Structural determination of the polar glycoglycerolipids from thermophilic bacteria Meiothermus taiwanensis.

The polar glycolipids were isolated from the thermophilic bacteria Meiothermus taiwanensis ATCC BAA-400 by ethanol extraction and purified by Sephadex LH-20 and silica gel column chromatography. The fatty acid composition of O-acyl groups in the glycolipids was obtained by gas chromatography mass spectroscopy analysis on their methyl esters derived from methanolysis and was made mainly of C(15:0) (34.0%) and C(17:0) (42.3%) fatty acids, with the majority as branched fatty acids (over 80%). Removal of O-acyl groups under mild basic conditions provided two glycolipids, which differ only in N-acyl substitution on a hexosamine. Electrospray mass spectroscopy analysis revealed that one has a C(17:0) N-acyl group and the other hydroxy C(17:0) in a ratio of about 1 : 3.5. Furthermore, complete de-lipidation with strong base followed by selective N-acetylation resulted in a homogeneous tetraglycosyl glycerol. The linkages and configurations of the carbohydrate moiety were then elucidated by MS and various NMR analyses. Thus, the major glycolipid from M. taiwanensis ATCC BAA-400 was determined with the following structure: alpha-Galp(1-6)-beta-Galp(1-6)-beta-GalNAcyl(1,2)-alpha-Glc(1,1)-Gro diester, where N-acyl is C(17:0) or hydroxy C(17:0) fatty acid and the glycerol esters were mainly iso- and anteisobranched C(15:0) and C(17:0).     

Enzymes involved in starch synthesis in the developing mung bean seed

The levels of free sugars, starch and enzymes involved in starch metabolism—sucrose synthetase, UDP and ADP glucose pyrophosphorylase, phosphorylase and starch synthetase—were assayed during seed development of three cultivars of mung bean (Vigna radiata). Free sugars and starch increased with increasing seed weight. Changes in levels of sucrose synthetase, UDP- and ADP-glucose pyrophosphorylases, and phosphorylase were paralleled by changes in starch accumulation. After the maximum activity levels of these enzymes had been reached, maximum activities of soluble starch synthetase and starch granule-bound starch synthetase occurred. There were high activities of sucrose synthetase and phosphorylase at maximum rates of starch accumulation. Thus, starch could be synthesized via the ADP glucose pathway in mung bean seeds. However, phosphorylase may account for the starch accumulation in the early stages of mung bean seed development.     

Effect of enzyme and ligand protonation on the binding of folates to recombinant human dihydrofolate reductase: implications for the evolution of eukaryotic enzyme efficiency.

There is marked pH dependence of the rate constant (koff) for tetrahydrofolate (H4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31----Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH approximately 5, and this accounts for the marked decrease in kcat for this variant as the pH is decreased from 7 to 5. This decreased kcat at low pH is not seen for most DHFRs. koff for dissociation of folate, dihydrofolate (H2folate), and H4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of koff in the range pH 5-7 is well described by a pKa of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), koff increases further as the pH is raised, and this relation is governed by a second pKa which is close to the pKa for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases koff. Evidence is presented that the dependence of pH on koff for hDHFR accounts for the shape of the kcat versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.     

Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase.

Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain. Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone. Southern analysis showed that ERG8 was a single-copy gene. Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame. The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases. Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential. Expression of the ERG8 gene in S. cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity. Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells.     

A device to simplify the assay of ligand binding to cell surfaces

A sensitive and reproducible technique for characterization of the binding of ¹²⁵I-labeled protein ligands to cell surfaces is described. The physical separation of cell-bound and free ligand was accomplished by centrifugation-filtration using an assembly of plastic micro test tubes. This assembly allowed rapid and efficient separation of free and cell-bound ligands with minimal manipulation of the cells.     

