M-phase regulation of the recruitment of mRNAs onto polysomes using the CDK1/cyclin B inhibitor aminopurvalanol

Translation under the control of the universal cell cycle regulator CDK1/cyclin B was investigated during the first cell cycle in sea urchin embryos. The CDK1/cyclin B inhibitor aminopurvalanol arrested embryos at the G2/M transition. Polysomal mRNAs were purified from control and arrested embryos, and screened for specific mRNA recruitment or release at M-phase by subtractive hybridization. The polysomal repartition of clones issued from this screen was analyzed. Three specific mRNAs were selectively recruited onto polysomes at M-phase. Conversely, two other specific mRNAs were released from polysomes. The isolation of these translationally regulated mRNAs gives now important tools for insights into the regulation of protein synthesis by the cell cycle regulator CDK1-cyclin B.     

Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan

Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln¹⁰⁶ is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.     

Supplemental Effects of Arginine and Methionine on Growth,and on Formations of Urea and Creatine of Adrenalectomized Rats Fed High Glycine Diets

The effects of adrenalectomy on growth, some enzyme activities in the liver and kidney, and urinary excretion of urea, creatinine and creatine were investigated in rats fed the 10% casein diets containing 7% glycine with or without l-arginine and l-methionine (10C, 10C7G and 10C7ArgMet).

Body weight gains of the intact 10C and 10C7GArgMet groups were almost same as the corresponding adrenalectomized groups. The body weight of the adrenalectomized 10C7G group was extremely decreased though that of the intact 10C7G group was maintained almost constant; but the decrease was recovered by the administration of hydrocortisone. The activities of liver arginase and carbamylphosphate synthetase were not affected by those diets. Liver serine dehydratase and ornithine δ-aminotransferase activities were increased in the intact 10C7G and 10C7GArgMet groups, but these increases were depressed by adrenalectomy. Glutamate-pyruvate transminase activities in the liver of intact 10C7G and 10C7GArgMet groups were also enhanced, but were extremely decreased in the corresponding adrenalectomized groups. Kidney transamidinase activity was not affected by adrenalectomy. The amount of urinary excreted urea was almost unchanged by adrenalectomy, but was increased by hydrocortisone administration. The amounts of excreted creatine of the adrenalectomized groups were generally larger than the corresponding intact groups, but slightly decreased by the administration of hydrocortisone. The amount of excreted creatinine was not generally affected by adrenalectomy.     

Purifications and Enzymatic Properties of β-Amylase and Pullulanase from Bacillus cereus var. mycoides

A β-amylase and a pullulanase produced by Bacillus cereus var. mycoides were purified by means of ammonium sulfate fractionation, adsorption on starch and celite and Sephadex G–100 column chromatography. The purified enzymes were homogeneous in disc electrophoresis.

The β-amylase released only maltose from amylose, amylopectin, starch and glycogen, and the released maltose was in β-form. The pullulanase released maltose, maltotriose and maltotetraose from β-limit dextrin and maltotriose from pullulan, but not amylose-like substance from amylopectin.

The optimum pHs of β-amylase and pullulanase were about 7 and 6~6.5, respectively. The optimum temperatures of the enzymes were about 50°C. The enzymes were inhibited by the sulfhydryl reagents such as mercuric chloride and p-chloromercuribenzoate, and the inhibitions with p-chloromercuribenzoate were restored by the addition of cysteine. The molecular weights of β-amylase and pullulanase were estimated to be 35,000±5,000 and 110,000±20,000, respectively.     

Aerobic secondary utilization of a non-growth and inhibitory substrate 2,4,6-trichlorophenol by Sphingopyxis chilensis S37 and sphingopyxis-like strain S32

This paper reports 2,4,6-trichlorophenol (246TCP) degradation bySphingopyxis chilensis S37 and Sphingopyxis chilensis-like strain S32,which were unable to use 246TCP as the sole carbon and energy source. In R2A broth, the strainsdegraded 246TCP up to 0.5 mM. Results with mixtures of different 246TCP and glucose concentrations in mineral salt media demonstrated dependence on glucose to allow bacterial growth and degradation of 246TCP. Strain S32 degraded halophenol up to 0.2 mM when 5.33 mM glucose was simultaneously added, while strain S37 degraded the compound up to 0.1 mM when 1.33 mM glucose was added. These 246TCP concentrations were lethal for inocula in absence of glucose. Stoichiometricreleases of chloride and analysis by HPLC, GC-ECD and GC-MS indicated 246TCP mineralisation by both strains. To our knowledge, this is the first report of bacteriaable to mineralize a chlorophenol as a non-growth and inhibitory substrate. The concept of secondary utilization instead of cometabolism is proposed for this activity.     

On the same cell type GPI-anchored normal cellular prion and DAF protein exhibit different biological properties

Normal cellular prion protein (PrP(C)) and decay-accelerating factor (DAF) are glycoproteins linked to the cell surface by glycosylphosphatidylinositol (GPI) anchors. Both PrP(C) and DAF reside in detergent insoluble complex that can be isolated from human peripheral blood mononuclear cells. However, these two GPI-anchored proteins possess different cell biological properties. The GPI anchor of DAF is markedly more sensitive to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) than that of PrP(C). Conversely, PrP(C) has a shorter cell surface half-life than DAF, possibly due to the fact that PrP(C) but not DAF is shed from the cell surface. This is the first demonstration that on the surface of the same cell type two GPI-anchored proteins differ in their cell biological properties.     

