Characterization of DNA end-binding activities in higher plants.

DNA double-strand-breaks (DSB) are the most severe lesion in cells exposing to ionizing radiation and many other stress environments. Repair of DNA DSB is therefore critical to cellular survival. In this work, we observed the double-stranded DNA end-binding (DEB) like activities in rice (Oryza sativa L. cv. TN5) suspension cells and hypocotyls from etiolated mung bean (Vigna radiata L. TN5) seedlings. Higher plant DEB-like protein binds primarily to linearized double-stranded DNA ends. Competition of unlabeled probe was examined in double-stranded DEB assay of cell extracts from rice and mung bean. DEB-like activities of higher plants did not depend on sequence and types of double-stranded DNA ends. Distinct electrophoretic mobility shift patterns and binding features further indicate that DEB-like factors from various sources might not share identical structure and function, and probably belong to different types of DEB proteins from higher plants. Our evidence suggests that DEB proteins are certainly ubiquitous in all organisms probably for repairing and processing double-stranded DNA breaks from formidable lethal lesion.     

Factor 420-dependent tyridine nucleotide-linked hydrogenase system of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.     

High productivity tank fermentation for gramicidin S synthetases

A high productivity tank fermentation for gramicidin S synthetases has been developed to supply biocatalyst for a preparative-scale ATP-driven cell-free enzymatic synthesis employing the polypeptide antibiotic, gramicidin S, as a model product. A rich, complex medium supports rapid and dense growth of the enzyme-producing microorganism, Bacillus brevis ATCC 9999, accompanied by the appearance of excellentenzyme activities. Under conditions used, the two enzyme fractions of the gramicidin S synthesizing system, as well as the total enzymatic activity for synthesis of gramicidin S, all reach their maxima simultaneously at the point where growth enters the stationary phase. Successful batch enzyme fermentations have been performed at the bench (14 liter) and pilot (180 liter)scales.     

The neurovirulent determinants of ts1, a paralytogenic mutant of Moloney murine leukemia virus TB, are localized in at least two functionally distinct regions of the genome.

To better understand the molecular mechanism involved in retrovirus ts1-induced paralytic disease in mice, we constructed a panel of recombinant viruses between ts1 and the wild-type viruses Moloney murine leukemia virus (MoMuLV) and MoMuLV-TB, a strain of MoMuLV. These recombinant viruses were constructed in an attempt to identify the sequence(s) in the genome of ts1 which contains the critical mutation(s) responsible for the neurovirulence of ts1. Two functionally distinct sequences in the genome of ts1 were found to be responsible for its paralytogenic ability. One of these sequences, the 0.77-kilobase-pair XbaI-BamHI (nucleotides 5765 to 6537) fragment which encodes the 5' half of gp70 and 11 base pairs upstream of the env gene coding sequence, determines the inability of ts1 to process Pr80env. The other sequence, the 2.30-kilobase-pair BamHI-PstI (nucleotides 538 to 8264 and 1 to 567) fragment, which comprises nearly two-thirds of the env gene, the long terminal repeat, and the 5' noncoding sequence, determines the enhanced neurotropism of ts1. Replacement of any one of these two regions with the homologous region from either one of the two wild-type viruses resulted in recombinant viruses which either totally failed to induce paralysis or induced a greatly attenuated form of paresis in some of the infected mice.     

Specific binding of three neurotoxins with phospholipase A2 activity to synaptosomal membrane preparations from the guinea pig brain

Snake presynaptic toxins such as crotoxin, -bungarotoxin and taipoxin block neuromuscular transmission through inhibiting the release of acetylcholine by their phospholipase A₂ activities. On the other hand, many other phospholipase A₂s show little neurotoxicity. It is likely that the difference lies in whether high affinity binding to nerve cell membranes exists or not. To test this idea, crotoxin, -bungarotoxin and taipoxin were first radioactively labeled with Na(¹²⁵I) without loss of their neurotoxicity. Using the radioactive toxins we have found that each of the three showed specific binding to synaptosomal membranes from guinea pig brain. In contrast, we could not detect specific binding of a non-neurotoxic pancreatic phospholipase A₂. Crotoxin and taipoxin, but not -bungarotoxin, also bound specifically to membrane preparation from other tissues. The binding of each toxin was not greatly affected by the other two toxins. The photoaffinity labeling technique has been used to obtain further information about the components which bind crotoxin. For this purpose, (¹²⁵I) crotoxin was derivatized with N-hydroxysuccinimidyl-4-azidobenzoate. Autoradiographic analysis of the membranes following photoirradiation in the presence of the modified crotoxin revealed that an 85K dalton component was preferentially covalently conjugated with the crotoxin analogue in a specific manner.On leave from Department of Biochemistry and Biophysics, University of Hawaii, School of Medicine, Honolulu, Hawaii.     

