Mercury-Induced Inhibition of Photosystem II Activity and Changes in the Emission of Fluorescence from Phycobilisomes in Intact Cells of the Cyanobacterium, Spirulina platensis

Addition of low concentrations of mercury chloride (HgCl₂ tointact cells of the cyanobacterium, Spirulina platensis causedan enhancement in the intensity of fluorescence emitted fromphycocyanin at room temperature and induced blue shifts in theemission peak suggestive of changes in energy transfer withinthe phycobilisomes. HgCl₂ also suppressed the whole-chain electrontransport activity (H₂O methylviologen) at much lower concentrationsthan that required to inhibit Hill activity supported by para-benzoquinone.The extent of inhibition of Hill activity was much higher underhigh-intensity light than that under low-intensity light. Ourresults indicate that mercury ions at low concentrations affectthe transfer of energy within phycobilisomes and at high concentrationsthey inhibit electron transport in this cyanobacterium. (Received February 21, 1989; Accepted October 2, 1989)     

Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.     

Non-equivalence of YEPD and synthetic complete media in yeast reversion studies

In yeast reversion studies, assay of the total number of cells is made by plating irradiated cells on agar plates containing yeast extract, peptone and dextrose (YEPD) medium. The number of revertants are scored by plating cells on synthetic complete (SC) medium deficient in the particular nutrient for which the reversion is tested. In this procedure equivalence for cell survival between the YEPD and the SC media is always assumed. However it is shown in this paper that this assumption is valid only up to dose levels where cell killing is not significant. At high doses, survivals on the two media differ significantly from each other for both high and low LET radiations. This difference influences the slope of the reversion frequency curve at high doses. Since the reversion frequency is expressed with reference to the number of survivors after a given radiation dose, it is essential to see that the same chance of survival is offered to the reverted and unreverted cells.Even though reversion is reported to vary linearly with dose, it is found that this linearity is restricted only to dose levels where cell killing is not significant. At higher doses, the reversion frequency varies in a very complex manner with dose for both high and low LET radiations. The complexity depends further on the reference medium chosen.     

Some differences in the properties of carnitine palmitoyltransferase activities of the mitochondrial outer and inner membranes.

Recent evidence has shown that the outer, overt, malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPTo) activity resides in the mitochondrial outer membrane [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382]. A comparison of CPTo activity of rat liver mitochondria with the inner, initially latent, carnitine palmitoyltransferase (CPTi) of the mitochondrial inner membrane has revealed that the presence of digitonin and several other detergents inactivates CPTo activity. The CPTi activity, in contrast, was markedly stimulated by various detergents and phospholipid liposomes. These findings explain why in previous studies, which used digitonin or other detergents to expose, separate and purify the CPT activities, the inferences were drawn that (a) the ratio of latent to overt CPT was quite high, (b) both the CPT activities could be ascribed to one active protein recovered, and (c) the observed lack of malonyl-CoA inhibition indicated possible loss/separation of a putative malonyl-CoA-inhibition-conferring protein. Although both CPTo and CPTi were found to catalyse the forward and the backward reactions, CPTo showed greater capacity for the forward reaction and CPTi for the backward reaction. The easily solubilizable CPT, released on sonication of mitoplasts or of intact mitochondria under hypo-osmotic conditions, resembled CPTi in its properties. When octyl glucoside was used under appropriate conditions, 40-50% of the CPTo of outer membranes became solubilized, but it showed limited stability and decreased malonyl-CoA sensitivity. Malonyl-CoA-inhibitability of CPTo was decreased also on exposure of outer membranes to phospholipase C. When outer membranes that had been exposed to octyl glucoside or to phospholipase C were subjected to a reconstitution procedure using asolectin liposomes, the malonyl-CoA-inhibitability of CPTo was restored. A role of phospholipids in the malonyl-CoA sensitivity of CPTo is thus indicated.     