Molecular network and functional implications of macromolecular tRNA synthetase complex

Understanding the complex network and multi-functionality of proteins is one of the main objectives of post-genome research. Aminoacyl-tRNA synthetases (ARSs) are the family of enzymes that are essential for cellular protein synthesis and viability that catalyze the attachment of specific amino acids to their cognate tRNAs. However, a lot of evidence has shown that these enzymes are multi-functional proteins that are involved in diverse cellular processes, such as tRNA processing, RNA splicing and trafficking, rRNA synthesis, apoptosis, angiogenesis, and inflammation. In addition, mammalian ARSs form a macromolecular complex with three auxiliary factors or with the elongation factor complex. Although the functional meaning and physiological significance of these complexes are poorly understood, recent data on the molecular interactions among the components for the multi-ARS complex are beginning to provide insights into the structural organization and cellular functions. In this review, the molecular mechanism for the assembly and functional implications of the multi-ARS complex will be discussed.     

Increased level of glycoxidation product N(epsilon)-(carboxymethyl)lysine in rat serum and urine proteins with aging: link with glycoxidative damage accumulation in kidney

Accumulation of carboxymethylated proteins (CML-proteins) is taken as a biomarker of glycoxidative stress which is thought to contribute to the age-related impairment in tissue and cell function. To investigate the occurrence and extent of glycoxidative damage with aging in rat kidney, serum and urine, we have prepared a polyclonal antibody against CML-modified bovine serum albumin. We subsequently used it for immunolocalization and in enzyme-linked immunosorbent assays to evaluate CML-protein content. In the serum, CML-protein level was 1.43+/-0.14 pmol CML/micrograms protein at 3 months and significantly increased by 50% from 10 to 27 months (1.50+/-0.14 pmol CML/micrograms protein vs 2.27+/-0.26 pmol CML/micrograms protein), albumin and transferrin being the main modified proteins. In the urine, CML-protein level was 2.50+/-0.14 pmol CML/micrograms protein at 3 months and markedly increased from 10 months (2.99+/-0.24 pmol CML/micrograms protein) to 27 months (3.76+/-0.25 pmol CML/micrograms protein), with albumin as the main excreted modified protein. Immunolocalization of CML-proteins in kidney provided evidence for an age-dependent increased accumulation in extracellular matrices. Intense staining of the glomerular basement membrane (GBM), Bowman's capsule, and the tubular basement membrane was found. Additionally, the CML content for collagen from GBM was 195.85+/-28.95 pmol CML/microgrms OHPro at 3 months and significantly increased from 10 months (187.61+/-21.99 pmol CML/micrograms OHPro) to 27 months (334.55+/-62.21 pmol CML/micrograms OHPro). These data show that circulating CML-protein level in serum and urine and CML accumulation in nephron extracellular matrices with aging are increasing in parallel. The CML-protein measurement in serum and urine may thus be used as an index for the assessment of age-associated glycoxidative kidney damage.     

Substrate and product inhibition of hydrogen production by the extreme thermophile,Caldicellulosiruptor saccharolyticus

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H(2) in the gas phase (identical with partial hydrogen pressure (pH(2)) of (1-2) x 10(4) Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H(2) (identical with pH(2) of 5.6 x 10(4) Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70 degrees C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins.     

Effect of regular training on the myocardial and plasma concentrations of taurine andα-amino acids in thoroughbred horses

Summary Exercise induces significant changes in the free intracellular amino acid pool in skeletal muscle but little is known of whether such changes also occur in cardiac muscle. In this study the effect of regular exercise on the size and the constituents of the free amino acid pool in the hearts and in the plasma of thoroughbred horses was investigated. The total free intracellular amino acid pool in the hearts of control horses was 30.9 ± 1.2mol/g wet weight (n = 6). Glutamine but not taurine was present at the highest concentration (13.5 ± 0.9 and 7.7 ± 0.69mol/g wet weight for glutamine and taurine respectively). As for the rest of the amino acids in the pool, only glutamate and alanine were present at levels greater than 1mol/g wet weight (4.6 ± 0.25 and 1.7 ± 0.14 for glutamate and alanine respectively). The tissue to plasma ratio was highest for taurine at 155, followed by glutamate at 111, aspartate and glutamine at 37, alanine at 5.8 and ratios of less than 3 for the rest of the amino acids. The total free intracellular amino acid pool in the hearts of exercised horses was slightly but not significantly lower than control (28.1 ±1.1mol/g wet weight, n = 6). Regular exercise increased the intracellular concentration of threonine, valine, isoleucine, leucine and phenylalanine but was only significant (p < 0.05) for threonine. This work has documented the profile of taurine and protein amino acids in the heart and in the plasma of thoroughbred horses and showed that in contrast to skeletal muscle, heart muscle does not show major changes in amino acids during regular exercise.