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.     

Structure determination of the constitutive 20S proteasome from bovine liver at 2.75 A resolution

The crystal structure of the 20S proteasome from bovine liver was determined by the molecular replacement method using the structure of the 20S proteasome from the yeast Sacccharomyces cerevisiae. The initial phases were refined by density modification coupled with non-crystallographic symmetry averaging. The structural model was refined with the program CNS. The final R-factor and R(free) were 0.25 and 0.29, respectively. The constitutive proteasome without any contamination by the immunoproteasome was identified in the crystal structure.     

Preparation of poly(L-lactide)-based microspheres having a cationic or anionic surface using biodegradable surfactants

Poly(L-lactide)-based microspheres having cationic or anionic surfaces were prepared using polydepsipeptide-block-poly(L-lactide)s as surfactants. Polydepsipeptide-block-poly(L-lactide)s having amino or carboxylic acid groups on their side chains were synthesized through anionic ring-opening polymerizations of L-lactide using the corresponding protected polydepsipeptides as macroinitiators and consequent deprotections. Since these amphiphilic copolymers consisting of hydrophobic segments and hydrophilic segments with amino or carboxylic acid groups could be converted to cationic or anionic block copolymers, they could act as surfactants preparing poly(L-lactide)-based microspheres by an oil-in-water emulsion method. The amount of ionic groups located on the surfaces of the obtained microspheres was found to increase with increasing the feed of charged polydepsipeptide-block-poly(L-lactide)s in the blend of poly(L-lactide) and block copolymers. The average diameters of the dried microspheres estimated by scanning electron microscopy were found to decrease with an increase in feed of block copolymers in polymer blends.     

Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning,biochemical properties and developmentally regulated expression

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.     

Model of the alphaLbeta2 integrin I-domain/ICAM-1 DI interface suggests that subtle changes in loop orientation determine ligand specificity

The interaction of the alphaLbeta2 integrin with its cellular ligand the intercellular adhesion molecule-1 (ICAM-1) is critical for the tight binding interaction between most leukocytes and the vascular endothelium before transendothelial migration to the sites of inflammation. In this article we have modeled the alphaL subunit I-domain in its active form, which was computationally docked with the D1 domain of the ICAM-1 to probe potential protein-protein interactions. The experimentally observed key interaction between the carboxylate of Glu 34 in the ICAM-1 D1 domain and the metal ion-dependent adhesion site (MIDAS) in the open alphaL I-domain was consistently reproduced by our calculations. The calculations reveal the nature of the alphaLbeta2/ICAM-1 interaction and suggest an explanation for the increased ligand-binding affinity in the "open" versus the "closed" conformation of the alphaL I-domain. A mechanism for substrate selectivity among alphaL, alphaM, and alpha2 I-domains is suggested whereby the orientation of the loops within the I-domain is critical in mediating the interaction of the Glu 34 carboxylate of ICAM-1 D1 with the MIDAS.     

Activation of glucuronidation through reduction of a disulfide bond in rat UDP-glucuronosyltransferase 1A6

