The discovery of acylated beta-amino acids as potent and orally bioavailable VLA-4 antagonists

Acylated beta-amino acids are described as potent, specific and orally bioavailable antagonists of VLA-4. The initial lead was identified from a combinatorial library. Subsequent optimization using a traditional medicinal chemistry approach led to significant improvement in potency (up to 8-fold) while maintaining good pharmacokinetic properties.     

Distinctive KIR and HLA diversity in a panel of north Indian Hindus

HLA and KIR are diverse and rapidly evolving gene complexes that work together in human immunity mediated by cytolytic lymphocytes. Understanding their complex immunogenetic interaction requires study of both HLA and KIR diversity in the same human population. Here a panel of 72 unrelated north Indian Hindus was analyzed. HLA- A, B, C, DRB1, DQA1, and DQB1 alleles and their frequencies were determined by sequencing or high-resolution typing of genomic DNA; KIR genotypes were determined by gene-specific typing and by allele-level DNA typing for KIR2DL1, 2DL3, 2DL5, 3DL1, and 3DL2. From HLA analysis, the north Indian population is seen to have several characteristics shared either with Caucasian or East Asian populations, consistent with the demographic history of north India, as well as specific features, including several alleles at high frequency that are rare or absent in other populations. A majority of the north Indian KIR gene profiles have not been seen in Caucasian and Asian populations. Most striking is a higher frequency of the B group of KIR haplotypes, resulting in equal frequencies for A and B group haplotypes in north Indians. All 72 members of the north Indian panel have different HLA genotype and different KIR genotype.     

Mechanism of generation of spontaneous miniature outward currents (SMOCs) in retinal amacrine cells

A subtype of retinal amacrine cells displayed a distinctive array of K(+) currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of -60 to -40 mV. Depolarizations above approximately -40 mV were associated with the disappearance of SMOCs and the appearance of transient (I(to)) and sustained (I(so)) outward K(+) currents. I(to) appeared at about -40 mV and its apparent magnitude was biphasic with voltage, whereas I(so) appeared near -30 mV and increased linearly. SMOCs, I(to), and a component of I(so) were Ca(2+) dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of -20 mV and above. Their mean amplitude was Nernstian with respect to [K(+)](ext) and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca(2+)-free solutions, indicative of BK-type Ca(2+)-activated K(+) currents. Dihydropyridine Ca(2+) channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca(2+) permeation through the kainic acid receptor had no effect. Blockade of organelle Ca(2+) channels by ryanodine, or intracellular Ca(2+) store depletion with caffeine, eradicated SMOCs. Internal Ca(2+) chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca(2+) influx through L-type Ca(2+) channels and its subsequent amplification by Ca(2+)-induced Ca(2+) release via the ryanodine receptor leads to a localized elevation of internal Ca(2+). This amplified Ca(2+) signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.     

Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors

The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model. Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.     

Mechanistic implications for Escherichia coli cofactor-dependent phosphoglycerate mutase based on the high-resolution crystal structure of a vanadate complex

The structure of Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), complexed with the potent inhibitor vanadate, has been determined to a resolution of 1.30 A (R-factor 0.159; R-free 0.213). The inhibitor is present in the active site, principally as divanadate, but with evidence of additional vanadate moieties at either end, and representing a different binding mode to that observed in the structural homologue prostatic acid phosphatase. The analysis reveals the enzyme-ligand interactions involved in inhibition of the mutase activity by vanadate and identifies a water molecule, observed in the native E.coli dPGM structure which, once activated by vanadate, may dephosphorylate the active protein. Rather than reflecting the active conformation previously observed for E.coli dPGM, the inhibited protein's conformation resembles that of the inactive dephosphorylated Saccharomyces cerevisiae dPGM. The provision of a high-resolution structure of both active and inactive forms of dPGM from a single organism, in conjunction with computational modelling of substrate molecules in the active site provides insight into the binding of substrates and the specific interactions necessary for three different activities, mutase, synthase and phosphatase, within a single active site. The sequence similarity of E.coli and human dPGMs allows us to correlate structure with clinical pathology.     

