Mechanistic studies of methane biogenesis by methyl-coenzyme M reductase: evidence that coenzyme B participates in cleaving the C-S bond of methyl-coenzyme M.

Methyl-coenzyme M reductase (MCR), the key enzyme in methanogenesis, catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). Steady-state and presteady-state kinetics have been used to test two mechanistic models that contrast in the role of CoBSH in the MCR-catalyzed reaction. In class 1 mechanisms, CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage. On the other hand, in class 2 mechanisms, methane is formed in the absence of CoBSH, which functions to regenerate active MCR after methane is released. Steady-state kinetic studies are most consistent with a ternary complex mechanism in which CoBSH binds before methane is formed, as found earlier [Bonacker et al. (1993) Eur. J. Biochem. 217, 587-595]. Presteady-state kinetic experiments at high MCR concentrations are complicated by the presence of tightly bound CoBSH in the purified enzyme. Chemical quench studies in which (14)CH(3)-SCoM is rapidly reacted with active MCRred1 in the presence versus the absence of added CoBSH indicate that CoBSH is required for a single-turnover of methyl-SCoM to methane. Similar single turnover studies using a CoBSH analogue leads to the same conclusion. The results are consistent with class 1 mechanisms in which CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage and are inconsistent with class 2 mechanisms in which CoBSH binds after methane is formed. These are the first reported pre-steady-state kinetic studies of MCR.     

A mobile quorum-sensing system in Serratia marcescens

Quorum-sensing systems that have been widely identified in bacteria play important roles in the regulation of bacterial multicellular behavior by which bacteria sense population density to control various biological functions, including virulence. One characteristic of the luxIR quorum-sensing genes is their diverse and discontinuous distribution among proteobacteria. Here we report that the spnIR quorum-sensing system identified in the enterobacterium Serratia marcescens strain SS-1 is carried in a transposon, TnTIR, which has common characteristics of Tn3 family transposons and is mobile between chromosomes and plasmids of different enterobacterial hosts. SpnIR functions in the new host and was shown to negatively regulate the TnTIR transposition frequency. This finding may help reveal the horizontal transfer and evolutionary mechanism of quorum-sensing genes and alter the way that we perceive regulation of bacterial multicellular behavior.     

The control of mitochondrial respiration in yeast: A possible role of the outer mitochondrial membrane

Mitochondrial respiration in yeast (S. cerevisiae) is regulated by the level of glucose in the medium. Glucose is known to inhibit respiration by repressing key enzymes in the respiratory chain. We present evidence that the early events in this inhibition include the closure of VDAC channels, the primary pathway for metabolite flow across the outer membrane. Aluminum hydroxide is known to inhibit the closure of VDAC. Addition of aluminum acetylacetonate to yeast cells, which should elevate the aluminum hydroxide concentrations in the cytoplasm, caused the inhibition of cell respiration by glucose to be delayed for up to 100 min. No significant effect of aluminum was observed in cells grown on glycerol. Yeast cells lacking the VDAC gene were also unresponsive to the addition of aluminum salt in the presence of glucose. Therefore, the closure of VDAC channels may be an early step in the inhibition of the respiration of yeast by glucose.     

Mutagenesis and transformation of C3H 10T12 cells treated with ethyl methanesulfonate

Survival, mutagenesis and transformation were measured in mouse embryo C3H $\text{10}\text{T}\text{1}\text{2}$ cells following treatment with ethyl methanesulfonate (EMS). Ouabain-resistant cells and transformed cells were isolated, and reconstruction experiments were carried out to determine the optimum conditions for the measurement of mutation and transformation frequencies. Survival was measured by plating efficiency; mutagenesis was measured in terms of the induction of cells able to form colonies in the presence of ouabain; and transformation was measured by the induction of cells forming either morphologically altered colonies on a monolayer of contact-inhibited cells or of cells capable of forming colonies in semi-solid media. When confluent monolayers were incubated for 4 h after treatment with EMS, to allow excision repair before the resumption of DNA synthesis, survival as well as the frequencies of both mutation and transformation increased. When this repair (or holding) period was extended to 24 h, the frequencies of mutation and transformation both decreased as compared to the 4-h holding period. Thus, the holding periods affect the frequencies of EMS-induced mutagenesis and transformation similarly.     

Anion exchanger 1b, but not sodium-bicarbonate cotransporter 1b, plays a role in transport functions of zebrafish H+-ATPase-rich cells

Similar to mammalian proximal tubular cells, H(+)-ATPase rich (HR) cells in zebrafish skin and gills are also responsible for Na(+) uptake and acid secretion functions. However, the basolateral transport pathways in HR cells are still unclear. In the present study, we tested the hypothesis if there are specific slc4 members involved in basolateral ion transport pathways in HR cells. Fourteen isoforms were identified in the zebrafish(z) slc4 family, and the full-length cDNAs of two novel isoforms, zslc4a1b (anion exchanger, zAE1b) and zslc4a4b (Na(+)/HCO(3)(-) cotransporter, zNBCe1b), were sequenced. mRNA signals of zslc4a1b and zslc4a4b were mainly detected in certain groups of ionocytes in zebrafish skin/gills. Further double immunocytochemistry or in situ hybridization demonstrated that zAE1b, but not zNBCe1b, was localized to basolateral membranes of HR cells. Acclimation to low-Na(+) or acidic environments stimulated the mRNA expression of zslc4a1b in zebrafish gills, and loss-of-function of zslc4a1b with specific morpholinos caused significant decreases in both the whole body Na(+) content and the skin H(+) activity in the morphants. On the basis of these results, it was concluded that zAE1b, but not zNBCe1b, is involved in the basolateral transport pathways in Na(+) uptake/acid secretion mechanisms in zebrafish HR cells.     