UDP-glucuronosyltransferase- (UGT-) dependent glucuronidation is an important detoxification process for many endogenous and exogenous compounds in mammals. Treatment of rat hepatic microsomes with the reducing reagent dithiothreitol (DTT) resulted in a significant increase in p-nitrophenol (p-NP) glucuronidation in a time- and concentration-dependent manner. The DTT-dependent activation of glucuronidation was specific for planar phenols but not for bilirubin or testosterone without membrane perturbation of the microsomes. p-NP glucuronidation in Gunn rat hepatic microsomes lacking UGT1 isozymes was not affected by DTT, indicating that UGT1A6 in the microsomes is mainly involved in the activation. The DTT-dependent activation was inhibited by 1,6-bis(maleimido)hexane (BMH) but not by N-ethylmaleimide, indicating that cross-linking between cysteine residues in UGT1A6 is responsible for the activation. Immunoblot analysis of rat hepatic microsomes on nonreducing SDS-PAGE gels revealed that most of the UGT1A6 migrated as a monomer, suggesting that DTT could affect an intramolecular disulfide bond in the UGT1A6 that may be responsible for the activation. To identify which of the ten cysteines in UGT1A6 are involved in the disulfide bond, rat UGT1A6 wild type and a set of mutants, each with a cysteine to serine substitution, were constructed and expressed in COS cells. Treatment of COS microsomes with DTT had no effect on the activity of the wild type but BMH showed significant inhibition, suggesting that UGT1A6 expressed in COS cells may be in the reduced and activated state. Replacement of either Cys 121 or Cys 125 with serine showed insensitivity to the BMH-dependent inhibition. These results demonstrate that both Cys 121 and Cys 125 are responsible for the activation of the activity through the disulfide bond in rat UGT1A6.     

The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of the nascent peptide during translation termination

The ribosomal peptidyl transferase center is responsible for two fundamental reactions, peptide bond formation and nascent peptide release, during the elongation and termination phases of protein synthesis, respectively. We used in vitro genetics to investigate the functional importance of conserved 23S rRNA nucleotides located in the peptidyl transferase active site for transpeptidation and peptidyl-tRNA hydrolysis. While mutations at A2451, U2585, and C2063 (E. coli numbering) did not significantly affect either of the reactions, substitution of A2602 with C or its deletion abolished the ribosome ability to promote peptide release but had little effect on transpeptidation. This indicates that the mechanism of peptide release is distinct from that of peptide bond formation, with A2602 playing a critical role in peptide release during translation termination.     

Importance of renal mitochondria in the reduction of TEMPOL,a nitroxide radical

Spin probing methods using an electron spin resonance (ESR) spectrometer are used extensively and bring us a lot of information about in vivo redox mechanisms. However, the in vivo reducing mechanisms of exogenous nitroxide radicals, which serve as typical spin probing reagents are not clear. To clarify this, we examined the sequential kinetics of a spin probe, 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) in the in vivo organs, tissue homogenates and subcellular fractions of kidney and liver using an in vivo and X-band ESR spectrometers. As a parameter of reducing activity, we calculated the half-life of TEMPOL from the decay curve of ESR signal intensity. The half-life of TEMPOL in the whole organs and homogenates of the kidney was significantly shorter than that of the liver, this indicates that the kidney has more reducing activity against TEMPOL as compared to the liver. Subcellular fractional studies revealed that this reducing activity of the kidney mainly exists in the mitochondria. Contrarily, in addition to reduction in the mitochondria, TEMPOL in the liver was reduced by the microsome and cytosol.     

Apoptosis in the aging process

Although many hypotheses have been proposed to explain the aging process, the exact mechanisms are not well defined. Recent accumulating evidence indicates that dysregulation of the apoptotic process may be involved in some aging processes; however, it is still debatable how exactly apoptosis is expressed during aging in vivo. In this review, we discuss recent findings related to apoptosis of individual organs during aging and their significance. We demonstrate that aging enhances apoptosis and susceptibility to apoptosis in several types of intact cells. In contrast, in certain genetically damaged, initiated, and preneoplastic cells, aging suppresses these age-associated apoptotic changes. In various cells, apoptosis enhances the elimination of damaged and dysfunctional cells presumably caused by oxidative stress, glycation, and DNA damage. In these cases, the incidence of apoptosis correlates with the level of accumulated injury. It is concluded that apoptosis plays an important role in the aging process and tumorigenesis in vivo probably as an inherent protective mechanism against age-associated tumorigenesis.     

Structure and function of the fourth subunit (Dpb4p) of DNA polymerase epsilon in Saccharomyces cerevisiae

DNA polymerase (Pol) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS–PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Pol. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (Δdpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the Δdpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying Δdpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Pol in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S.cerevisiae Pol is conserved in eukaryotes.