Analogs of adenosine 3',5'-cyclic phosphate. II. Synthesis and enzymatic activity of derivatives of 1,N6-ethenoadenosine 3',5'-cyclic phosphate

The reactions of chloroacetaldehyde with adenosine 3′,5′-cyclic phosphate, and with several analogs modified at C₈ of the purine ring or C₅, of the sugar, lead to the corresponding 1,N⁶-etheno derivatives^d. Similar reactions using other 2-bromoaldehydes or phenacyl bromide give 1,N⁶-ethenonucleotides substituted at the α- or β-positions of the etheno bridge respectively. The ability of these compounds to activate the protein kinases from rabbit muscle and calf brain has been evaluated over a wide range of concentrations. While no derivative proved to be more active than adenosine 3′,5′-cyclic phosphate itself using the enzyme from rabbit muscle, a wide spectrum of activities was found using that from calf brain.     

Role of astrocytes in neurovascular coupling

Neural activity is intimately tied to blood flow in the brain. This coupling is specific enough in space and time that modern imaging methods use local hemodynamics as a measure of brain activity. In this review, we discuss recent evidence indicating that neuronal activity is coupled to local blood flow changes through an intermediary, the astrocyte. We highlight unresolved issues regarding the role of astrocytes and propose ways to address them using novel techniques. Our focus is on cellular level analysis in?vivo, but we also relate mechanistic insights gained from ex?vivo experiments to native tissue. We also review some strategies to harness advances in optical and genetic methods to study neurovascular coupling in the intact brain.     

Template‐free protein structure prediction and quality assessment with an all‐atom free‐energy model

Biophysical forcefields have contributed less than originally anticipated to recent progress in protein structure prediction. Here, we have investigated the selectivity of a recently developed all‐atom free‐energy forcefield for protein structure prediction and quality assessment (QA). Using a heuristic method, but excluding homology, we generated decoy‐sets for all targets of the CASP7 protein structure prediction assessment with <150 amino acids. The decoys in each set were then ranked by energy in short relaxation simulations and the best low‐energy cluster was submitted as a prediction. For four of nine template‐free targets, this approach generated high‐ranking predictions within the top 10 models submitted in CASP7 for the respective targets. For these targets, our de‐novo predictions had an average GDT_S score of 42.81, significantly above the average of all groups. The refinement protocol has difficulty for oligomeric targets and when no near‐native decoys are generated in the decoy library. For targets with high‐quality decoy sets the refinement approach was highly selective. Motivated by this observation, we rescored all server submissions up to 200 amino acids using a similar refinement protocol, but using no clustering, in a QA exercise. We found an excellent correlation between the best server models and those with the lowest energy in the forcefield. The free‐energy refinement protocol may thus be an efficient tool for relative QA and protein structure prediction. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Biosynthesis and turnover of a 34-kDa protein growth factor in human cytotrophoblasts

Recently we isolated a new protein growth factor of 34 kDa from synctial membranes of human placenta. In its polypeptide molecular mass, antigenic structure, receptor binding specificity and partial amino acid sequence, it is unlike several known growth factors, hormones and other proteins. Here we report studies on its biosynthesis and turnover in cultured cytotrophoblasts from term human placenta. Expression of the 34-kDa protein in these cells was studied by immunoprecipitation and Western blot analyses using a highly specific antibody. The experiments have produced the following results. a) Immunostaining and Western blot analyses have demonstrated the presence of immunoreactive 34-kDa protein in isolated cytotrophoblasts. The protein is present in both freshly isolated cells and in cells that have fused in culture to form multinuclear syncytiotrophoblasts. b) Trophoblastic biosynthesis of the protein has been demonstrated by in vitro translation of cellular mRNA and by metabolic labelling experiments with intact cells. c) Pulse-chase experiments show that biosynthesis of the protein does not involve any detectable precursors of higher or lower molecular mass. d) Studies on turnover indicate that the synthesized protein is unusually stable with a half-life of 50-70 h.