Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase,in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.     

Cyclin-dependent kinase 5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3

Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5(-/-) and p35(-/-) mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival are unknown. Here, we show that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis.     

A Caenorhabditis elegans TGF-beta, DBL-1, controls the expression of LON-1, a PR-related protein, that regulates polyploidization and body length

Using cDNA-based array analysis combined with double-stranded RNA interference (dsRNAi), we have identified yk298h6 as a target gene of Caenorhabditis elegans TGF-beta signaling. Worms overexpressing dbl-1, a TGF-beta ligand, are 16% longer than wild type. Array analysis shows yk298h6 to be one of several genes suppressed in such worms. Disruption of yk298h6 function by dsRNAi also resulted in long worms, suggesting that it is a negative regulator of body length. yk298h6 was then mapped to, and shown to be identical to, lon-1, a known gene that affects body length. lon-1 encodes a 312 amino acid protein with a motif sequence that is conserved from plants to humans. Expression studies confirm that LON-1 is repressed by DBL-1, suggesting that LON-1 is a novel downstream component of the C.elegans TGF-beta growth regulation pathway. Consistent with this, LON-1 is expressed mainly in the larval and adult hypodermis and has dose-dependent effects on body length associated with changes in hypodermal ploidy, but not hypodermal cell proliferation.     

Allele frequencies of single nucleotide polymorphisms in the second exon of the myoglobin gene among the Japanese

The difference in the allele frequencies of two single nucleotide polymorphisms (SNPs) in the second exon of the myoglobin gene between Japanese and other populations is reported. These SNPs are the substitutions of (A79G) and (T109C), and they were investigated by a single polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis followed by direct sequencing. The substitutions were always linked and two alleles were found in the samples used: the A-T allele with no substitution at positions (79A) and (109T) and the G-C allele with substitutions of (79G) and (109C). The frequencies of these alleles were 0.755 and 0.245, respectively, and they were found to be in Hardy-Weinberg equilibrium. The distribution of alleles in the Japanese population was significantly different from that reported among whites, blacks, and Hispanics (p < 0.0001).     

Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase,a multifunctional polyketide biosynthetic enzyme,by free coenzyme A

6-Hydroxymellein (6HM) synthase is a multifunctional polyketide enzyme induced in carrot cells, whose fully active homodimer catalyzes condensation of acyl-CoAs and the NADPH-dependent ketoreduction of the enzyme-bound intermediate. 6HM-forming activity of the synthase was markedly decreased when the reaction mixture pH was adjusted from 7.5 to 6.0. However, under these slightly acidic conditions, the acyl condensation catalyzed by the dissociated monomer enzyme was appreciably stimulated by addition of free coenzyme A (CoA). In contrast, the condensation reaction at pH 6.0 was significantly inhibited in the presence of CoA when the reaction was carried out with the NADPH-omitted dimer synthase. Among the kinetic parameters of the acyl condensation, velocity of the monomer-catalyzing reaction at the acidic pH was appreciably increased upon addition of CoA while K(m)s did not show any significant change in the presence and absence of the compound. These results suggest that CoA associates with a specific site in the dissociated monomeric form of 6HM synthase, and the velocity of the acyl condensation reaction catalyzed by the CoA-synthase complex appreciably increases in acidic conditions.     

Terpenoids from Tripterygium doianum (Celastraceae)

The extract of Tripterygium doianum (Celastraceae) afforded three triterpenoids [3beta-acetoxy-11-ursen-13alpha,30-olide, 25-chloro-24-hydroxytirucall-7-en-3-one and tirucall-7-en-3,24-dione], two sesquiterpenoids [5alpha-acetoxy-1beta,8alpha-bis-cinnamoyl-4alpha-hydroxydihydroagarofuran and 5alpha-acetoxy-1beta-benzoyl-8alpha-cinnamoyl-4alpha-hydroxydihydroagarofuran] and nine known triterpenoids. Their structures were established based on spectroscopic studies.     

Effects of Cl2MDP-encapsulating liposomes in a murine model of Pseudomonas aeruginosa-induced sepsis

Pseudomonas aeruginosa is a pathogen that frequently causes acute lung injury, bacteremia and sepsis in critically ill patients. As tissue macrophages are a major producer of inflammatory mediators that contribute to septic physiology, and are essential for eliminating bacteria from the circulation, we investigated the role of tissue macrophages in the generation of both inflammatory and anti-inflammatory cytokines in septic shock by using our mouse model of P. aeruginosa pneumonia. To see the effects of tissue macrophage depletion, we intravenously injected dichloromethylene-diphosphonate (Cl2MDP)-encapsulating liposomes in mice. Two days after the liposome injection, we instilled cytotoxic P. aeruginosa (PA103) into the lung that disseminates and causes septic shock. After the infection, we collected blood and bronchoalveolar lavage fluids. The samples were then analyzed for TNF-alpha, MIP-2, and IL-10 concentration. We compared these results to control mice that received either liposomes without Cl2MDP or phosphate buffered saline alone. Plasma TNF-alpha, MIP-2, and IL-10 levels were significantly decreased in the tissue macrophage-depleted mice compared to the control groups of mice. Although depletion of tissue macrophages by Cl2MDP-liposome administration did not affect the severity of bacteremia or the survival of infected mice, these results imply that tissue macrophages have a major role in the production of both proinflammatory and anti-inflammatory cytokines in the circulation and in the causing septic physiology associated with P. aeruginosa pneumonia.     

Feedback regulation of pancreatic secretion by peptide YY

The present status of our understanding of the feedback regulation of pancreatic secretion by peptide YY (PYY) released from the distal intestine is reviewed. Exocrine pancreatic secretion is primarily controlled by the cephalic (the vagus nerve), gastric (acid and pepsin secretion, and nutrients delivered into the duodenum by gastric emptying), and intestinal (secretin and CCK) mechanisms. PYY acts on the multiple sites in the brain and gut, and inhibits pancreatic secretion by regulating these primary control mechanisms. The involvement of Y(1) and Y(2) receptors has been suggested in the regulation of pancreatic secretion. However, it remains to be studied which site of action or receptor subtype is physiologically most important for this regulation.