Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B(2) and M1 muscarinic acetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (< EKWAP), a member of this structurally related peptide family, was essential for the development of captopril, the first site-directed ACE inhibitor used for the treatment of human hypertension. Nowadays, more Bj-PROs have been identified with higher ACE inhibition potency compared to Bj-PRO-5a. However, despite its modest inhibitory effect of ACE inhibition, Bj-PRO-5a reveals strong bradykinin-potentiating activity, suggesting the participation of other mechanisms for this peptide. In the present study, we have shown that Bj-PRO-5a induced nitric oxide (NO) production depended on muscarinic acetylcholine receptor M1 subtype (mAchR-M1) and bradykinin B(2) receptor activation, as measured by a chemiluminescence assay using a NO analyzer. Intravital microscopy based on transillumination of mice cremaster muscle also showed that both bradykinin B(2) receptor and mAchR-M1 contributed to the vasodilatation induced by Bj-PRO-5a. Moreover, Bj-PRO-5a-mediated vasodilatation was completely blocked in the presence of a NO synthase inhibitor. The importance of this work lies in the definition of novel targets for Bj-PRO-5a in addition to ACE, the structural model for captopril development. (C) 2011 Elsevier Inc. All rights reserved.

The Snake Venom Peptide Bj-PRO-7a Is a M1 Muscarinic Acetylcholine Receptor Agonist

Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensin-converting enzyme-independent mechanisms. Here, we show that Bj-PRO-7a (Bj-PRO-7a reached the maximum response in the presence of 3 mu M Bj-PRO-7a (EC(50) = 0.25 +/- 0.07 mu M). The variation observed by calcium imaging in these cells ranged from 52 to 1218 nM (EC(50) = 0.31 +/- 0.12 mu M). [Ca(2+)](i) responses in CHO-M1 cells were largely inhibited by pirenzepine, a specific M1 antagonist. Neural-differentiated P19 cells expressing endogenous M1 receptors were also responsive to Bj-PRO-7a application, whereas no such response was observed in undifferentiated P19 cells not expressing muscarinic receptors. As further support for its specific action on M1 receptors, the peptide did not activate M3 subtypes in transfected CHO cells. Our findings provide a novel M1 muscarinic receptor agonist that could be used for basic research and even for pharmacological applications. (C) 2010 International Society for Advancement of Cytometry

Subcellular localization of proteasomes and their regulatory complexes in mammalian cells

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable c-interferoninducible catalytic b-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit speci®c antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core a-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some signi®cant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immuno¯uorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following cinterferon treatment of cultured cells but c-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

Proteasome activities decrease during dexamethasone-induced apoptosis of thymocytes

The induction of apoptosis in thymocytes by the glucocorticoid dexamethasone was used as a model system to investigate whether there are changes in 20 S and 26 S proteasome activities during apoptosis. We observed that thymocytes contain high concentrations of proteasomes and that following treatment with dexamethasone, cell extracts showed a decrease in proteasome chymotrypsin-like activity which correlated with the degree of apoptosis observed. The decrease in chymotrypsin-like activity of 20 S and 26S proteasomes was still apparent after these complexes had been partially puri®ed from apoptotic thymocyte extracts and was therefore not due to competition resulting from a general increase in protein turnover. The trypsin-like and peptidylglutamylpeptide hydrolase activities of proteasome complexes were also observed to decrease during apoptosis, but these decreases were reversed by the inhibition of apoptosis by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-¯uoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased further in the presence of the apoptosis inhibitor. Val-Ala-Asp-¯uoromethylketone was found to inhibit the chymotrypsin- and trypsin-like activity of 26 S proteasomes in .itro. The decrease in proteasome activities in apoptosis did not appear to be due to a decrease in the concentration of total cellular proteasomes. Thus, the early decreases in 20 S and 26 S proteasome activities during apoptosis appear to be due to a down-regulation of their proteolytic activities and not to a decrease in their protein concentration. These data suggest that proteasomes may be responsible, in thymocytes, for the turnover of a protein that functions as a positive regulator of apoptosis.

