Pressure-jump studies of the folding/unfolding of trp repressor.

The dimeric protein, trp apo-repressor of Escherichia coli has been subjected to high hydrostatic pressure under a variety of conditions, and the effects have been monitored by fluorescence spectroscopic and infra-red absorption techniques. Under conditions of micromolar protein concentration and low, non-denaturing concentrations of guanidinium hydrochloride (GuHCl), tryptophan and 8-anilino-1-naphthalene sulfonate (ANS) fluorescence detected high pressure profiles demonstrate that pressures below 3 kbar result in dissociation of the dimer to a monomeric species that presents no hydrophobic binding sites for ANS. The FTIR-detected high pressure profile obtained under significantly different solution conditions (30 mM trp repressor in absence of denaturant) exhibits a much smaller pressure dependence than the fluorescence detected profiles. The pressure-denatured form obtained under the FTIR conditions retains about 50 % alpha-helical structure. From this we conclude that the secondary structure present in the high pressure state achieved under the conditions of the fluorescence experiments is at least as disrupted as that achieved under FTIR conditions. Fluorescence-detected pressure-jump relaxation studies in the presence of non-denaturing concentrations of GuHCl reveal a positive activation volume for the association/folding reaction and a negative activation volume for dissociation/unfolding reaction, implicating dehydration as the rate-limiting step for association/folding and hydration as the rate-limiting step for unfolding. The GuHCl concentration dependence of the kinetic parameters place the transition state at least half-way along the reaction coordinate between the unfolded and folded states. The temperature dependence of the pressure-jump fluorescence-detected dissociation/unfolding reaction in the presence of non-denaturing GuHCl suggests that the curvature in the temperature dependence of the stability arises from non-Arrhenius behavior of the folding rate constant, consistent with a large decrease in heat capacity upon formation of the transition state from the unfolded state. The decrease in the equilibrium volume change for folding with increasing temperature (due to differences in thermal expansivity of the folded and unfolded states) arises from a decrease in the absolute value for the activation volume for unfolding, thus indicating that the thermal expansivity of the transition state is similar to that of the unfolded state.     

Anillin and the septins promote asymmetric ingression of the cytokinetic furrow

During cytokinesis, constriction of a cortical contractile ring generates a furrow that partitions one cell into two. The contractile ring contains three filament systems: actin, bipolar myosin II filaments, and septins, GTP-binding hetero-oligomers that polymerize to form a membrane-associated lattice. The contractile ring also contains a potential filament crosslinker, Anillin, that binds all three filament types. Here, we show that the contractile ring possesses an intrinsic symmetry-breaking mechanism that promotes asymmetric furrowing. Asymmetric ingression requires Anillin and the septins, which promote the coalescence of components on one side of the contractile ring, but is insensitive to a 10-fold reduction in myosin II levels. When asymmetry is disrupted, cytokinesis becomes sensitive to partial inhibition of contractility. Thus, asymmetric furrow ingression, a prevalent but previously unexplored feature of cell division in metazoans, is generated by the action of two conserved furrow components and serves a mechanical function that makes cytokinesis robust.     

Analysis of microtubule polymerization in vitro and during the cell cycle in Xenopus egg extracts

Microtubules are dynamic polymers that participate in multiple cellular processes such as vesicular transport and cell division. Microtubule dynamics alter dramatically during the cell cycle. An excellent system to study microtubule dynamics is Xenopus egg extracts since it is a system that is open to manipulation. The extracts can be cycled between mitosis and interphase allowing the study of microtubules in these phases as well as during cell cycle transitions. Here, we provide simple assays to study microtubules in extracts and in vitro using purified components. Protocols are provided for the purification of frog tubulin, microtubule pelleting from extracts and in vitro, assembly of microtubule structures in extracts, and isolation of microtubule-associated proteins from extract. These methods can be used to analyze the effect of a protein of interest on the microtubule cytoskeleton.     

Synthesis of pyrazole encompassing 2-pyridone derivatives as antibacterial agents

A series of novel compounds 6-amino-1-((1,3-diphenyl-1H-pyrazole-4-yl)methyleneamino)-4-(aryl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitriles (4a–t) were synthesized and characterized by IR, ¹H NMR, ¹³C NMR and mass spectral data. These compounds were screened for their in vitro antibacterial activity against Staphylococcus aureus, Streptococcus pyogenes (Gram positive), Escherichia coli, Pseudomonas aeruginosa (Gram negative) by serial broth dilution and cytotoxic activity (NIH 3T3 & HeLa) by MTT assay. The results indicated that compounds 4g, 4i, 4m, 4o, 4r and 4t exhibit potent antibacterial activity against bacterial strains at non-cytotoxic concentrations.     

Protective effect of L-arginine against lipid peroxidation in goat epididymal spermatozoa

L-arginine plays an important role in physiology of spermatozoa and is shown to enhance the metabolism of these cells. We report here the effect of L-arginine on membrane lipid peroxidation of goat epididymal spermatozoa. Both natural peroxidation as well as that induced by UV radiation, freezing and oxidizing agents have been studied. Irrespective of the nature of induction of peroxidation, L-arginine reduces the extent of lipid peroxidation in a concentration dependent manner. Both L-arginine and alpha-tocopherol act synergistically in protecting against lipid peroxidation induced by the above methods. Thus, in order to provide protection against lipid peroxidation, L-arginine may be added in media used to preserve spermatozoa.     

Oral Antibiotic Treatment of Mice Exacerbates the Disease Severity of Multiple Flavivirus Infections

1. Download high-res image (163KB)
2. Download full-size image

     

Alkaloid production byClaviceps sp. SD-58; Involvement of phosphatase isozymes

Simultaneous reduction in alkaloid yield and level of phosphatases by high concentrations of phosphate was observed inClaviceps sp. SD-58. Tryptophan-induced culture showed an increase in alkaloid yield and the level of phosphatases. Phosphate caused repression of both acid phosphatase (isoenzyme I) and alkaline phosphatase (isoenzymes III and V).     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Oral Delivery of Peptide Formulations and Their Cellular Evaluation

International Journal of Peptide Research and Therapeutics - The development of peptide-based formulations presents numerous challenges to the formulator due to their complexity, delicate structure...     

The adenosine dimethyltransferase KsgA recognizes a specific conformational state of the 30S ribosomal subunit

The methyltransferase KsgA modifies two adjacent adenosines in 16S rRNA by adding two methyl groups to the N(6) position of each nucleotide. Unlike nearly all other rRNA modifications, these modifications and the responsible enzyme are highly conserved phylogenetically, suggesting that the modification system has an important role in ribosome biogenesis. It has been known for some time that KsgA recognizes a complex pre-30S substrate in vitro, but there is disagreement in the literature as to what that substrate can be. That disagreement is resolved in this report; KsgA is unable to methylate 30S subunits in the translationally active conformation, but rather can modify 30S when in an experimentally well established translationally inactive conformation. Recent 30S crystal structures provide some basis for explaining why it is impossible for KsgA to methylate 30S in the translationally active conformation. Previous work identified one set of ribosomal proteins important for efficient methylation by KsgA and another set refractory methylation. With the exception of S21 the recent crystal structures of 30S also instructs that the proteins important for KsgA activity all exert their influence indirectly. Unfortunately, S21, which is inhibitory to KsgA activity, has not had its position determined by X-ray crystallography. A reevaluation of published biophysical data on the location also suggests that the refractory nature of S21 is also indirect. Therefore, it appears that KsgA solely senses the conformation 16S rRNA when carrying out its enzymatic activity.