Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

Sleep loss reduces the DNA-binding of BMAL1, CLOCK, and NPAS2 to specific clock genes in the mouse cerebral cortex.

We have previously demonstrated that clock genes contribute to the homeostatic aspect of sleep regulation. Indeed, mutations in some clock genes modify the markers of sleep homeostasis and an increase in homeostatic sleep drive alters clock gene expression in the forebrain. Here, we investigate a possible mechanism by which sleep deprivation (SD) could alter clock gene expression by quantifying DNA-binding of the core-clock transcription factors CLOCK, NPAS2, and BMAL1 to the cis-regulatory sequences of target clock genes in mice. Using chromatin immunoprecipitation (ChIP), we first showed that, as reported for the liver, DNA-binding of CLOCK and BMAL1 to target clock genes changes in function of time-of-day in the cerebral cortex. Tissue extracts were collected at ZT0 (light onset), -6, -12, and -18, and DNA enrichment of E-box or E'-box containing sequences was measured by qPCR. CLOCK and BMAL1 binding to Cry1, Dbp, Per1, and Per2 depended on time-of-day, with maximum values reached at around ZT6. We then observed that SD, performed between ZT0 and -6, significantly decreased DNA-binding of CLOCK and BMAL1 to Dbp, consistent with the observed decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was also decreased by SD, although SD is known to increase Per2 expression in the cortex. DNA-binding to Per1 and Cry1 was not affected by SD. Our results show that the sleep-wake history can affect the clock molecular machinery directly at the level of chromatin binding thereby altering the cortical expression of Dbp and Per2 and likely other targets. Although the precise dynamics of the relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive, the results also suggest that part of the reported circadian changes in DNA-binding of core clock components in tissues peripheral to the suprachiasmatic nuclei could, in fact, be sleep-wake driven.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the β(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated β(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the β(2) tail. This was shown by molecular modeling of the β(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.

Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents

Slime and proteinase activity of 54 strains consisting of 19 Candida parapsilosis and 35 C. albicans strains isolated from blood samples were investigated in this study. Ketoconazole, amphothericin B, and fluconazole susceptibility of Candida species were compared with slime production and proteinase activity of these species. For both Candida species, no correlation was detected between the slime activity and minimum inhibitory concentration (MIC) values of the three antifungal agents. For both Candida species no correlation was detected between the proteinase activity and the MIC values of amphothericin B, and fluconazole however, statistically significant difference, was determined between the proteinase activity and MIC values of ketoconazole (p = 0.007). Slime production was determined by using modified Christensen macrotube method and proteinase activity was measured by the method of Staib. Antifungal susceptibility was determined through the guidelines of National Committee for Laboratory Standards (NCCLS M27-A).

Intracellular polyamine pools, oligopeptide-binding protein A expression, and resistance to aminoglycosides in Escherichia coli

The role of intracellular free polyamine (putrescine and spermidine) pools in multiple resistance to aminoglycoside antibiotics was investigated among in vitro selected kanamycin-resistant Escherichia coli J53 mutants expressing diminished oligopeptide-binding protein (OppA) levels and/or defective ornithine decarboxylase (ODC) activity. The results suggest that diminished OppA content, but not defective ODC activity expression, increased the relative concentration of free spermidine as compared to the wild type strain. Moreover, by adding exogenous polyamines or polyamine synthesis inhibitors to cultures with different mutant strains, a direct relationship between the intracellular OppA levels and resistance to kanamycin was revealed. Collectively these results further suggest a complex relation among OppA expression, aminoglycoside resistance and polyamine metabolism.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity.

The Xenopus vitellogenin (vit) gene B1 estrogen-inducible enhancer is formed by two closely adjacent 13 bp imperfect palindromic estrogen-responsive elements (EREs), i.e. ERE-2 and ERE-1, having one and two base substitutions respectively, when compared to the perfect palindromic consensus ERE (GGTCANNNTGACC). Gene transfer experiments indicate that these degenerated elements, on their own, have a low or no regulatory capacity at all, but in vivo act together synergistically to confer high receptor- and hormone-dependent transcription activation to the heterologous HSV thymidine kinase promoter. Thus, the DNA region upstream of the vitB1 gene comprising these two imperfect EREs separated by 7 bp, was called the vitB1 estrogen-responsive unit (vitB1 ERU). Using in vitro protein-DNA interaction techniques, we demonstrate that estrogen receptor dimers bind cooperatively to the imperfect EREs of the vitB1 ERU. Binding of a first receptor dimer to the more conserved ERE-2 increases approximately 4- to 8-fold the binding affinity of the receptor to the adjacent less conserved ERE-1. Thus, we suggest that the observed synergistic estrogen-dependent transcription activation conferred by the pair of hormone-responsive DNA elements of the vit B1 ERU is the result of cooperative binding of two estrogen receptor dimers to these two adjacent imperfect EREs.

