Detection of antibody to hepatitis C E2/NS1 protein in patients with type C hepatitis.

Putative E2/NS1 sequence of hepatitis C virus was expressed in E. coli as a fusion protein with maltose binding protein. Approximately 80 kDa protein was obtained containing 38 kDa E2/NS1 protein. The antibody to this protein was detectable in the same serum from which the sequence was amplified. It was also detectable in none of 7 acute hepatitis, in 2 of 12 chronic persistent hepatitis, in 3 of 25 chronic active hepatitis, and in 2 of 4 cirrhosis. It was detectable in none of 10 normal subjects. In 3 cases who were positive for the antibody before the interferon treatment, it became undetectable after the treatment. Thus, it seems that the antibody is not a neutralizing antibody and is related to active viral replication.     

Genetically polymorphic α-l-fucosidase (FUCA1) isozymes detected in blood plasma

The main isozyme patterns of desialylated blood plasma or serum -l-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl--l-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA11 ^* and FUCA12 ^* alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+SD) of the three phenotypes were 318.8 ± 116.7 nmol/ml per h for type 1, 268.0 ± 108.3 nmol/ml per h for type 2-1, and 233.2 ± 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA11 ^* gene product in plasma has about 1.4 times the activity of FUCA12 ^*.     

Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC7942

Summary The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1–254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O₂ evolution (photosynthetic O₂ evolution coupled to nitrate reduction) in wild-type cells measured in the presence of l-methionine d,l-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 M), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O₂ evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system.     

cis-Elements and Protein Factors Related to Regulation of Transcription of Arylsulfatase Gene during Sea Urchin Development

Eight restriction fragments (I–VIII) were prepared to cover a whole span of the enhancer region in the upstream of the Ars gene of the sea urchin, Hemicentrotus pulcherrimus , and their abilities to influence on the Ars gene expression were estimated by CAT assay. Only three fragments (III, IV and V) encompassing a 0.6 kb region between −2.8 kb and −2.2 kb stimulated CAT expression. By mobility shift assays, it was found that the Ars enhancer region is composed of multiple cis -acting elements that interact with nuclear proteins in a sequence-specific manner. Among them, two sequences, a G-string and a GATCTCCCC, were determined by DNA footprinting as sites of protein-DNA interaction. The DNA-binding factor prevalence changed ontogenically in three different patterns. Possible activation of DNA-binding proteins through their modification is discussed.     

Structure, Function and Regulation of the Nitrate Transport System of the Cyanobacterium Synechococcus sp. PCC7942

The active nitrate transport system of the cyanobacterium Synechococcussp. PCC7942 is encoded by the four genes nrtA, nrtB, nrtC andnrtD. It is essential for the growth of the cyanobacterium atphysiological concentrations of nitrate and has been shown tobe involved in the active transport of nitrite as well. Thededuced amino acid sequences of the NrtB, NrtC and NrtD proteinsindicate that the transporter is a member of the ABC (ATP-bindingcassette) superfamily of active transporters. Among the prokaryoticABC transporters, the cyanobacterial nitrate/nitrite transporteris unique in having a membrane-bound protein NrtA and an NrtA-likeextra domain linked to one of the ATP-binding subunits (C-terminaldomain of NrtC). Molecular biological, biochemical and physiologicalstudies suggest that NrtA is the substrate-binding protein requiredfor the transport of nitrate/nitrite and that the C-terminaldomain of NrtC has a regulatory role. Comparison of the structuresof nitrate transporters from eukaryotic and prokaryotic, photosyntheticand non-photosynthetic organisms indicate that the nrt nitrate/nitritetransporter represents a prokaryotic nitrate transporter distinctfrom the nitrate transporters of eukaryotes. ¹Recipient of the JSPP Young Investigator Award, 1994.     

The Lipid Phase of Thylakoid and Cytoplasmic Membranes from the Blue-Green Algae (Cyanobacteria), Anacystis nidulans and Anabaena variabilis

The lipid phases of the thylakoid and cytoplasmic membranesfrom the blue-green alga, Anacystis nidulans, were studied bya spin-probe method using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl.The thylakoid and cytoplasmic membranes of this alga were bothin the liquid crystalline state at growth temperature, and inthe phase separation state at about 0?C. The thylakoid membranesentered the phase separation state at a temperature higher thanthe cytoplasmic membranes. The lipid phase of the thylakoidmembranes from Anabaena variabilis was studied in a similarway, and these membranes were found also to undergo the phasetransition. The temperature for the onset of the phase separationand the fluidity of the membrane lipids of both algae dependedon the growth temperature of the culture. (Received April 9, 1984; Accepted June 1, 1984)     

Synthesis of L(+) Tartaric Acid from 5-Keto-D-gluconic Acid in Pelargonium

5-Keto-D-[1-¹⁴C]gluconic acid, the most effective precursorof L(+)tartaric acid among all labeled compounds which haveever been tested in grapes, was found to be a good precursorof L(+)tartaric acid in a species of Pelargonium. The synthesisof labeled L(+)tartaric acid from D-[1-¹⁴C]glucose in Pelargoniumwas remarkably depressed when a 0.5% solution of D-gluconateor 5-keto-D-gluconate was administered continuously to leavestogether with D-[1-¹⁴C]glucose. Our results provide strong evidence that D-[1-¹⁴C]glucose ismetabolized in Pelargonium to give labeled L(+)tartaric acidvia (probably D-gluconic acid and) 5-keto-D-gluconic acid withoutpassing through L-ascorbic acid. Labeled L-idonic acid was found in young leaves of Pelargoniumwhich had been labeled with L-[U-¹⁴C]ascorbic acid. The synthesisof the labeled L-idonic acid increased when a 0.1% solutionof L-threonate was administered continuously to leaves togetherwith L-[U-¹⁴C]ascorbic acid. Specifically labeled compounds, recognized as the members ofthe synthetic pathway for L(+)tartaric acid from L-ascorbicacid via L-idonic acid in grapes, were administered to youngleaves of Pelargonium. Each compound (2-keto-L-[U-¹⁴C]idonicacid, L-[U-¹⁴C]idonic acid, 5-keto-D-[1-¹⁴C]gluconic acid and5-keto-D-[6-¹⁴C]gluconic acid) was partly metabolized, as ingrapes. The metabolic pathway starting from L-ascorbic acidto L(+)tartaric acid via L-idonic acid, however, did not actuallycontribute to the synthesis of L(+)tartaric acid in Pelargoniumprobably because the activity of each metabolic step was muchlower than that observed in grapes. (Received May 28, 1984; Accepted July 30, 1984)     

Photochemical reaction of 9-cis-retro-γ-rhodopsin at low temperatures

9-cis-Retro-γ;rhodopsin (λ_max = 420 nm) was prepared from 9-cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.     

Increase of pancreatic somatostatin concentration in early phases of streptozotocin diabetes in rats

A small yet significant increase of immunoassayable pancreatic somatostatin concentration (0.107 +/- 0.005 vs. 0.156 +/- 0.017 microgram/g at 24 hr, p less than 0.05) was found in rats, 24 hr as well as 7 days after treatment with a diabetogenic dose of streptozotocin (65 mg/kg BW). These animals were characterized by marked decreases of insulin in the pancreas without any significant changes in pancreatic glucagon concentration. These results suggest that an abrupt deprivation of insulin from islets results in an elevation of pancreatic somatostatin concentration, and that glucagon in the pancreas plays a minor role in determining pancreatic somatostatin concentration in rats with insulin-deprived diabetes of short duration.