Low-pressure microwave-induced helium plasma emission spectrometry: Vaporization characteristics of metal chlorides,nitrates, and sulfates

A low-pressure microwave-induced helium plasma serves as an excitation source for metal chlorides, nitrates, and sulfates vaporized from a filament, resulting in fractional vaporization and differential sensitivities of detection of the elements depending on the vapor pressures of their salts. The shapes of the single emission peaks, which are simple in the presence of potassium chloride, become complex and may double in number.     

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

Transblot identification of biotin-containing proteins in rat liver

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.     

NK-like cytotoxicity of allospecific gamma delta TCR+ T cell clones]

We recently generated a series of human alloantigen-specific, CD3+, gamma delta- TCR+ clones by stimulating CD3+, CD4-, CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). These clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Most but not all of these clones express the NK cell associated marker, CD57, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not NK-resistant line, Raji. Gamma delta clones which lacked expression of CD57 had no detectable NK activity. The allospecific cytotoxicity of CD57+ and CD57- clones was inhibited by mAb to CD3 or the TCR delta- chain. In contrast, the NK-like activity of the CD57+ clones was enhanced by these antibodies over a wide range of antibody concentration. An HLA class I framework-specific mAb had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the gamma delta- TCR+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+, TCR- gamma delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Cloning and sequencing of a ligninase gene from a lignindegrading basidiomycete,Phanerochaete chrysosporium

Summary A ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3 and 5 intron boundaries. The putative eukaryotic regulatory sequences, i.e. CAAT and TATA box-like sequences, are present in the 5 flanking region.     

The amplified long genomic sequence (ALGS) located in the centromeric regions of mouse chromosomes.

We reported previously that the haploid genome of standard strains of laboratory mice contains approximately 70 copies of an amplified long genomic sequence, designated ALGS, that includes a retroposon of the gene for elongation factor 2 (MER). The length of each repeating unit is more than 60 kb, and the sequence of the unit is highly conserved among the repeats. In the present study, Southern blot analysis of the genomes of wild rodents demonstrated that the ALGS is present in all subspecies of Mus musculus and is abundant in M. spicilegus, whereas it is absent in M. spretus as well as in Rattus and other closely related genera. This result indicates that the amplification occurred after the species differentiation with the genus Mus and at least prior to the differentiation of subspecies of M. musculus. To locate chromosomal positions of the ALGS, in situ hybridization was carried out with laboratory strains and wild mice. It appears that the ALGS is located in the centromeric regions of most chromosomes in laboratory mice, M. musculus and M. spicilegus, whereas no positive signals were observed with M. spretus, in accordance with the results from the Southern blotting analysis.     

Antitumor effect of RBS (rice bran saccharide) on ENNG-induced carcinogenesis

We examined whether orally administered RBS (rice bran saccharide), prepared from rice bran by hot water extraction, increases immunocompetence, inhibits gastrointestinal carcinogenesis with N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) or shows an antitumor effect. After the administration of RBS, phytohemagglutinin (PHA)- and pokeweed mitogen (PWM)-stimulated blastogenesis of lymphocytes derived from the mesenteric lymph nodes and peripheral blood was enhanced, and the helper/ suppressor T-cell ratio was elevated, and migration activity of peritoneal macrophages was also increased in rats treated continuously with ENNG. ENNG-induced gastrointestinal carcinomas were observed in 43% of those administered RBS (ENNG-RBS) as compared with 88% in the control (ENNG) and 94% in the prednisolone (PRD) group (ENNG-PRD). The 12-month survival rate of rats bearing gastrointestinal cancer was 58% in the ENNG-RBS group as compared with 25% in the ENNG group and 15% in the ENNG-PRD group. RBS prevented the reduction in immunocompetence in the course of carcinogenesis, suppressed carcinogenesis, and prolonged the survival of rats with gastrointestinal cancer. Antitumor activities of RBS are thought to be a kind of host mediated action. The growth inhibition ratio of transplantable ENNG-induced cancer in Wistar rats was 42.1% in the RBS and 51.8% in the 5-FU group. Since little is known about the potent antitumor activity of -glucan, it would be interesting to consider the relationship between the structure and the biological activities of polysaccharides.