META060 inhibits multiple kinases in the NF-kappaB pathway and suppresses LPS--mediated inflammation in vitro and ex vivo.

OBJECTIVE: We investigated whether a novel candidate META060 targeted the inflammatory signal transduction without affecting constitutive COX-2 enzymatic activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We also investigated its bioavailability in humans and its anti-inflammatory effect ex vivo. METHODS: We measured prostaglandin E(2), nitric oxide, TNFalpha and IL-6 by ELISA, COX-2 protein by Western blot, NF-kappaB nuclear binding by electrophoretic mobility shift assays, and NF-kappaB activation by luciferase assay. Kinase inhibitions were measured by cell-free assays. Bioavailability was tested in 4 human subjects consuming 940 mg META060. LPS-activated TNFalpha and IL-6 were measured in peripheral blood mononuclear cells (PBMC) isolated from 1 subject up to 6 hours post administration. RESULTS: META060 dose-dependently inhibited prostaglandin E(2) and nitric oxide formation, COX-2 abundance, and NF-kappaB activation. In cell-free assays, META060 inhibited multiple kinases in the NF-kappaB signaling pathway, including BTK, PI3K, and GSK3. META060 was detected in the plasma of the subjects; isolated PBMC were resistant to LPS-stimulated TNFalpha and IL-6 production. CONCLUSION: Without inhibiting COX-2 enzyme, META060 reduces the inflammation by inhibiting multiple kinases involved in NF-kappaB pathway, and may have potential as a safe anti-inflammatory therapeutic.

Clinical implications for four drugs of the DSM-IV distinction between substance dependence with and without a physiological component.

OBJECTIVE: The DSM-IV work group asked researchers and clinicians to subtype substance dependent individuals according to the presence or absence of physiological symptoms. A recent report from the Collaborative Study on the Genetics of Alcoholism demonstrated that among alcohol-dependent men and women, a history of tolerance or withdrawal was associated with a more severe clinical course, especially for individuals with histories of alcohol withdrawal. This article evaluates similar distinctions among subjects in the collaborative study who were dependent on marijuana, cocaine, amphetamines, or opiates. METHOD: Structured interviews gathered information from 1,457 individuals with a lifetime diagnosis of marijuana dependence, 1,262 with histories of cocaine dependence, 647 with amphetamine dependence, and 368 subjects with opiate dependence. For each drug, the clinical course was compared for subjects whose dependence included a history of withdrawal (group 1), those dependent on each drug who denied withdrawal but reported tolerance (group 2), and those who denied both tolerance and withdrawal (group 3). RESULTS: The proportion of dependent individuals who denied tolerance or withdrawal (group 3) ranged from 30% for marijuana to 4% for opiates. For each substance, individuals in groups 1 and 2 evidenced more severe substance-related problems and at least a trend for greater intensities of exposure to the drug; those reporting withdrawal (group 1) showed the greatest intensity of problems. CONCLUSIONS: The designation of dependence in the context of tolerance or withdrawal identifies individuals with more severe clinical histories. These results support the importance of the designation of a physiological component to dependence, especially for people who have experienced a withdrawal syndrome.

Cloning and characterization of NSP1, a locus encoding a component of a CDC25-dependent, nutrient-responsive pathway in Saccharomyces cerevisiae.

The NSP1 gene in Saccharomyces cerevisiae has been identified by its ability, when expressed at high levels, to bypass the CDC25 requirement for growth. Sequence analysis of the cloned NSP1 locus suggests that the NSP1 product contains 269 amino acids and has a membrane-spanning domain at its carboxyl terminus. The NSP1 protein does not have sequence similarity to other known proteins, and is not related to the CDC25 protein, or to any of the previously described suppressors of CDC25 mutants. Phosphoprotein analysis of NSP1-suppressed cells indicates that the NSP1 product controls the phosphorylation of two 31 kD proteins whose phosphorylation and dephosphorylation are strongly correlated with cell-cycle arrest and proliferation, respectively, and suggests that the NSP1 product is an important downstream element of a CDC25-dependent, nutrient-responsive, phosphorylation pathway.

Identification of a 31 kDa protein in Saccharomyces cerevisiae whose phosphorylation is controlled negatively by the CDC25 gene product.

Phosphoprotein patterns in two mutants of Saccharomyces cerevisiae, cdc25-20(ts) and cdc25-20(ts) bcy1, were analysed by two-dimensional polyacrylamide gel electrophoresis. Comparison with the phosphoprotein patterns of the mutants cyr1-2(ts) and bcy1, analysed in a previous study, demonstrated not only that the CDC25 gene product is a positive element in the regulation of adenylyl cyclase activity, as suggested by recent studies, but that it is also a negative element in the phosphorylation of a 31 kDa protein (p31c and p31d), a protein whose phosphorylation is correlated with cell cycle arrest, and dephosphorylation with cell cycle initiation, respectively. Moreover, the phosphorylation phenotype of p31c and p31d suggests that the activity of the CDC25 protein is subject to feedback regulation by cAMP-dependent protein kinase, and that the CDC25 protein is a key element in an ammonium (NH+4) signal-response system.

