Preparation of stereoselectively-deuterated NADH and NADPH by coupling of glutamate racemase and glutamate dehydrogenase

Summary We have developed a simple method for preparation of [4R²H] and [4S-²H]-NAD(P)H by combination of glutamate racemase, glutamate dehydrogenase, and 2-aminoethanesulfonate: 2-oxoglutarate aminotransferase. In this new enzymatic procedure, no deuterated substrates were used.     

Differential Responses in Activity of Antioxidant Enzymes to Different Environmental Stresses in Arabidopsis thaliana

Arabidopsis thaliana . Three-week-old plants were exposed to a high temperature (30 C), an enhanced light intensity (200 μE/m²/sec), water deficiency (water deprivation for 2 days), a chilling temperature (5 C), or ultraviolet-B (UV-B) radiation (0.25 or 0.094 W/m²) for 1 week (except for water deficiency). The high temperature and enhanced light treatments increased only dehydroascorbate reductase (DHAR) activity. Water deficiency enhanced the activities of DHAR and guaiacol peroxidase (PER). Chilling temperature increased the activities of ascorbate peroxidase (APX) and glutathione reductase (GR), whereas it decreased catalase (CAT) activity. UV-B at an intensity of 0.25 W/m² elevated the activities of APX, monodehydroascorbate reductase (MDHAR), GR, PER and superoxide dismutase (SOD). It was suggested that the amounts of phenylpropanoid compounds increased during treatments of plants with enhanced light intensity, chilling temperature, and UV-B. These results suggest that some differences exist among the oxidative stress conditions caused by the different treatments, although all of these treatments seem to be related to active oxygen production. We propose that in A. thaliana, environmental stresses may be classified into those which induce DHAR activity and those which induce APX activity. Received 11 January 1999/ Accepted in revised form 22 April 1999     

Possible role of the duodenum in the entero-PP axis

The plasma pancreatic polypeptide response to a meal was compared in 6 healthy controls and 30 patients with gastric cancer who had undergone either subtotal gastrectomy or total gastrectomy with radical lymph node dissection including sympathectomy. Twelve patients were reconstructed with Billroth I, 9 patients with Billroth II, 6 patients with a double tract, and 3 patients with Roux-en-Y. Ten patients with a gastric ulcer who had undergone Billroth I gastrectomy including pyloric ring preservation also were examined. Impaired pancreatic polypeptide secretion was noted only in Billroth II and Roux-en-Y patients, where the duodenum is not affected by the passage of meals.Billroth I and double tract patients, in contrast, and an enhanced pancreatic polypeptide secretion. However, in BI patients with pyloric ring preservation the PP response to a meal was almost normal. These findings suggest an important role of the duodenum in the entero-PP axis in man.     

Transfection and expression of a gene coding for a human melanoma antigen recognized by autologous cytolytic T lymphocytes

Human melanoma line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocytes (CTL). As a first step towards the cloning of the gene coding for one of these antigens, we tried to obtain transfectants expressing the antigen. The DNA recipient cell was a variant of MZ2-MEL which had been selected with a CTL clone for the loss of antigen E. It was cotransfected with genomic DNA of the original melanoma line and with selective plasmid pSVtkneo. Geneticin-resistant transfectants were obtained at a frequency of 2 × 10^–4. These transfectants were then screened for their ability to stimulate the production of tumor necrosis factor by the anti-E CTL clone. One transfectant expressing antigen E was identified among 70 000 drug-resistant transfectants. Its sensitivity to lysis by the anti-E CTL was equal to that of the original melanoma cell line. When this transfectant was submitted to immunoselection with the anti-E CTL clone, the resulting antigen-loss variants were found to have lost several of the transfected pSVtkneo sequences. This indicated that the gene coding for the antigen had been integrated in the vicinity of pSVtkneo sequences, as expected for cotransfected DNA. Address correspondence and offprint requests to: T. Boon.     

