Effect of chromium on photosystem 2 in the unicellular green alga, <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

We investigated the effect of chromium (20–40 g m⁻³, 8–72 h) on the photosystem 2 (PS2) activities of Chlorella pyrenoidosa cells. By using chlorophyll fluorescence transients, thermoluminescence, oxygen polarography, and Western blot analysis for D1 protein we found that inhibition of PS2 can be accounted for by the enhanced photodestruction of the reaction centres in the cells cultivated in the presence of Cr(VI) at 25 °C in “white light” (18 W m⁻²). Hence photodestruction of D1 is caused by an enhanced oxidative stress and lipid peroxidation, as indicated by the appearance of a high-temperature thermoluminescence band.     

Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases

Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.     

87Rb, 23Na and 31P nuclear magnetic resonance spectroscopy of the perfused rat kidney

87Rb, 23Na and 31P nuclear magnetic resonance (NMR) were used to monitor changes in renal cations and energetics during the induction of hypoxia in the isolated perfused rat kidney. The NMR-determined unidirectional Rb+ flux in normoxic kidneys was shown to be a good measure of net intracellular K+ influx in the perfused rat kidney model. The changes in 87Rb, 23Na and 31P spectra following the induction of hypoxia are consistent with hypoxic depletion of intracellular adenosine triphosphate (ATP) and a subsequent decrease in Na-K-ATPase transport activity. The exponential rate constant for 87Rb+ efflux measured during Rb+ uptake in normoxic kidneys (0.12 +/- 0.01 min-1) was not significantly different to the rate constant for 87Rb+ efflux during the induction of hypoxia (0.16 +/- 0.07 min-1). We conclude that there is no direct effect of hypoxia on renal cellular membrane integrity and that renal cell sensitivity to hypoxia is due to an inability to sustain cellular ion gradients following depletion of intracellular ATP.     

ATP interactions of the tau and gamma subunits of DNA polymerase III holoenzyme of Escherichia coli

The tau and gamma subunits of the DNA polymerase III holoenzyme of Escherichia coli were each isolated in large quantities as oligomers from overproducing cells in which their genes (dnaZ and X) were under the control of a T7 phage promoter. The 52-kDa gamma subunit (encoded by the dnaZ sequence) contains three-forths of the N-terminal residues of the 71-kDa tau subunit (encoded by the dnaX sequence). Both gamma and tau share a binding site for ATP (or dATP). A DNA-dependent ATPase activity (Lee, S.H., and Walker, J.R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2713-2717) exhibited only by the tau subunit, presumably requires a DNA-binding site in the C-terminal domain lacking in the gamma subunit. Among ATPases dependent on single-stranded DNA, the tau activity is remarkable in the failure of homopolymers (e.g. poly(dA) or poly(dT)) to replace natural DNAs. The presumed need for certain secondary structures may reflect a feature of template binding in the crucial contribution that tau makes to the high processivity of polymerase III holoenzyme. Limited tryptic digestion of tau generates a fragment that resembles gamma in: (i) size, (ii) binding of ATP without ATPase activity, and (iii) a level of complementing holoenzyme activity in extracts of dnaZ-mutant cells that is higher than that of tau.     

Levels of Hexachlorocyclohexanes and Dichloro-Dipheny-Trichloroethanes in Penguin Droppings Collected from Ardley Island,Maritime Antarctic

This article reports the concentrations of hexachlorocyclohexanes (HCHs) and dichloro-diphenyl-trichloroethanes (DDTs) in fresh Gentoo penguin (Pygoscelis Papua) droppings deposited on the Ardley Island (62°13′S, 58°56′W), West Antarctica, and those in the island's two lake cores. In the fresh penguin droppings, metabolites were the predominant constituents of the DDT group, most likely due to the ban on DDT use in agriculture and the higher stability of the metabolites in the environment. In contrast, γ-HCH comprised 66% of the total HCHs, apparently due to the continuous use of Lindane in the Southern Hemisphere. The concentrations of HCHs and DDTs in the fresh droppings are many times higher than those in penguin plume, the latter currently being used as biomaterial for monitoring coastal contaminations in Antarctica. Results of this study indicate that penguin droppings have a good potential for use as another biomonitoring material in detecting the organochlorine pesticides and other contaminants in the maritime Antarctic. Because penguin droppings can be well preserved in lake sediments, they also provide a good and continuous historical record of these contaminants. The slight increase of DDTs since the 1990s, as observed in the aged penguin droppings in the sediment cores, suggests there have been new inputs of DDT into the Antarctic coastal ecosystem, probably from the illegal use of DDT in the Southern Hemisphere.     

Both low and high concentrations of staurosporine induce G1 arrest through down-regulation of cyclin E and cdk2 expression

Staurosporine has been reported to cause arrest of cells in G1 phase at low concentration and in G2 phase at high concentration. This raises the question of why the effects of staurosporine on the cell cycle depend on the applied concentration. In order to verify these multiple functions of staurosporine in Meth-A cells, we used cyclin E as a landmark of G1/S transition, cyclin B as a landmark of G2/M transition and MPM2 as a hallmark of M phase. We found that staurosporine arrested cells in G1 phase at a low concentration (20 nM) and in G2/M phase at a high concentration (200 nM). However, 200 nM staurosporine increased the expression of cyclin B and cdc2 proteins, suggesting that the cells progressed through the G2/M transition, and increased the expression of MPM2 protein, indicating that the cells entered M phase. Moreover, 200 nM staurosporine increased the expression of p53 and p21 proteins and inhibited the expression of cyclin E and cdk2 proteins, suggesting that the cells were arrested in the G1 phase of the next cycle. Morphological observation showed similar results as well. These data suggest that the G2/M accumulation induced by 200 nM staurosporine does not reflect G2 arrest, but rather results from M phase arrest, followed by progression from M phase to the G1 phase of the next cycle without cytokinesis, and finally arrest of the cells in G1 phase.     

Genomic mapping of chromosomal region 2p15-p21 (D2S378-D2S391): integration of Genemap'98 within a framework of yeast and bacterial artificial chromosomes

The region of chromosome 2 encompassed by the polymorphic markers D2S378 (centromeric) and D2S391 (telomeric) spans an approximately 10-cM distance in cytogenetic bands 2p15-p21. This area is frequently involved in cytogenetic alterations in human cancers. It also harbors the genes for several genetic disorders, including Type I hereditary nonpolyposis colorectal cancer (HNPCC), familial male precocious puberty (FMPP), Carney complex (CNC), Doyne's honeycomb retinal dystrophy (DHRD), and one form of familial dyslexia (DYX-3). Only a handful of known genes have been mapped to 2p16. These include MSH2, which is responsible for HNPCC, FSHR, the gene responsible for FMPP, EFEMP-1, the gene mutated in DHRD, GTBP, a DNA repair gene, and SPTBN1, nonerythryocytic beta-spectrin. The genes for CNC and DYX-3 remain unknown, due to lack of a contig of this region and its underrepresentation in the existing maps. This report presents a yeast- and bacterial-artificial chromosome (YAC and BAC, respectively) resource for the construction of a sequence-ready map of 2p15-p21 between the markers D2S378 and D2S391 at the centromeric and telomeric ends, respectively. The recently published Genemap'98 lists 146 expressed sequence tags (ESTs) in this region; we have used our YAC-BAC map to place each of these ESTs within a framework of 40 known and 3 newly cloned polymorphic markers and 37 new sequence-tagged sites. This map provides an integration of genetic, radiation hybrid, and physical mapping information for the region corresponding to cytogenetic bands 2p15-p21 and is expected to facilitate the identification of disease genes from the area.