Mutations in the SPARC-Related Modular Calcium-Binding Protein 1 Gene, SMOC1, Cause Waardenburg Anophthalmia Syndrome.

Waardenburg anophthalmia syndrome, also known as microphthalmia with limb anomalies, ophthalmoacromelic syndrome, and anophthalmia-syndactyly, is a rare autosomal-recessive developmental disorder that has been mapped to 10p11.23. Here we show that this disease is heterogeneous by reporting on a consanguineous family, not linked to the 10p11.23 locus, whose two affected children have a homozygous mutation in SMOC1. Knockdown experiments of the zebrafish smoc1 revealed that smoc1 is important in eye development and that it is expressed in many organs, including brain and somites.

Binding of d-methadone, l-methadone, and dl-methadone to proteins in plasma of healthy volunteers: role of the variants of alpha 1-acid glycoprotein.

The plasma concentrations of alpha 1-acid glycoprotein (AAG), albumin, triglycerides, cholesterol, and total proteins, as well as the plasma binding of racemic, d-methadone, and l-methadone were measured in 45 healthy subjects. The AAG phenotypes and the concentrations of AAG variants were also determined. The measured free fractions for racemic, d-methadone, and l-methadone were, respectively, 12.7% +/- 3.3%, 10.0% +/- 2.9%, and 14.2% +/- 3.2% (mean +/- SD). A significant correlation was obtained between the binding ratio (B/F) for dl-methadone and the total AAG concentration (r = 0.724; p less than 0.001). A multiple stepwise regression analysis showed that AAG was the main explanatory variable for the binding of the racemate. When concentrations of AAG variants were considered, a significant correlation was obtained between the binding ratio of dl-methadone and orosomucoid2 A concentration (r = 0.715; p less than 0.001), a weak correlation between dl-methadone and orosomucoid1 S concentration (r = 0.494; p less than 0.001), and no correlation between dl-methadone and orosomucoid1 F1 concentration (r = 0.049; not significant). Similar findings were obtained with the enantiomers. This study shows the importance of considering not only total AAG but also concentrations of AAG variants when measuring the binding of methadone and possibly of other drugs in plasma.

Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner

We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s) from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.

Improving binding affinity and stability of peptide ligands by substituting glycines with D-amino acids.

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.

Multiple adenosine 3',5'-cyclic [corrected] monophosphate response element DNA-binding proteins generated by gene diversification and alternative exon splicing.

The protein sequence deduced from the open reading frame of a human placental cDNA encoding a cAMP-responsive enhancer (CRE)-binding protein (CREB-327) has structural features characteristic of several other transcriptional transactivator proteins including jun, fos, C/EBP, myc, and CRE-BP1. Results of Southwestern analysis of nuclear extracts from several different cell lines show that there are multiple CRE-binding proteins, which vary in size in cell lines derived from different tissues and animal species. To examine the molecular diversity of CREB-327 and related proteins at the nucleic acid level, we used labeled cDNAs from human placenta that encode two different CRE-binding proteins (CREB-327 and CRE-BP1) to probe Northern and Southern blots. Both probes hybridized to multiple fragments on Southern blots of genomic DNA from various species. Alternatively, when a human placental c-jun probe was hybridized to the same blot, a single fragment was detected in most cases, consistent with the intronless nature of the human c-jun gene. The CREB-327 probe hybridized to multiple mRNAs, derived from human placenta, ranging in size from 2-9 kilobases. In contrast, the CRE-BP1 probe identified a single 4-kilobase mRNA. Sequence analyses of several overlapping human genomic cosmid clones containing CREB-327 sequences in conjunction with polymerase chain reaction indicates that the CREB-327/341 cDNAs are composed of at least eight or nine exons, and analyses of human placental cDNAs provide direct evidence for at least one alternatively spliced exon. Analyses of mouse/hamster-human hybridoma DNAs by Southern blotting and polymerase chain reaction localizes the CREB-327/341 gene to human chromosome 2. The results indicate that there is a dichotomy of CREB-like proteins, those that are related by overall structure and DNA-binding specificity as well as those that are related by close similarities of primary sequences.