Identification of phosphoproteins correlated with proliferation and cell cycle arrest in Saccharomyces cerevisiae: positive and negative regulation by cAMP-dependent protein kinase.

Recent genetic and biochemical studies of two mutants of the cAMP pathway in yeast, cyr1 and bcy1, have demonstrated that cAMP-dependent protein phosphorylation plays a major regulatory role in the control of proliferation and differentiation. As a first step in examining this regulatory system in more detail and in identifying the protein substrates of cAMP-dependent protein kinase, we have analyzed phosphoprotein patterns in the mutants cyr1-2(ts) and bcy1 by two-dimensional polyacrylamide gel electrophoresis. Our analysis has revealed several proteins whose phosphorylation is controlled positively or negatively by the cAMP pathway in yeast. The presence of some of these phosphoproteins was directly associated with proliferation (positive regulation), while that of others was correlated with cell cycle arrest (negative regulation). The phosphoprotein patterns of cyr1-2(ts) temperature-arrested cells, and nitrogen (NH+4)-starved cells, were strikingly similar, suggesting that response to NH+4 is mediated in part by adenylate cyclase. Phosphoproteins whose presence correlated with cell cycle arrest were found to be phosphorylated on serine and threonine residues, while the major phosphoproteins present predominantly in proliferating cells were phosphorylated only on serine residues. None of the greater than 20 phosphoproteins we examined contained phosphotyrosine under either growth condition.

Developmentally regulated phosphoproteins associated with chromosome complexes in yeast.

Previous studies on our laboratory have shown that nuclear DNA from Saccharomyces cerevisiae can be isolated in the form of fast-sedimenting chromosome complexes (FSCC). In cycling cells, three FSCC forms, denoted g1 and g2, can be distinguished by their characteristic sedimentation velocities and are found correspondingly in cells in G1, S and G2 of the cell cycle, respectively. A fourth form, denoted go, is found exclusively in stationary-phase and nitrogen-starved cells and defines the non-cycling state, Go. We used the differing sedimentation velocities of the FSCC of proliferating and non-cycling cells as a way to isolate and examine their associated proteins. We report here a two-dimensional polyacrylamide gel electrophoresis analysis of [35S]methionine and 32PO4-labelled proteins extracted from FSCC isolated from cycling cells (g1, r and g2, collectively denoted 'cycling FSCC'), and nutritionally arrested cells (go FSCC). Among the 120 35S-labelled FSCC-associated polypeptides detected, 25 were unique to go FSCC and 7 were unique to cycling FSCC. Among the 84 32P-labelled FSCC-associated polypeptides detected, 52 were unique to go FSCC and 7 were unique to cycling FSCC. Comigrating 35S and 32P-labelled polypeptides were matched in 34 of the 84 phosphorylated polypeptides, and 21 of these showed the same specificity of association to either cycling or go FSCC. This analysis demonstrates that there are major differences in the proteins associated with FSCC from cycling and nutritionally arrested cells, and indicates that a relationship exists between the growth state of the yeast cell, protein phosphorylation and chromosome-complex structure.

Control of the cAMP pathway by the cell cycle start function, CDC25, in Saccharomyces cerevisiae.

We investigated the relationship in Saccharomyces cerevisiae between the cell cycle start function, CDC25, and two mutants defining components of the cAMP pathway. The thermolabile adenylate cyclase mutant cyr1-2(ts) is phenotypically similar to the temperature-sensitive mutant cdc25(ts) in that both mutants, when shifted to the restrictive temperature, arrest in G1 of the cell cycle and permit the initiation of meiosis and sporulation. The mutant bcy1 [a lesion resulting in a low level of regulatory (R) subunit and a high level of active, catalytic (C) subunit of the cAMP-dependent protein kinase] suppresses the temperature-sensitive phenotype of cyr1-2(ts) and confers an asporogenous phenotype. We found that cdc25(ts) complemented cyr1-2(ts), and, unlike cyr1-2(ts), was not suppressible by bcy1, demonstrating that CYR1 and CDC25 must encode different functions. Also our results indicate that CDC25 does not encode the R subunit of the cAMP-dependent protein kinase. In addition, although the cdc25(ts)bcy1 double mutant was temperature sensitive like cdc25(ts), we found that the cdc25(ts)bcy1 homozygous diploid was asporogenous like bcy1/bcy1. The inability of the cdc25(ts)bcy1 double mutant to sporulate demonstrated that CDC25 does not encode the C subunit of the cAMP kinase, and indicated that the CDC25 function modulates the cAMP pathway to control meiosis and sporulation. Further, the temperature-sensitive phenotype of the double mutant, and hence the inability of bcy1 to suppress cdc25(ts), suggested that a second CDC25 cell cycle function exists which is independent of the cAMP pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative pharmacokinetics of antibiotics in newborn calves: chloramphenicol, lincomycin, and tylosin.

