Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.     

Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture

5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles.     

Differential responses of Brassica napus and Petunia hybrida to leaf protoplast isolation stress

Changes in the response to abiotic stress during the isolation of leaf protoplasts were compared between a recalcitrant species of Brassica napus and regenerating species of Petunia hybrida . Initially, levels of soluble free putrescine (put), spermidine (spd) and spermine (spm) in leaves and protoplasts were determined. The sum of these three polyamines increased in petunia and B. napus leaf protoplasts by 1.6-fold and 1.1-fold, respectively. The soluble free fraction of spd and spm decreased in B. napus but not in petunia protoplasts. During the isolation of leaf protoplasts from B. napus , the ratio of soluble free put to the total PAs almost doubled, but that of spd and spm declined significantly. Petunia leaf protoplasts treated with cyclohexylamine (CHA), an inhibitor of spermidine synthase, accumulated ammonia and soluble putrescine, but lost the soluble spermidine. The soluble polyamine levels of CHA-treated petunia leaf protoplasts corresponded with those in B. napus . Leaves were subjected to abiotic stress during the isolation of protoplasts, namely wounding and osmotic stress which changed soluble free polyamine levels in B. napus and petunia, respectively. Both B. napus and petunia leaf protoplasts showed an increase in ammonia, but total free amino acid content and activation of proteases were only enhanced in B. napus leaf protoplasts. These results suggest that in B. napus wounding initiated senescence of leaf protoplasts during their isolation, leading to a constant production of ethylene early in the culture.     

Estimation of effective population size of HIV-1 within a host: a pseudomaximum-likelihood approach

Using pseudomaximum-likelihood approaches to phylogenetic inference and coalescent theory, we develop a computationally tractable method of estimating effective population size from serially sampled viral data. We show that the variance of the maximum-likelihood estimator of effective population size depends on the serial sampling design only because internal node times on a coalescent genealogy can be better estimated with some designs than with others. Given the internal node times and the number of sequences sampled, the variance of the maximum-likelihood estimator is independent of the serial sampling design. We then estimate the effective size of the HIV-1 population within nine hosts. If we assume that the mutation rate is 2.5 x 10(-5) substitutions/generation and is the same in all patients, estimated generation lengths vary from 0.73 to 2.43 days/generation and the mean (1.47) is similar to the generation lengths estimated by other researchers. If we assume that generation length is 1.47 days and is the same in all patients, mutation rate estimates vary from 1.52 x 10(-5) to 5.02 x 10(-5). Our results indicate that effective viral population size and evolutionary rate per year are negatively correlated among HIV-1 patients.     

The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro

To elucidate the function of protein disulfide isomerase (PDI), we screened for PDI-binding proteins in a bovine liver extract using affinity column chromatography. One of the binding proteins was identified by SDS-PAGE and N-terminal amino acid sequence analysis to be cyclophilin B (Cyp B). Use of the BIACORE system revealed that purified bovine Cyp B bound specifically to bovine PDI with a K(D) value of 1.19 x 10(-5) M. Interestingly, the binding affinity between PDI and Cyp B was strengthened by preincubation of the Cyp B with cyclosporin A (CsA), yielding a K(D) value of 3.67 x 10(-6) M. Although the interaction between PDI and Cyp B affected neither the isomerase activity of PDI nor the peptidyl-prolyl cis-trans isomerase activity of Cyp B, Cyp B increased the chaperone activity of PDI. However, the complex of Cyp B and CsA completely inhibited the chaperone activity of PDI. Thus, PDI and Cyp B appear to cooperate with each other to regulate the functional expression of proteins in vivo.     

Evolution of Human Polyomavirus JC: Implications for the Population History of Humans

The polyomavirus JC virus (JCV), the etiological agent of progressive multifocal leukoencephalopathy, is ubiquitous in the human population, infecting children asymptomatically, then persisting in the kidney. The main mode of transmission of JCV is from parents to children through long-term cohabitation. Twelve JCV subtypes that occupy unique domains in Europe, Africa, and Asia have been identified. Here, we attempted to elucidate the evolutionary relationships among JCV strains worldwide using the whole-genome approach with which a highly reliable phylogeny of JCV strains can be reconstructed. Sixty-five complete JCV DNA sequences, derived from various geographical regions and belonging to 11 of the 12 known subtypes, were subjected to phylogenetic analysis using three independent methods: the neighbor-joining, maximum parsimony, and maximum likelihood methods. The trees obtained with these methods consistently indicated that ancestral JCVs were divided into three superclusters, designated as Types A, B, and C. A split in Type A generated two subtypes, EU-a and -b, mainly containing European and Mediterranean strains. The first split in Type B generated Af2 (the major African subtype). Subsequent splits in Type B generated B1-c (a minor European subtype) and all seven Asian subtypes (B1-a, -b, -d, B2, MY, CY, and SC). Type C generated a single subtype (Af1), consisting of strains derived from western Africa. While the present findings provided a basis on which to classify JCV into types or subtypes, they have several implications for the divergence and migration of human populations. Received: 4 April 2001 / Accepted: 31 July 2001     