AvGI,an index of genes transcribed in the salivary glands of the ixodid tick Amblyomma variegatum

Random clones from a cDNA library made from mRNA purified from dissected salivary glands of feeding female Amblyomma variegatum ticks were subjected to single pass sequence analysis. A total of 3992 sequences with an average read length of 580 nucleotides have been used to construct a gene index called AvGI that consists of 2109 non-redundant sequences. A provisional gene identity has been assigned to 39% of the database entries by sequence similarity searches against a non-redundant amino acid database and a protein database that has been assigned gene ontology terms. Homologs of genes encoding basic cellular functions including previously characterised enzyme activities, such as stearoyl CoA saturase and protein phosphatase, of ixodid tick salivary glands were found. Several families of abundant cDNA sequences that may code for protein components of tick cement and A. variegatum proteins which may contribute to anti-haemostatic and anti-inflammatory responses, and, one with potential immunosuppressive activity, were also identified. Interference with the function of such proteins might disrupt the life cycle of A. variegatum and help to control this ectoparasite or to reduce its ability to transmit disease causing organisms. AvGI represents an electronic knowledge base, which can be used to launch investigations of the biology of the salivary glands of this tick species. The database may be accessed via the World Wide Web at http://www.tigr.org/tdb/tgi.shtml.     

Evidence for a global inhibitor-induced conformation change on the Ca(2+)-ATPase of sarcoplasmic reticulum from paired inhibitor studies

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by a variety of hydrophobic, hydroxy-containing molecules. A kinetic method has been used to study competition between binding of pairs of inhibitors to the ATPase. The presence of 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ) decreases the affinity of the ATPase for 2,5-dipropyl-1,4-dihydroxybenzene (PHQ), suggesting that PHQ and BHQ bind to the same site on the ATPase. In contrast, the presence of BHQ increases the affinity of the ATPase for curcumin and vice versa. This suggests that BHQ and curcumin bind to separate sites on the ATPase and that binding of the first inhibitor to the ATPase results in a change to a conformation with higher affinity for the second inhibitor. This is consistent with previous experiments with BHQ and thapsigargin suggesting a conformation change on inhibitor binding, E2 + I <--> 2; E2I <--> 2; E2(A)I, with E2(A)I having a higher affinity for the second inhibitor than E2. The affinity for BHQ is also increased by binding of diethylstilbesterol, ellagic acid, or nonylphenol, and the affinity for curcumin is also increased by ellagic acid. These results showing that binding of a variety of inhibitors of very different structures all result in a general increase in inhibitor affinity point to a global conformational change on the Ca(2+)-ATPase caused by inhibitor binding, as well as any local, inhibitor-specific changes in conformation.     

Anisakis simplex: dangerous--dead and alive?

The risk of infection with Anisakis simplex and related parasites of fish has been recognized for some time, but it is now emerging that ingestion of material from dead parasites in food is also potentially dangerous. The resulting allergic reactions range from rapid onset and potentially lethal anaphylactic reactions to chronic, debilitating conditions. This review discusses the problems and clinical implications associated with A. simplex, other related conditions, and the way in which disease manifestations vary from person to person.     

CDC42 is required for polarized growth in human pathogen Candida albicans

Cdc42p is a member of the RAS superfamily of GTPases and plays an essential role in polarized growth in many eukaryotic cells. We cloned the Candida albicans CaCDC42 by functional complementation in Saccharomyces cerevisiae and analyzed its function in C. albicans. A double deletion of CaCDC42 was made in a C. albicans strain containing CaCDC42 under the control of the PCK1 promoter. When expression of the heterologous copy of CaCDC42 was repressed in this strain, the cells ceased proliferation. These arrested cells were large, round, and unbudded and contained predominantly two nuclei. The PCK1-mediated overexpression of wild-type CaCdc42p had no effect on cells. However, in cells overexpressing CaCdc42p containing the dominant-negative D118A substitution, proliferation was blocked and the arrested cells were large, round, unbudded, and multinucleated, similar to the phenotype of the cdc42 double-deletion strain. Cells overexpressing CaCdc42p containing the hyperactive G12V substitution also ceased proliferation in yeast growth medium; in this case the arrested cells were multinucleated and multibudded. An intact CAAX box is essential for the phenotypes associated with either CaCdc42p^G12V or CaCdc42p^D118A ectopic expression, suggesting that membrane attachment is involved in CaCdc42p function. In addition, the lethality caused by ectopic expression of CaCdc42p^G12V was suppressed by deletion of CST20 but not by deletion of CaCLA4. CaCdc42p function was also examined under hypha-inducing conditions. Cdc42p depletion prior to hyphal induction trapped cells in a round, unbudded state, while depletion triggered at the same time as hyphal induction permitted the initiation of germ tubes that failed to be extended. Ectopic expression of either the G12V or D118A substitution protein modified hyphal formation in a CAAX box-dependent manner. Thus, CaCdc42p function appears important for polarized growth of both the yeast and hyphal forms of C. albicans.