The pharmacokinetics of 3 antibiotics--chloramphenicol, lincomycin, and tylosin--were determined in newborn calves. The kinetic determinations, using 2-compartment open models, were made at increasing ages from 1 day to 42 days and compared with those made from 9-month-old calves. Although all 3 antibiotics require a degree of hepatic metabolism, there were marked differences in the development of elimination processes for the individual drugs. Elimination of tylosin, which was slow in the calves at birth, increased rapidly and was equal to that in older calves by the end of 1 week. Lincomycin elimination was not markedly reduced in calves at birth. Chloramphenicol elimination was slow in calves at birth and only by 4 to 6 weeks attained the rate found in older calves. Dosage adjustments would not be required for the antibiotics tylosin or lincomycin when given to newborn calves, but may be necessary for chloramphenicol given to calves before they are 4 to 6 weeks old.

Glucose-initiated germination of mucor racemosus sporangiospores.

Treatments leading to the initiation of germination of Mucor racemosus sporangiospores were examined. The results support the hypothesis that glucose is a specific trigger molecule for the initiation of Mucor racemosus sporangiospores. Glucose and some of the glucose analogues tested could initiate germination, mannose, 3-O-methyl-D-glucose, 5-thio-D-glucose and 6-deoxy-D-glucose being the most effective. The initiation event appeared to depend on the concentration of the initiator, with glucose and 3-O-methyl-D-glucose exhibiting nearly identical kinetic constants. Spores accumulated not only glucose and 3-O-methyl-D-glucose, but also the 1-O-methyl-D-glucose analogue, which did not initiate germination. The accumulated 3-O-methyl-D-glucose was not metabolized. The initiation sequence appeared to require the continued presence of the initiator as well as protein synthesis.

Role of amino acids and endogenous protein in the germination of Mucor racemosus sporangiospores.

The role of amino acids as triggers of Mucor racemosus sporangiospore germination was investigated. No single amino acid was effective as glucose or peptone at triggering germination. Germination induced by glucose or peptone was pH-independent, whereas germination induced by glutamate was pH-dependent. The composition of the free amino acid pools of M-spores (those unable to germinate on glutamate) was qualitatively and quantitatively similar to that of C-spores (those capable of germinating on glutamate) with the exceptions of hydroxyproline and methionine and methionine whose concentrations were several-fold higher in C-spores. Glutamate and leucine were taken up by germinating and nongerminating spores; however, significant protein synthesis occurred only in germinating spores. Spores not triggered by 3-O-methyl-D-glucose (M-spores) contained about one-half the protein of those triggered by 3-O-methyl-D-glucose (C-spores). C-spores initiated to germinate by 3-O-methyl-D-glucose decreased in total organic carbon and protein over a 6 h period; removal of the 3-O-methyl-D-glucose resulted in an immediate halt of protein degradation and spore swelling. These results suggest that protein is a major endogenous reserve in M. racemosus sporangiospores and that its turnover is a necessary event for glucose-triggered germination.

Relationship between sporulation medium and germination ability of Mucor racemosus sporangiospores.

Asexual sporangiospores of Mucor racemosus produced on a minimal sporulation medium (M spores) germinated only if glucose, mannose or a complex substrate such as peptone, yeast extract or Casamino acids was present. Once germinated, growth was supported by a wide range of substrates including amino acids, carbohydrates or organic acids. Sporangiospores produced on a nutritionally complex sporulation medium (C-spores) germinated on a wide range of carbon sources. C-spore phenotype was pleiotropic in that sporangiospores capable of germinating on cellobiose could always germinate on glutamate or xylose; but C-spores capable of germinating on xylose or glutamate did not always germinate on cellobiose. There was a hierarchy of substrates capable of initiating germination with glucose = mannose greater than xylose greater than glutamate greater than cellobiose. C-spores also differed from M-spores by initiating germination in the presence of the non-metabolizable glucose analogue 3-O-methylglucose. These results suggest that at least two sporangiospore phenotypes are produced depending upon the concentration and type of ingredients present in the sporulation medium.