Universal crack closure integral for SIF estimation

Numerical analysis of cracked structures often involves numerical estimation of stress intensity factors (SIFs) at a crack tip/front. A newly developed formulation called universal crack closure integral (UCCI) for the evaluation of potential energy release rates (PERRs) and the corresponding SIFs is presented in this paper. Unlike the existing element dedicated forms of crack closure integrals (MCCI, VCCI) with application limited to finite element analysis, this new numerical SIF/PERR estimation technique is independent of the basic stress analysis procedure, making it universally applicable. The second merit of this procedure is that it avoids the generally error-producing zones close to the crack tip/front singularity. The UCCI procedure, based on Irwin's original CCI, is formulated and explored using a simple 2D problem of a straight crack in an infinite sheet. It is then applied to some three-dimensional crack geometries with the stresses and displacements obtained from a boundary element program.

Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn(341), Tyr(60) and Phe(351) in tetrahydrofolate binding

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10-methylene THE SHMT also catalyses several THF-independent side reactions such as cleavage of P-hydroxy amino acids, trans-amination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe(351) to glycine did not have any effect oil either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a Mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three Mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.

Triploid plants from endosperm cultures of sandalwood by experimental embryogenesis

Embryogenesis has been induced from endosperm callus cultures of sandalwood (Santalum album L.). Viable plantlets developed from the embryoids on subculture to White's basal medium supplemented with 0.5 mg/l of indole acetic acid. Chromosomal analysis of the root tips showed the triploid number 3n = 30.

Cytochrome c oxidase is preferentially synthesized in the rough endoplasmic reticulum--mitochondrion complex in rat liver

The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum--mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum--mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.

CLXXX. Vegetable Proteins. I. The Proteins of Dolichos Lab Lab

The field bean (Dolichos lab lab ; Tamil name, Mochai ; Kanarese, Avarai) is a legume which is widely cultivated in South India often as a mixed crop with cereals. The kernel of the seed enters into the diet of may South Indian households, and in the Mysore State the seed are used as a delicacy when they are green for over four months in the year. The haulm, husk and pods are commonly used a fodder. As the kernel which is widely used as an article of food and considered to be very nutritious, contains about 24% of protein hitherto uninvestigated and as the quality of protein plays an important role in nutrition, the present work was undertaken.

Incompressible boundary layer for longitudinal flow over a cylinder with an applied magnetic field

The flow, heat and mass transfer problem for a steady laminar incompressible boundary layer flow in an electrically conducting fluid over a longitudinal cylinder with an applied magnetic field has been studied. The partial differential equations governing the flow have been solved numerically using an implicit finite-difference scheme. The results are found to be strongly dependent on the magnetic field and dissipation parameter. The effect of the mass transfer is more pronounced on the skin friction than on the heat transfer. The results have been compared with those of the series solution, the asymptotic solution, the Glauert and Lighthill's solution, local similarity, local nonsimilarity and difference-differential methods. Good agreement is found with all of them, except with the results of the local similarity and series solution methods.

Localization of testis-variant histones in rat testis chromatin

Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.

Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.

The formation of carbon nanostructures by in situ TEM mechanical nanoscale fatigue and fracture of carbon thin films

A technique to quantify in real time the microstructural changes occurring during mechanical nanoscale fatigue of ultrathin surface coatings has been developed. Cyclic nanoscale loading, with amplitudes less than 100 nm, is achieved with a mechanical probe miniaturized to fit inside a transmission electron microscope (TEM). The TEM tribological probe can be used for nanofriction and nanofatigue testing, with 3D control of the loading direction and simultaneous TEM imaging of the nano-objects. It is demonstrated that fracture of 10-20 nm thick amorphous carbon films on sharp gold asperities, by a single nanoscale shear impact, results in the formation of < 10 nm diameter amorphous carbon filaments. Failure of the same carbon films after cyclic nanofatigue, however, results in the formation of carbon nanostructures with a significant degree of graphitic ordering, including a carbon onion.