Generation of amyloid beta protein from a presenilin-1 and betaAPP complex

Presenilin-1 (PS1) is a causative gene in early onset familial Alzheimer's disease (FAD). FAD-linked mutant PS1s significantly increased Abeta40 and Abeta42(43) levels (P < 0.001) and decreased the production of an 11.4 kD (beta-stub) and an 8.7 kD (alpha-stub) carboxyl-terminal fragment of amyloid beta precursor protein (betaAPP-CTFs) (P < 0.01). In the 2% CHAPS extracted lysates, the complex containing the amino-terminal fragment of PS1 (PS1-NTF), the carboxyl-terminal fragments of PS1 (PS1-CTF), and betaAPP-CTFs was identified. Incubation of this isolated complex at pH 6.4 showed the direct generation of Abeta40 and gamma-stub from this complex. This reaction was inhibited by a gamma-secretase inhibitor. The degrading rate of a co-precipitated beta-stub was facilitated under the presence of FAD-linked mutant PS1s. This findings suggest that the direct generation of Abeta from the complex may play an important role in the pathogenesis of Alzheimer's disease.     

Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings

Employing classical two-dimensional electrophoresis (2-DE), amino acid sequencing and immunoblot analysis, we examine for the first time the effect of ozone, a highly notorious environmental pollutant, on rice seedling proteins. Drastic visible necrotic damage to leaf by ozone and consequent increase in ascorbate peroxidase protein(s) was accompanied by rapid changes in the 2-DE protein profiles, over controls. Out of a total of 56 proteins investigated, which were reproducible in repeated experiments, 52 protein spots were visually identified as differentially expressed over controls. Six proteins were N-terminally blocked, and the sequence of 14 proteins could not be determined, whereas 36 proteins were N-terminally and one was internally sequenced. Ozone caused drastic reductions in the major leaf photosynthetic proteins, including the abundantly present ribulose-1, 5-bisphosphate carboxylase/oxygenase, and induction of various defense/stress related proteins. Most prominent change in leaves, within 24 h post-treatment with ozone, was the induced accumulation of a pathogenesis related (PR) class 5 protein, three PR 10 class proteins, ascorbate peroxidase(s), superoxide dismutase, calcium-binding protein, calreticulin, a novel ATP-dependent CLP protease, and an unknown protein. Present results demonstrate the highly damaging effect of ozone on rice seedlings at the level of the proteome.     

Phosphorylation of retinoblastoma-related protein by the cyclin D/cyclin-dependent kinase complex is activated at the G1/S-phase transition in tobacco

In mammals, D-type cyclin-associated kinases mainly regulate the G1/S transition by phosphorylating the retinoblastoma (Rb) protein. We previously demonstrated that in tobacco, cyclin D (Nicta; CycD3;3) is complexed with the PSTAIRE-containing cyclin-dependent kinase (CDKA) from tobacco. Here, we show that Nicta; CycD3;3-associated kinases phosphorylate both the tobacco Rb-related protein (NtRb1) and histone H1. Although NtRb1 kinase activity was detected only during the middle G1- to early S-phase, histone H1 kinase activity was observed as two peaks in G1- to S-phase and G2/M- to M-phase. Importantly, we show that the proportion of cells in the G1-phase was reduced in transgenic Bright Yellow-2 cells overexpressing Nicta; CycD3;3-GFP. Mutational analyses revealed that phosphorylation of Thr-191 in Nicta; CycD3;3 possibly is required for both full kinase activity and localization predominantly to the nucleus. These data suggest that Nicta; CycD3;3 acts as a rate-limiting regulator in the G1/S transition by forming active complexes with CDKA or its related kinases to phosphorylate Rb-related protein and potentially plays a novel role during G2/M and mitosis.     

Effect of electrical stimulation on musculoskeletal systems; a meta-analysis of controlled clinical trials

This study was a meta-analysis to examine whether electrical stimulation has specific effects in the healing of musculoskeletal repair process and in the diminution of symptoms with bone and joint disorders. Using MEDLINE (1966-1999) and EMBASE (1985-1999) a search for articles was carried out with four medical subject headings. Data were extracted from all the accessed articles and additionally collected from appropriate journal lists. A total of 20 randomized controlled trials on bones was identified which assessed healing of fractures, bone graft, and other conditions; and 29 randomized controlled trials on soft tissues and joints were also found, dealing with healing of skin wounds or dermal ulcers, soft tissue injury, and other conditions. Using criteria through which the quality of studies was assessed, the content of the articles was reorganized into a tabular form. The majority of the identified articles reported positive findings, but all the trials showed methodological flaws to some extent. Because of heterogeneity of the studies and the various outcome measurements, pooling of only part of the data was performed. The combined results of 12 trials on bones and 16 trials on soft tissues, the cases in which major endpoints were mainly union or healing rate, revealed statistically significant effects. The studies in this review had some methodological limitations, and the selected pooled trials do not constitute acceptable proof that electrical stimulation has specific effects on health. However, one cannot ignore the statistically significant positive findings reported in the trials, from which extracted data were able to be combined.