Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Synthesis of sex-specific forms of cytochrome P-450 in rat liver is transiently suppressed by hepatic monooxygenase inducers

The effects of phenobarbital (PB), 3-methylcholanthrene (MC), and alpha-naphthoflavone (alpha-NF) on the synthesis of drug-inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), and sex-specific forms of cytochrome P-450, P-450(M-1), and P-450(F-1), in male and female rats were studied. Whereas P-450(PB-1) and P-450(MC-1) in liver microsomes were markedly induced in both sexes by treatment with PB and MC, respectively, the contents of P-450(M-1) and P-450(F-1) were significantly decreased by the treatments. alpha-NF, which is not a P-450 inducer, did not change the contents of sex-specific forms of cytochrome P-450. The translatable mRNAs of the P-450s were also determined by using an in vitro translation system. The mRNAs coding for P-450(PB-1) and P-450(MC-1) were increased by drug administrations. On the other hand, the mRNAs coding for P-450(M-1) and P-450(F-1) were transiently decreased by the drugs, and then returned to the normal levels. The time courses of the induction of the drug-inducible P-450s and the repression of the sex-specific P-450s showed no close correlation. alpha-NF had no effect on the synthesis of P-450(M-1) and P-450(F-1). We also found that the synthesis of P-450(M-1) in the livers of untreated rats showed no diurnal variations.     

Potential fragmentation and loss of thermal habitats for charrs in the Japanese archipelago due to climatic warming

1. The upper thermal limits of the present distributions of two charr species, Dolly Varden, Salvelinus malma , and white-spotted charr, S. leucomaenis , in streams of the Japanese archipelago were examined using groundwater temperature as an index of thermal condition. The lower limits of the altitudinal distributions of Dolly Varden and white-spotted charr were delineated, respectively, by 8 and 16 °C groundwater isotherms.
2. The potential impact of future climatic warming on the geographical distribution, habitat extent and population fragmentation of each species was predicted at both the full archipelago and individual catchment levels.
3. For Dolly Varden, analysis at the full archipelago level indicated a loss of 27.6, 67.2, 79.6 and 89.6% of the current geographical range, respectively, for a 1, 2, 3 and 4 °C increase in mean annual air temperature. The present distribution area of white-spotted charr would likewise reduce by 4.1, 20.5, 33.8 and 45.6%, respectively.
4. Based on the analyses of three individual catchments, one for Dolly Varden and two for white-spotted charr, the lower habitat boundaries for the two charr species could be expected to rise increasingly to higher elevations in each catchment as warming proceeded. As a consequence, there would be large reductions in mean habitat area, with increasing habitat fragmentation followed by localized extinctions of the two species.     

Additive Induction of Egr-1 (zif/268) mRNA Expression in Neuroblastoma × Glioma Hybrid NG108-15 Cells via Cholinergic Muscarinic, α2-Adrenergic, and Bradykinin Receptors

Abstract: Administration of carbachol, noradrenaline, and bradykinin induced Egr-1 mRNA expression within 1 h in mouse neuroblastoma × rat gliomahybrid NG108–15 cells. With specific receptor antagonists, the Egr-1 inductions by carbachol and noradrenaline were shown to be mediated via cholinergic muscarinic and α₂-adrenergic receptors, respectively. At their saturation levels for Egr-1 induction, the two agonists had additive effects when added together, but no prolongation of the effect on Egr-1 induction was observed. Addition of carbachol or noradrenaline 6 h after primary stimulation with carbachol or noradrenaline did not result in secondary Egr-1 induction, probably because of receptor desensitization. On the other hand, bradykinin consistently had an additive effect on Egr-1 induction, irrespective of the time of its addition, suggesting that the signal pathways for Egr-1 induction by carbachol or noradrenaline and by bradykinin are different. Treatment of cells with pertussis toxin or cholera toxin strongly inhibited Egr-1 induction by carbachol or noradrenaline but only partially inhibited the induction by bradykinin. Thus, the signals transduced in NG108–15 cells by different neurotransmitter receptors appear to have different effects on Egr-1 induction, depending on the times of stimulation and the combinations of receptors stimulated.     

Potential fragmentation and loss of thermal habitats for charrs in the Japanese archipelago due to climatic warming

1. The upper thermal limits of the present distributions of two charr species, Dolly Varden, Salvelinus malma , and white-spotted charr, S. leucomaenis , in streams of the Japanese archipelago were examined using groundwater temperature as an index of thermal condition. The lower limits of the altitudinal distributions of Dolly Varden and white-spotted charr were delineated, respectively, by 8 and 16 °C groundwater isotherms.
2. The potential impact of future climatic warming on the geographical distribution, habitat extent and population fragmentation of each species was predicted at both the full archipelago and individual catchment levels.
3. For Dolly Varden, analysis at the full archipelago level indicated a loss of 27.6, 67.2, 79.6 and 89.6% of the current geographical range, respectively, for a 1, 2, 3 and 4 °C increase in mean annual air temperature. The present distribution area of white-spotted charr would likewise reduce by 4.1, 20.5, 33.8 and 45.6%, respectively.
4. Based on the analyses of three individual catchments, one for Dolly Varden and two for white-spotted charr, the lower habitat boundaries for the two charr species could be expected to rise increasingly to higher elevations in each catchment as warming proceeded. As a consequence, there would be large reductions in mean habitat area, with increasing habitat fragmentation followed by localized extinctions of the two species.     

Reversed-phase HPLC determination of chlorophyll a' and phylloquinone in Photosystem I of oxygenic photosynthetic organisms. Universal existence of one chlorophyll a' molecule in Photosystem I.

