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.     

Enzymatic isomerization and epimerization of D-erythrose 4-phosphate and its quantitative analysis by gas chromatography/mass spectrometry

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of D-erythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon D-erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and D-erythrose 4-phosphate. At the equilibrium, about 90% of the tetrose 4-phosphate existed in the form of D-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.     

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of ¹⁵O, ¹³N, and ¹¹C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.     

Production,Isolation and Pharmacological Studies of AI-77s

Two potent gastroprotective substances against experimental gastric ulcers in rats induced by stress and five of their analogues were isolated from the culture broth of bacterial strain AI-77 which was classified taxonomically as Bacillus pumilus. Physico-chemical properties and pharmacological activities of the seven compounds were examined and compared with each other.     

Highly frequent single amino acid substitution in mammalian elongation factor 2 (EF-2) results in expression of resistance to EF-2-ADP-ribosylating toxins

Toxin-resistant polypeptide chain elongation factor 2 cDNA has been cloned from a mutant hamster cell line with only non-ADP-ribosylatable elongation factor 2. The mutation conferring resistance to diphtheria toxin and Pseudomonas aeruginosa exotoxin A is a G-to-A transition in the first nucleotide of codon 717. Codon 715 encodes a histidine residue that is modified post-translationally to diphthamide, which is the target amino acid for ADP-ribosylation by both toxins. Transfection of mouse L cells with a recombinant elongation factor 2 cDNA differing from the wild-type only by this G-to-A transition confers resistance to P. aeruginosa exotoxin A. The degrees of toxin-resistant protein synthesis of stable transfectants are dependent on the ratio of non-ADP-ribosylated elongation factor 2 to wild-type elongation factor 2, not the amount of non-ADP-ribosylated elongation factor 2. The mutation creates a new Mbo II restriction site in the elongation factor 2 gene. Several independently isolated diphtheria toxin-resistant Chinese hamster ovary cell lines show the same alteration in the Mbo II restriction pattern.     

Seasonal changes in vertical distribution, biomass and faecal production of tubificids in the profundal region of a shallow Japanese lake

Two tubificid species Limnodrilus hoffmeisteri and L. claparedeianus formed more than 93% of the total number of oligochaetes in the profundal. Limnodrilus spp. worms were found down to 33 cm in the sediment but in great numbers in the upper zone in June and October. Worms confined to the top 15 cm of sediment accounted for 53-92% of the total number. There were two annual maxima in population density and biomass, one in late spring (66000 inds m⁻², 17 g wet wt m⁻²) and the other in mid autumn (97000 inds m⁻², 176 g wet wt m⁻²). Two regression lines describing the effect of temperature on faecal production rate were obtained; Log F = 0.0604 T (°C) −0.7660 (below 15°C), Log F = 0.0266 T – 0.2170 (above 15°C). In total 26.8 kg dry wt m⁻² of sediment was defecated annually by Limnodrilus spp. The sediment in the 0–10 cm stratum may pass through the guts of the worms 2.3 times a year. Sedimentation rates in profundal region were very low with respect to the faecal production rates of the tubificids.     

Decreased intracellular free magnesium in erythrocytes of spontaneously hypertensive rats

Using 31p-NMR (the phosphorus nuclear magnetic resonance) spectroscopy, we measured intracellular free Mg levels in the erythrocytes of untreated (n = 7) and diltiazem-treated spontaneously hypertensive rats (SHR) (n = 8), and compared them with age-matched Wistar-Kyoto rats (WKY) (n = 10). The intracellular free Mg levels were significantly (p less than 0.01) decreased in untreated SHR compared with those in control WKY. A successful antihypertensive treatment with diltiazem increased the intracellular free Mg levels compared with untreated SHR (p less than 0.05). Furthermore, an inverse correlation was observed between intracellular free Mg levels and blood pressure levels in all groups (r = -0.48, p less than 0.01, n = 25). These observations suggest that abnormalities of intracellular Mg metabolism may be, in part, related to the development or the maintenance of hypertension in SHR.     

The direct activation of human multidrug resistance gene (MDR1) by anticancer agents

Enhanced expression of a multidrug-resistance gene (MDR1) is observed in some cancer patient, but any regulatory mechanisms of MDR1 gene expression in this phenomenon is not yet known. In this study, the regulation of MDR1 gene was analysed by transient expression assays in the presence of anticancer agents. We found that MDR1 promoter could be activated directly on the addition of anticancer agents including vincristine, daunomycin, adriamycin and colchicine. The results suggest that the level of MDR1 mRNA expression is associated with previous chemotherapy, including drugs that select the multidrug resistance phenotype.     

Induction of manganese superoxide dismutase by tumor necrosis factor in human breast cancer MCF-7 cell line and its TNF-resistant variant

Tumor necrosis factor (TNF)-resistant variant of human mammary cancer MCF-7 cell line was isolated by stepwise selection. The final TNF-resistant variant Tnf-1000 showed more than 100-fold higher resistance than the parental MCF-7 cell. Saturation kinetics for 125I-TNF binding showed that TNF-1000 cells had similar TNF receptor numbers as MCF-7 cells, but of a lower affinity. Induction of superoxide dismutase (SOD) was compared between MCF-7 and Tnf-1000 cells treated with TNF: SOD scavenges potentially toxic superoxide radicals. TNF induced more mitochondrial manganese SOD (SODm) in MCF-7 than in Tnf-1000 whereas there appeared to be no significant induction of cytosolic copper/zinc SOD (SODc) by TNF in both MCF-7 and Tnf-1000 cell lines. Acquirement of TNF-resistance in MCF-7 cells might be correlated with expression of SODm.