Methylation status of ribosomal RNA gene clusters in the flow-sorted human acrocentric chromosomes

Southern blot analysis of the human acrocentric chromosomes that were flow-sorted from B-lymphoblastoid cell line GM130B revealed that the sensitivity of the ribosomal RNA (rDNA) gene clusters to the restriction enzyme NotI differs among these rDNA-containing chromosomes: the rDNA clusters of Chromosomes (Chr) 13, 14, and 15 are much more sensitive to NotI digestion than those of Chrs 21 and 22 in this particular cell line. Detailed analysis by use of methylation-sensitive enzymes HpaII and HhaI and methylation-insensitive enzyme MspI confirmed the significant variation in the methylation status of rDNA clusters among these chromosomes. Quantitative analysis by fluorescent in situ hybridization (FISH) indicated that copy number of rDNA varies among individual chromosomes, but the average copy number in the acrocentric Chrs 21 and 22 is significantly greater than that of the Chrs 13, 14, and 15 in GM130B cells. Similar analysis reveals that the methylation status of rDNA clusters in another B-lymphoblastoid cell line GM131 was different from that of GM130B. These data together indicate that the copy number and methylation patterns of rDNA clusters differ among individual acrocentric chromosomes in a given cell line, and they are different among cell lines.     

Degradation of Mitochondrial Phospholipids During Experimental Cerebral Ischemia in Rats

Changes in content of brain mitochondrial phospholipids were examined in rats after 30 and 60 min of decapitation ischemia compared with controls, to explore the degradation of the mitochondrial membrane and its relation to dysfunction of mitochondria. Activities of respiratory functions and respiratory enzymes (cytochrome c oxidase; F0F1-ATPase) decreased significantly during ischemia. Considerable decreases in cardiolipin and phosphatidylinositol content were observed after 60 min, and other phospholipids showed similar but nonsignificant decreases in content. The amount of polyunsaturated fatty acids chains, such as arachidonic and docosahexaenoic acids, was reduced in each phospholipid, in some cases significantly, after 30 and 60 min of ischemia in time-dependent manners. Degradation of mitochondrial phospholipids during ischemia associated with the deterioration of mitochondrial respiratory functions suggested the significance of such changes in phospholipid content in disintegration of cellular energy metabolism during cerebral ischemia.     

DNA diagnosis of hydatidiform mole using the polymerase chain reaction

Summary We have used the polymerase chain reaction (PCR) technique for the diagnosis of hydatidiform mole, a trophoblastic disease. For this, we targeted the hypervariable 3 flanking region of the APOB gene (APOB/ VNTR) because of its high heterozygosity index (0.61) in the Japanese population. We examined seven clinical cases which were tentatively diagnosed as hydatidiform moles. Five of these revealed DNA segments unique to the paternal APOB allele, allowing us to diagnose a complete mole. The PCR technique for targeting the APOB/VNTR appears useful for early diagnosis of hydatidiform mole.     

Expression in Escherichia coli of the Extrinsic 18-kDa Protein of Photosystem II of Spinach

A cDNA encoding the precursor for the 18-kDa protein of PSIIof spinach was expressed in Escherichia coli. When the celllysate was incubated at 7°C, the precursor was degradedby proteases of E. coli to a polypeptide of 18 kDa (P18) thatconsisted of the mature protein moiety plus the last four residuesof the transit peptide. P18 was able to reconstitute the water-oxidizingcomplex of NaCl-treated PSII membranes supplemented with the23-kDa protein. Moreover, P18 was cleaved by the prolyl endoproteinaseof spinach specifically at the Pro-12-Leu-13 bond, as was theauthentic 18-kDa protein. These properties of P18 indicate thatthe present expression system is potentially useful for studiesof the substrate specificity of the endoproteinase, as wellas of the structure-function relationships of the 18-kDa protein. (Received November 12, 1994; Accepted January 11, 1995)