Chlorophyll (Chl) a', the C132-epimer of Chl a, is a constituent of the primary electron donor (P700) of Photosystem (PS) I of a thermophilic cyanobacterium Synechococcus (Thermosynechococcus) elongatus, as was recently demonstrated by X-ray crystallography. To determine whether PS I of oxygenic photosynthetic organisms universally contains one molecule of Chl a', pigment compositions of thylakoid membranes and PS I complexes isolated from the cyanobacteria T. elongatus and Synechocystis sp. PCC 6803, the green alga Chlamydomonas reinhardtii, and the green plant spinach, were examined by simultaneous detection of phylloquinone (the secondary electron acceptor of PS I) and Chl a' by reversed-phase HPLC. The results were compared with the Chl a/P700 ratio determined spectrophotometrically. The Chl a'/PS I ratios of thylakoid membranes and PS I were about 1 for all the organisms examined, and one Chl a' molecule was found in PS I even after most of the peripheral subunits were removed. Chl a' showed a characteristic extraction behaviour significantly different from the bulk Chl a in acetone/methanol extraction upon varying the mixing ratio. These findings confirm that a single Chl a' molecule in P700 is the universal feature of PS I of the Chl a-based oxygenic photosynthetic organisms.     

In vivo and in vitro characterization of Ser477X mutations in polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. 61-3: effects of beneficial mutations on enzymatic activity, substrate specificity, and molecular weight of PHA

Evolutionary engineered polyhydroxyalkanoate (PHA) synthases from Pseudomonas sp. 61-3 enhance PHA accumulation and enable the monomer composition of PHAs to be regulated. We characterized a newly screened Ser477Arg (S477R) mutant of PHA synthase by in vivo analyses of P(3-hydroxybutyrate) [P(3HB)] homopolymer and P(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] copolymer productions in the recombinants of Escherichia coli. The results indicated that the S477R mutation contributed to a shift in substrate specificity to smaller monomers containing a 3HB unit rather than to an enhancement in catalytic activity. Multiple mutations of S477R with other beneficial mutations, for example, Ser325Cys, exhibited synergistic effects on both an increase in PHA production (from 9 wt % to 21 wt %) and an alteration of substrate specificity. Furthermore, the effects of complete amino acid substitutions at position 477 were characterized in terms of in vivo PHA production and in vitro enzymatic activity. The five mutations, S477Ala(A)/Phe(F)/His(H)/Arg(R)/Tyr(Y), resulted in a shift in substrate specificity to smaller monomer units. The S477Gly(G) mutant greatly enhanced activity toward all different sizes of substrates with carbon numbers ranging from 4 to 12. These results indicated that the residue 477 contributes to both the catalytic activity and substrate specificity of PHA synthase. In recombinant E. coli, the S477A/F/G/H/R/Y mutations consistently led to increases (up to 6 times that of wild-type enzyme) in weight average molecular weights of P(3HB) homopolymers. On the basis of our studies, we created a structural feasibility accounting for the mutational effects on enzymatic activity and substrate specificity of PHA synthase.     

Polymorphisms of interferon-inducible genes OAS-1 and MxA associated with SARS in the Vietnamese population

We hypothesized that host antiviral genes induced by type I interferons might affect the natural course of severe acute respiratory syndrome (SARS). We analyzed single nucleotide polymorphisms (SNPs) of 2',5'-oligoadenylate synthetase 1 (OAS-1), myxovirus resistance-A (MxA), and double-stranded RNA-dependent protein kinase in 44 Vietnamese SARS patients with 103 controls. The G-allele of non-synonymous A/G SNP in exon 3 of OAS-1 gene showed association with SARS (p=0.0090). The G-allele in exon 3 of OAS-1 and the one in exon 6 were in strong linkage disequilibrium and both of them were associated with SARS infection. The GG genotype and G-allele of G/T SNP at position -88 in the MxA gene promoter were found more frequently in hypoxemic group than in non-hypoxemic group of SARS (p=0.0195). Our findings suggest that polymorphisms of two IFN-inducible genes OAS-1 and MxA might affect susceptibility to the disease and progression of SARS at each level.     

Mesorhizobium sp. J8 can establish symbiosis with Glycyrrhiza uralensis,increasing glycyrrhizin production

Licorice (Glycyrrhiza uralensis) is a medicinal plant that contains glycyrrhizin (GL), which has various pharmacological activities. Because licorice is a legume, it can establish a symbiotic relationship with nitrogen-fixing rhizobial bacteria. However, the effect of this symbiosis on GL production is unknown. Rhizobia were isolated from root nodules of Glycyrrhiza glabra, and a rhizobium that can form root nodules in G. uralensis was selected. Whole-genome analysis revealed a single circular chromosome of 6.7 Mbp. This rhizobium was classified as Mesorhizobium by phylogenetic analysis and was designated Mesorhizobium sp. J8. When G. uralensis plants grown from cuttings were inoculated with J8, root nodules formed. Shoot biomass and SPAD values of inoculated plants were significantly higher than those of uninoculated controls, and the GL content of the roots was 3.2 times that of controls. Because uninoculated plants from cuttings showed slight nodule formation, we grew plants from seeds in plant boxes filled with sterilized vermiculite, inoculated half of the seedlings with J8, and grew them with or without 100 µM KNO₃. The SPAD values of inoculated plants were significantly higher than those of uninoculated plants. Furthermore, the expression level of the CYP88D6 gene, which is a marker of GL synthesis, was 2.5 times higher than in inoculated plants. These results indicate that rhizobial symbiosis promotes both biomass and GL production in G. uralensis.