producers from our screening program. Monospore selections and mutagenic treatments succeeded to elevate the yield of EA over 40% to glucose supplied. Various cultural conditions were studied, and pH change during fermentation process was proved to be most important for favorable EA production. Over 80% yield could be obtained when washed mycelium was used in dilute glucose solution.

Abundant accumulation of EA by the strain FY 115, Penicillium sp., in fermentation broth was studied, and EA, both free and Na-salt, was obtained as crystal in the yield of about 45% to glucose supplied, in the media of 8% glucose by jar fermentor, in considering the inhibitory effect of some metal ion.

Extraction processes were improved to elevate the yield and was developed the continuous multi-bed extraction system of anion-exchange resin, which resulted in the yield of 90.9% of EA from fermentation broth in sum total.     

Polylysine Produced by Streptomyces

The xylitol dehydrogenase gene (xdh) of Bacillus pallidus was cloned and overexpressed in Escherichia coli using pQE60 vector, for the first time. The open reading frame of 759 bp encoded a 253 amino acid protein with a calculated molecular mass of 27,333 Da. The recombinant xylitol dehydrogenase (XDH) was purified to homogeneity by three-step column chromatography, producing a single SDS–PAGE band of 28 kDa apparent molecular mass. The enzyme exhibited maximal activity at 55 °C in glycine-NaOH buffer pH 11.0, with 66% of initial enzyme activity retained after incubation at 40 °C for 1 h. In further application of the recombinant bacterium to L-xylulose production from xylitol (initial concentration 5%) using a resting cell reaction, 35% L-xylulose was produced within 24 h. This result indicates that this recombinant XDH is applicable in the large-scale production of L-xylulose.     

Preferential association of kappa IIIb light chains with monoclonal human IgM kappa autoantibodies

The predominance of the relatively uncommon V region subgroup isotype kappa III among the light chains of human monoclonal (IgM kappa) anti-IgG antibodies, (i.e., rheumatoid factors), was further documented through sequence analyses of ten such autoantibodies isolated from IgM-anti-IgG cold-insoluble immune complexes (mixed cryoglobulins). The amino-terminal sequence of all ten kappa-chains was characteristic for kappa III proteins and virtually identical to that of a prototype kappa III light chain. Similar sequence identity was found for kappa-chains isolated from three IgM kappa autoantibodies that formed cold-insoluble immune complexes with low-density lipoprotein (LDL). The thirteen light chains were found to be virtually identical in sequence for the first framework region (FR); ten of these proteins sequenced through the first complementarity-determining region (CDR) and into the second FR were markedly similar. The second CDR of five proteins was almost identical in sequence to that of the prototype kappa III-chain. Concordance was also demonstrated between the structural classification of the light chains as kappa III and their immunochemical classification as members of this V region subgroup. Serologic analyses of light chains isolated from seven IgM kappa autoantibodies (six anti-IgG, one anti-LDL) and of one intact IgM kappa anti-LDL antibody showed that each had antigenic determinants common to kappa II proteins. These light chains also expressed the antigenic determinant(s) of a V-region sub-subgroup of kappa III proteins designated kappa IIIb. Our studies confirm the preferential association of kappa III (and kappa IIIb) light chains with IgM kappa anti-IgG antibodies and demonstrate a similar association for IgM kappa anti-LDL antibodies. The finding that these and other types of IgM kappa autoantibodies, e.g., cold agglutinins, have remarkably similar light chains suggests an inherent restriction in the immune response to self-antigens.     

Sexual dimorphism of body size and sternopleural bristle number: a comparison of geographic populations of an invasive cosmopolitan drosophilid

Zaprionus indianus is a cosmopolitan drosophilid, of Afrotropical origin, which has recently colonized South America. The sexual dimorphism (SD) of body size is low, males being almost as big as females. We investigated 10 natural populations, 5 from America and 5 from Africa, using the isofemale line technique. Three traits were measured on each fly: wing and thorax length and sternopleural bristle number. Two indices of SD were compared, and found to be highly correlated (r > 0.99). For the sake of simplicity, only the female/male (F/M) ratio was further considered. A significant genetic variability of SD was found in all cases, although with a low heritability (intra-class correlation of 0.13), about half the value found for the traits themselves. For size SD, we did not find any variation among continents or any latitudinal trend, and average values were 1.02 for wing length and 1.01 for thorax length. Bristle number SD was much greater (1.07). Among mass laboratory strains, SD was genetically much more variable than in recently collected populations, a likely consequence of laboratory drift. Altogether, SD, although genetically variable and prone to laboratory drift, is independent of size variations and presumably submitted to a stabilizing selection in nature.     

Mode of action on deacetylation of acetylated methyl glycoside by cellulose acetate esterase from Neisseria sicca SB

The regioselective deacetylation of purified cellulose acetate esterase from Neisseria sicca SB was investigated on methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside. The substrates were used as model compounds of cellulose acetate in order to estimate the mechanism for deacetylation of cellulose acetate by the enzyme. The enzyme rapidly deacetylated at position C-3 of methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside to accumulate 2,4,6-triacetate as the main initial reaction product in about 70% yield. Deacetylation was followed at position C-2, and generated 4,6-diacetate in 50% yield. The enzyme deacetylated the product at positions C-4 and C-6 at slower rates, and generated 4- and 6-monoacetates at a later reaction stage. Finally, it gave a completely deacetylated product. For 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, CA esterase deacetylated at positions C-3 and C-6 to give 2,4,6- and 2,3,4-triacetate. Deacetylation proceeded sequentially at positions C-3 and C-6 to accumulate 2,4-diacetate in 55% yield. The enzyme exhibited regioselectivity for the deacetylation of the acetylglycoside.     

Biophysics of sphingolipids I. Membrane properties of sphingosine, ceramides and other simple sphingolipids

Some of the simplest sphingolipids, namely sphingosine, ceramide, some closely related molecules (eicosasphingosine, phytosphingosine), and their phosphorylated compounds (sphingosine-1-phosphate, ceramide-1-phosphate), are potent metabolic regulators. Each of these lipids modifies in marked and specific ways the physical properties of the cell membranes, in what can be the basis for some of their physiological actions. This paper reviews the mechanisms by which these sphingolipid signals, sphingosine and ceramide in particular, are able to modify the properties of cell membranes.