Cenderitide-Eluting Film for Potential Cardiac Patch Applications

Cenderitide, also known as CD-NP, is a designer peptide developed by combining native mammalian c-type natriuretic peptide (CNP) and the C-terminus isolated from the dendroapis natriuretic peptide (DNP) of the venom from the green mamba. In early studies, intravenous and subcutaneous infusion of cenderitide was reported to reduce left ventricular (LV) mass and ameliorate cardiac remodelling. In this work, biodegradable polymeric films encapsulating CD-NP were developed and were investigated for their in vitro release and degradation characteristics. Subsequently, the bioactivity of released peptide and its effects on human cardiac fibroblast (HCF) were explored. We achieved sustained release from three films with low, intermediate and high release profiles for 30 days. Moreover, the bioactivity of released peptide was verified from the elevated production of cyclic guanosine monophospate (cGMP). The CD-NP released from films was able to inhibit the proliferation of hypertrophic HCF as well as suppress DNA synthesis in HCF. Furthermore, the sustained delivery from films showed comparable or superior suppressive actions on hypertrophic HCF compared to daily infusion of CD-NP. The results suggest that these films could be used to inhibit fibrosis and reduce cardiac remodelling via local delivery as cardiac patches.     

Contributions of cation-π interactions to the collagen triple helix stability

Cation–π interactions are found to be an important noncovalent force in proteins. Collagen is a right-handed triple helix composed of three left-handed PPII helices, in which (X–Y-Gly) repeats dominate in the sequence. Molecular modeling indicates that cation–π interactions could be formed between the X and Y positions in adjacent collagen strands. Here, we used a host–guest peptide system: (Pro-Hyp-Gly)₃-(Pro-Y-Gly-X-Hyp-Gly)-(Pro-Hyp-Gly)₃, where X is an aromatic residue and Y is a cationic residue, to study the cation–π interaction in the collagen triple helix. Circular dichroism (CD) measurements and T_m data analysis show that the cation–π interactions involving Arg have a larger contribution to the conformational stability than do those involving Lys, and Trp forms a weaker cation–π interaction with cationic residues than expected as a result of steric effects. The results also show that the formation of cation–π interactions between Arg and Phe depends on their relative positions in the strand. Moreover, the fluorinated and methylated Phe substitutions show that an electron-withdrawing or electron-donating substituent on the aromatic ring can modulate its π–electron density and the cation–π interaction in collagen. Our data demonstrate that the cation–π interaction could play an important role in stabilizing the collagen triple helix.     

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>10⁵ copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5′ untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.     

Impacts of terminal (4R)-fluoroproline and (4S)-fluoroproline residues on polyproline conformation

Many interests have been focused on prolyl cis–trans isomerization which is related to protein folding and isomer-specific biochemical recognition. Since polyproline can adopt either type I (PPI) helices with all cis amide bonds or type II (PPII) helices with all trans amide bonds, it has been a valuable model to study the prolyl isomerization. Recent studies have shown that stereoelectronic effects govern the stability of PPII structure and the rate of PPII → PPI conversion. To further explore the terminal stereoelectronic effects on polyproline conformation, herein we synthesized a series of host–guest peptides in which (2S,4S)-4-fluoroproline (flp) or (2S,4R)-4-fluoroproline (Flp) residues are incorporated into the C- or N-terminal end of a peptide and studied the thermodynamic and kinetic consequences on polyproline conformation. Circular dichroism measurements revealed that inserting 4-fluoroproline residues into the C terminus of a polyproline peptide induces a great stereoelectronic effect on PPII stability and PPII → PPI conversion rates. From the C terminus, a (Flp)₃ triplet stabilizes PPII structure and increases the transition barrier of PPII → PPI conversion by 1.53 kJ mol^?1 while a (flp)₃ triplet destabilizes PPII conformation and reduce the PPII → PPI transition barrier by 4.61 kJ mol^?1. In contrast, the 4-fluoroproline substitutions at the N terminus do not exhibit distinct stereoelectronic effects on PPII stability and PPII → PPI conversion rates. Our data demonstrate that the C-terminal stereoelectronic effects have a more dramatic impact on PPII stability and PPII → PPI conversion kinetics.     

Lek mating system and its impact on male annihilation technique

Simulation models were presented which describe the matings inside a lek. The size of the lek was assumed to follow a poisson distribution, the dominance factor of each of the males was drawn from a gamma distribution, and the mating probability for each male was determined by its mating effectiveness raised to female mating factor divided by the sum of all the mating effectivenesses raised to this power. The mating probatility for each male and the actual matings were determined by Monte Carlo simulation. Based on the simulation data, the effects of female mating factor and size of the lek on the frequency of actual matings accomplished by the males inside the lek were discussed. In the case of male annihilation, the mean number of effective matings per male in the lek, calculated from the frequency of actual matings weighted by the effective mating ratio, was used to evaluate the efficiency of male annihilation method.