Rainfall-driven sex-ratio genes in African buffalo suggested by correlations between Y-chromosomal haplotype frequencies and foetal sex ratio

Background  

The Y-chromosomal diversity in the African buffalo (Syncerus caffer) population of Kruger National Park (KNP) is characterized by rainfall-driven haplotype frequency shifts between year cohorts. Stable Y-chromosomal polymorphism is difficult to reconcile with haplotype frequency variations without assuming frequency-dependent selection or specific interactions in the population dynamics of X- and Y-chromosomal genes, since otherwise the fittest haplotype would inevitably sweep to fixation. Stable Y-chromosomal polymorphism due one of these factors only seems possible when there are Y-chromosomal distorters of an equal sex ratio, which act by negatively affecting X-gametes, or Y-chromosomal suppressors of a female-biased sex ratio. These sex-ratio (SR) genes modify (suppress) gamete transmission in their own favour at a fitness cost, allowing for stable polymorphism.     

Generation of lymphokine-activated killer (LAK) cells and possible implications in autologous bone marrow transplantation--a preliminary report

Lymphokine-activated killer (LAK) cells were generated successfully without mitogen from blood mononuclear cells obtained from 14 patients with varying malignancies and 2 normal donors. Cells from both groups showed a positive cytotoxicity by a 4-hour 51-Cr-release assay against a variety of target cells including natural killer (NK) sensitive K562 myeloid leukemia, NK-resistant Raji lymphoma cell lines, and fresh/cryopreserved leukemia cells from patients refractory to standard chemotherapy but not normal blood cells. Higher cytotoxic activity was obtained with a higher effector:target ratio at 100:1 greater than 50:1 greater than 25:1 (P less than 0.01) in each setting of different targets. Experiments involving cocultures of the LAK cells with either allogeneic (9) or autologous (3) bone marrow cells disclosed no detrimental effect on the committed hemopoietic stem cells by semisolid agar colony forming unit (CFU-GM) assay. The findings suggest that LAK cells may have a potential role for the in vitro purging of the residual leukemic cells from the marrow inoculum prepared for autologous bone marrow transplantation.     

Variation of biomass and kinetic parameters for nitrifying species in the TNCU3 process at different aerobic hydraulic retention times

In this study, the variation of biomass, kinetic parameters, and stoichiometric parameters for ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in TNCU3 process were explored at different aerobic hydraulic retention time (AHRT). The results indicated that the growth rate constants of AOB were 0.92, 0.88, and 0.95 days^?1, respectively, meanwhile, those of NOB were 2.58 1.41, and 1.40 days^?1, respectively, when AHRT was 5, 6, and 7 h. The lysis rate constants for AOB and NOB were 0.13 and 0.17 days^?1, respectively. When AHRT was 5, 6, and 7 h, the yield coefficients of AOB were 0.20, 0.23, and 0.28 g COD g^?1 N, respectively, meanwhile those of NOB were 0.23, 0.19, and 0.22 g COD g^?1 N, respectively. The average percentage of AOB was 0.44, 0.61, and 0.64%, respectively, while that of NOB was 0.46, 0.61, and 0.74%, respectively. The relation between the biomass percentage of AOB and AHRT was in a good agreement with first type hyperbolic curve. The relation between the biomass percentage of NOB and AHRT was in a good agreement with seven types of curve including simple exponential curve, power exponential curve, and first type hyperbolic curve etc. When the AHRT increased from 5 to 7 h, the removal efficiency of NH₄ ⁺–N increased from 80.2 to 94.8%, or by 14.6%. Meanwhile, the removal efficiency of total nitrogen increased from 63.6 to 70.9%, or by 7.3%.