Nitrogenase. VII. Effect of component ratio, ATP and H2 on the distribution of electrons to alternative substrates.

Some kinetic properties of purified component I (Mo-Fe protein) and component II (Fe protein) of nitrogenase (EC 1.7.99.2) from Azotobacter vinelandii have been examined. The apparent Km values for reducible substrates (0.1 atm for N2, 0.01 atm for acetylene) and dithionite (0.5 mM) are similar for osmotically shocked cell lysates and purified components. However, the ATP dependence of acetylene and N2 reduction varies sigmoidally with ATP concentration and as a function of the relative and absolute concentration of components I and II in the assay. Acetylene is reduced in preference to N2 in competitive assays when component I is in relative excess. Acetylene reduction is not as dependent upon ATP concentration as is N2 reduction, so that acetylene is also a preferred substrate at lower ATP levels. Hydrogen specifically inhibits N2 reduction, diverting electrons to acetylene when both substrates are present in the assay. We propose a model of the enzyme activity, in which the substrates for reduction are bound to component I with electrons being activated by component II. ATP may be involved in activating electrons and in maintaining the appropriate conformation or reduction state of components to allow effective reduction of substrates. The relative rate of reduction of alternative substrates is dependent on the concentration of the particular state(s) capable of reacting with each substrate. The concentration of a particular state of component I is a function of components I, II and ATPL     

Cytochrome oxidase and ascorbic acid in the normal and regenerating tail of the scincid lizard, Mabuya carinata. A histophysiological study.

The concentration of ascorbic acid (AA) and the histochemical distribution of this vitamin together with cytochrome oxidase have been investigated in the normal and regenerating tail of the Scincid lizard, Mabuya carinata. An interesting aspect of this investigation is the observation of a total lack of cytochrome oxidase in both the normal and regenerating tail of the lizard, except for the differentiating phase. On the other hand, AA has been found to be present in the normal and regenerating tail with above normal levels during wound healing (twofold) and differentiation (fivefold). In the light of the poor cytochrome oxidase activity, the higher content of AA noted during regeneration has been construed to play a possible role in the respiratory mechanics of the regenerating lizard tail. Further, the importance of AA in cellular metabolism and the wound healing and differentiative processes have also been discussed.     

SOMA: A Single Oligonucleotide Mutagenesis and Cloning Approach

Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.     

Impact of Histone H4 Lysine 20 Methylation on 53BP1 Responses to Chromosomal Double Strand Breaks

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.     

Promising applications of cold plasma for microbial safety,chemical decontamination and quality enhancement in fruits

Consumers’ demand is increasing for safe foods without impairing the phytochemical and sensory quality. In turn, it has increased research interest in the exploration of innovative food processing technologies. Cold plasma technology is getting popularity now days owing to its high efficacy in decontamination of microbes in fruit and fruit-based products. As a on-thermal approach, plasma processing maintains the quality of fruits and minimizes the thermal effects on nutritional properties. Cold plasma is also exploited for inactivating enzymes and degrading pesticides as both are directly related with quality loss and presently are most important concerns in fresh produce industry. The present review covers the influence of cold plasma technology on reducing microbial risks and enhancing the quality attributes in fruits.     

Assessment of differences in morphological and physiological leaf lodging characteristics between two cultivars of Hippeastrum rutilum

Kernel weight and morphology are important traits affecting cereal yields and quality. Dissecting the genetic basis of thousand kernel weight (TKW) and its related traits is an effective method to improve wheat yield. In this study, we performed quantitative trait loci (QTL) analysis using recombinant inbred lines derived from the cross ‘PuBing3228 × Gao8901’ (PG-RIL) to dissect the genetic basis of kernel traits. A total of 17 stable QTLs related to kernel traits were identified, notably, two stable QTLs QTkw.cas-1A.2 and QTkw.cas-4A explained the largest portion of the phenotypic variance for TKW and kernel length (KL), and the other two stable QTLs QTkw.cas-6A.1 and QTkw.cas-7D.2 contributed more effects on kernel width (KW). Conditional QTL analysis revealed that the stable QTLs for TKW were mainly affected by KW. The QTLs QTkw.cas-7D.2 and QKw.cas-7D.1 associated with TKW and KW were delimited to the physical interval of approximately 3.82 Mb harboring 47 candidate genes. Among them, the candidate gene TaFT-D1 had a 1 bp insertions/deletion (InDel) within the third exon, which might be the reason for diversity in TKW and KW between the two parents. A Kompetitive Allele-Specific PCR (KASP) marker of TaFT-D1 allele was developed and verified by PG-RIL and a natural population consisted of 141 cultivar/lines. It was found that the favorable TaFT-D1 (G)-allele has been positively selected during Chinese wheat breeding. Thus, these results can be used for further positional cloning and marker-assisted selection in wheat breeding programs. Seventeen stable QTLs related to kernel traits were identified. The stable QTLs for thousand kernel weight were mainly affected by kernel width. TaFT-D1 could be the candidate gene for QTLs QTkw.cas-7D.2 and QKw.cas-7D.1.