ROCKII Ser1366 phosphorylation reflects the activation status

ROCK (Rho-associated protein kinase), a downstream effector of RhoA, plays an important role in many cellular processes. Accumulating evidence has shown the involvement of ROCK activation in the pathogenesis of many diseases. However, a reagent capable of detecting ROCK activation directly is lacking. In the present study, we show autophosphorylation of ROCKII in an in vitro kinase reaction. The phosphorylation sites were identified by MS, and the major phosphorylation site was found to be at the highly conserved residue Ser1366. A phospho-specific antibody was generated that can specifically recognize ROCKII Ser1366 phosphorylation. We found that the extent of Ser1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that Ser1366 phosphorylation reflects its kinase activity. In addition, ROCKII Ser1366 phosphorylation could be detected in human breast tumours by immunohistochemical staining. The present study provides a new approach for revealing the ROCKII activation status by probing ROCKII Ser1366 phosphorylation directly in cells or tissues.     

Proteomic investigation of the impact of oxygen on the protein profiles of hyaluronic acid‐producing Streptococcus zooepidemicus

Hyaluronic acid (HA) is a linear and negatively charged polysaccharide regularly used in medicine and cosmetics. Recently Streptococcus zooepidemicus has been exploited in the fermentation industry to produce HA. Many studies showed that higher amounts of HA were produced under aerobic condition compared to anaerobic conditions. To explore the effect of oxygen on the HA synthesis in S. zooepidemicus, 2‐DE was used to compare the proteomes of aerobically and anaerobically fermented bacteria to identify proteins, which might be associated with the influence of oxygen on the HA synthesis. Totally nine pairs of 2‐DE gels collected from three batches were compared and nine overexpressed proteins were observed in aerobically fermented bacteria. These proteins were identified by LC/tandem MS as dihydrolipoamide dehydrogenase, UDP‐acetyl‐glucosamine pyrophosphoylase, dihydrolipoamide‐S‐acetyltransferase and acetoin dehydrogenase α and β chains, respectively. These upregulated proteins were involved in acetoin dissimilation, the central carbon metabolism and the HA anabolic pathway, implicating that oxygen might augment the expression of genes that are involved in central energy metabolism, acetoin reutilization and HA biosynthesis to enhance the amount of acetyl‐CoA as such that more acetyl‐CoA can be diverged from the central carbon metabolism to replenish acetyl‐CoA for the HA synthesis.     

Modulation of microglial immune responses by a novel thiourea derivative

Increasing evidence indicates that microglial activation plays an important role in the pathogenesis of Alzheimer's disease (AD). In AD, activated microglia may facilitate the clearance of β-amyloid (Aβ), a neurotoxic component in AD pathogenesis. However, microglial activation comes at the cost of triggering neuro-inflammation, which contributes to cerebral dysfunction. Thus, pharmacological approaches that can achieve a favorable combination of a reduced microglia-mediated neuro-inflammation, and an enhanced Aβ clearance may be beneficial for preventing the progression of the disease. Here, we show that some newly synthesized compounds may exert such a combination of functions. Using mouse primary microglia and RAW264.7 cells, we found that some thiourea derivatives significantly enhanced microglial Aβ phagocytosis and suppressed microglial immune responses, as evidenced by the reduced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Of note, some commercially available inhibitors for iNOS and/or COX-2, such as ibuprofen, dextromethorphan, and N^G-methyl-l-arginine (l-NMA), show negligible effects on microglial Aβ phagocytosis. Among the thiourea derivatives, our data show that a lead compound, designated as compound #326, (1-Naphthalen-1-yl-3-[5-(3-thioureido-phenoxy)-pentyl]-thiourea) appears to be the most potent in promoting Aβ phagocytosis and in inhibiting the LPS-induced expression of iNOS and COX-2 (when used at concentrations in the low μM range). The potency of compound #326 may have beneficial effects on modulating microglial activation in AD. The structure–activity relationship indicates that the thiourea group, alkyl linker, and the hydrophobic aryl group largely influence the dual functions of the compounds. These findings may indicate a structural basis for the improved design of future drug therapies for AD.