Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.     

Efficiency of serum copper/zinc ratio for differential diagnosis of patients with and without lung cancer

We examined serum copper (Cu), serum zinc (Zn), and the serum copper/zinc ratio (Cu/Zn) in 162 patients. All of them were seen to have an abnormal shadow in the chest X-ray films, that is, 109 patients with lung cancer (LC) and 53 patients with no lung cancer (NLC). The mean Cu and Cu/Zn in LC patients were significantly higher than those in NLC patients (p<0.05). In LC patients, Cu and Cu/Zn were higher and Zn was lower in advanced tumors than early ones. There was a significantly clear relation between Cu or Cu/Zn and the tumor (T) stages. When the relative risk (RR) of LC was estimated, it was seen that the higher Cu and Cu/Zn became, the higher RR became. Furthermore, we showed the sensitivity of the receiver operator characteristic of the test (ROC) curve for Cu, Cu/Zn, and carcinoembryonic antigen (CEA) to diagnose LC, as explained in a paragraph of methods.The determinations of Cu, Zn, and Cu/Zn are simple and inexpensive. They also appear to have a great diagnostic value in determining the local invasion of LC and as a screening test in the high-risk patients for LC.     

Genetic Enhancement of Cold Tolerance by Expression of a Gene for Chloroplast [omega]-3 Fatty Acid Desaturase in Transgenic Tobacco

The increased production of trienoic fatty acids, hexadecatrienoic (16:3) and linolenic (18:3) acids, is a response connected with cold acclimation of higher plants and is thought to protect plant cells against cold damage. Transgenic tobacco (Nicotiana tabacum cv SR1) plants that contain increased levels of 16:3 and 18:3 fatty acids, and correspondingly decreased levels of their precursors, hexadecadienoic and linoleic acids, were engineered by introduction of a chloroplast [omega]-3 fatty acid desaturase gene (the fad7 gene) isolated from Arabidopsis thaliana. When exposed to 1[deg]C for 7 d and then cultured at 25[deg]C, the suppression of leaf growth observed in the wild-type plants was significantly alleviated in the transgenic plants with the fad7 gene. The low-temperature- induced chlorosis was also much reduced in the plants transformed with the fad7 gene. These results indicate that increased levels of trienoic fatty acids in genetically engineered plants enhance cold tolerance.     

Analysis of simian immunodeficiency virus sequence variation in tissues of rhesus macaques with simian AIDS.

One rhesus macaque displayed severe encephalomyelitis and another displayed severe enterocolitis following infection with molecularly cloned simian immunodeficiency virus (SIV) strain SIVmac239. Little or no free anti-SIV antibody developed in these two macaques, and they died relatively quickly (4 to 6 months) after infection. Manifestation of the tissue-specific disease in these macaques was associated with the emergence of variants with high replicative capacity for macrophages and primary infection of tissue macrophages. The nature of sequence variation in the central region (vif, vpr, and vpx), the env gene, and the nef long terminal repeat (LTR) region in brain, colon, and other tissues was examined to see whether specific genetic changes were associated with SIV replication in brain or gut. Sequence analysis revealed strong conservation of the intergenic central region, nef, and the LTR. However, analysis of env sequences in these two macaques and one other revealed significant, interesting patterns of sequence variation. (i) Changes in env that were found previously to contribute to the replicative ability of SIVmac for macrophages in culture were present in the tissues of these animals. (ii) The greatest variability was located in the regions between V1 and V2 and from "V3" through C3 in gp120, which are different in location from the variable regions observed previously in animals with strong antibody responses and long-term persistent infection. (iii) The predominant sequence change of D-->N at position 385 in C3 is most surprising, since this change in both SIV and human immunodeficiency virus type 1 has been associated with dramatically diminished affinity for CD4 and replication in vitro. (iv) The nature of sequence changes at some positions (146, 178, 345, 385, and "V3") suggests that viral replication in brain and gut may be facilitated by specific sequence changes in env in addition to those that impart a general ability to replicate well in macrophages. These results demonstrate that complex selective pressures, including immune responses and varying cell and tissue specificity, can influence the nature of sequence changes in env.     

A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation

The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3' end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium , as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.     

Distribution of various nickel compounds in rat organs after oral administration

In this study, eight kinds of nickel (Ni) compounds were orally administered to Wistar male rats and the distribution of each compound was investigated 24 h after the administration. The Ni compounds used in this experiment were nickel metal [Ni−M], nickel oxide (green) [NiO(G)], nickel oxide (black) [NiO(B)], nickel subsulfide [Ni₃S₂], nickel sulfide [NiS], nickel sulfate [NiSO₄], nickel chloride [NiCl₂], and nickel nitrate [Ni(NO₃)₂]. The solubilities of the nickel compounds in saline solution were in the following order; [Ni(NO₃)₂>NiCl₂>NiSO₄]≫[NiS>Ni₃S₂]>[NiO(B)>Ni−M>NiO(G)]. The Ni level in the visceral organs was higher in the rats given soluble Ni compounds; Ni(NO₃)₂, NiCl₂, NiSO₄, than that in the rats receiving other compounds. In the rats to which soluble Ni compounds were administered, 80–90% of the recovered Ni amounts in the examined organs was detected in the kidneys. On the other hand, the Ni concentration in organs administered scarcely soluble Ni compounds; NiO(B), NiO(G), and Ni−M were very low. The estimated absorbed fraction of each Ni compounds was increased with the increase of the solubility. These results suggest that the kinetic behavior of Ni compounds administered orally is closely related with the solubility of Ni compounds, and that the solubility of Ni compounds is one of the important factors for determining the health effect of Ni compounds.     

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.