Paenibacillus amylolyticus 27C64 has a diverse set of carbohydrate-active enzymes and complete pectin deconstruction system

A draft genome of Paenibacillus amylolyticus 27C64 was assembled and a total of 314 putative CAZymes in 108 different families were identified. Comparison to well-studied polysaccharide-degrading organisms revealed that P. amylolyticus 27C64 has as many or more putative CAZymes than most of these organisms. Four different pectic substrates and xylan supported growth but cellulose was not utilized. Measurement of enzyme activities in culture supernatants revealed low levels of cellulase activity, high levels of xylanase activity, and pectinase activities that adapted to the specific polysaccharides provided. Relative expression levels of each putative pectinase in cells grown with and without three different pectic substrates were evaluated with RT-qPCR and distinct sets of genes upregulated in response to homogalacturonan, methylated homogalacturonan, and rhamnogalacturonan I were identified. It is also noted that this organism’s pectinolytic system differs from other well-studied systems and contains enzymes which are of value for further study.

     

Biochemical evidence for a deficiency of vitamin B6 in subjects reacting to monosodium L-glutamate by the Chinese Restaurant Syndrome

The concept of this research was that monosodium L-glutamate(MSG), an amino acid, might reveal a deficiency of vitamin B₆ by the neurological effects known as the Chinese Restaurant Syndrome (CRS). Tryptophan, an amino acid, is known to reveal a deficiency of B₆ by excretion products. Basal specific activities (S.A.'s) of EGOT were determined on 158 students on no B₆ supplement. Of 27 with extraordinarily low S.A.'s (<0.26 μmoles pyruvate/hr/10⁸ erythrocytes), 12 responded to MSG. By double blind, 3 of the 12 again revealed CRS after placebo to B₆; 8 of the other 9 showed no symptoms to MSG after supplemental B₆. These results show, p<0.01, that supplemental B₆ effectively prevents occurrence of CRS in responders to MSG.     

长治城市湿地公园滨岸区植物群落特征及其与土壤环境的关系

湿地生境的保护与恢复是当前城市湿地公园建设面临的重大挑战,而相关研究案例极少。通过对山西长治城市湿地公园滨岸区草本植物群落进行调查和多元分析,探讨物种分布与土壤因子之间的关系。结果如下:1)研究区植物共计20个种,以湿、中生植物为主,芦苇为建群种,优势种包括香蒲、早熟禾、扁杆藨草和苍耳,总体上物种丰富度较低,随着逐渐远离水域,物种丰富度、优势度和均匀度均呈递增趋势,物种组成上呈现水-湿生、湿-中生、中-旱生的梯度序列;2)CCA排序和Monte Carlo检验结果表明,土壤氮含量和水分含量是影响研究区植物群落分布的重要环境因子,二者分别可解释1.7%、1.4%的物种分布差异,较大的未被解释的部分表明研究区植物群落分布在很大程度上受到其他未选取因子的影响,如种间竞争、水位变化、人为干扰或随机因子。     

Catalytic influence of heparin on auramine O hydrolysis: A basis for differentiating heparin from other glycosaminoglycans based on its properties as a polyelectrolyte

The basis for heparin's ability to accelerate the conversion of auramine O (AuO) to Michlers ketone [P. Band & A. Lukton (1982) Anal. Biochem. 120 , 19–24] is here explained in terms of well-known polyelectrolyte phenomena. Acceleration of this acidcatalyzed hydrolysis appears to be due to colocalization of the cationic dye and hydronium ion within the microenvironment of heparin's electrostatic domain. The dependence of reaction rate on polymer, dye, and hydronium ion concentration, ionic strength, and various ratios of these parameters is consistent with what others have observed for polyelectrolyte catalysts. Dextran sulfate, polyvinyl sulfate, and polyanethole sulfonate likewise accelerate this reaction, thus precluding any explanation of the catalysis in terms of a specific catalytic site. A striking aspect about the polyanion–AuO system is the inability of all other natural glycosaminoglycans tested to catalyze this reaction, despite their analogous polyanionic nature. Manning's limiting laws describing counterion condensation in polyelectrolyte solutions provide a simple context within which to interpret these results. Calculation of the structural linear charge density parameter, ξ, based on analytically determined sulfate-to-hexosamine ratios, reveals that heparin is unique among the glycosaminoglycans in possessing an ξ value greater than unity under the conditions described. Thus, heparin's differential ability to catalyze AuO hydrolysis is a reflection of the fact that only heparin is sulfated to an extent great enough to maintain ξ > 1, even when the negative charge of carboxylate groups is neutralized. It is proposed that this distinction may be important to the unique biochemical attributes of heparin and that such considerations may prove useful in establishing structure–function relationships when comparing different glycosaminoglycan classes.     

Polyelectrolyte theory and chromatin-DNA quaternary structure: Role of ionic strength and Hl histone

Under the assumptions outlined in this paper and those of Manning's theory of polyelectrolyte screening the electrostatic interaction of histone 1 and DNA is examined and shown to lead to spontaneous bending of DNA. Critical variables for this bending are the ionic strength, the length of DNA interacting with each histone 1 molecule, and the fraction of DNA phosphate charges neutralized by the histone 1 molecule. This interaction is postulated as accounting for the observed folding of polynucleosomes into the “solenoid” regular structure. Wherever possible comparison is made between theoretical predictions and available experimental results. Finally, biological implications are briefly discussed.     

The dependence of proteins’ distribution within polyelectrolyte microcapsules on pH of the medium

The distribution of bovine serum albumin and ferritin within polyelectrolyte microcapsules was studied by transmission electron and confocal microscopy at the pH range 2–5. It was estimated that the protein’s distribution depends on the isoelectric point (pI) and first polyelectrolyte used for the preparation of the capsule shell. The peptide is placed in the bulk of capsule if the pH values of the medium are close to the isoelectric point of the protein and polycation was used as a first polyelectrolyte layer. If the first polyelectrolyte was polyanion, the protein is located near the internal surface of the shell. The protein is situated near the internal surface of the shell for both polyelectrolytes when pH is equal to pI.     

Regulation of the Hsp90 system

Hsp90 is a highly conserved and abundant chaperone. It participates in essential cellular activities by supporting the maturation process of its client proteins, many of which are protein kinases and steroid receptors. Client processing is achieved via extensive conformational changes within the dimeric chaperone. This requires an ATP hydrolysis activity that is controlled by auto-inhibitory mechanisms and several structurally diverse cofactors. Especially the client-specificity of Hsp90 depends on client-specific cofactors, which can adapt Hsp90's activities to the client requirements at different conditions and in different cell types. Additionally, post-translational modifications can influence almost every aspect of Hsp90's interactions and activities. In this review, we present these regulatory principles, discuss the factors that have an impact on Hsp90's function and elaborate the mechanisms that are responsible for regulating the Hsp90 machinery.     

Solvation effects upon the thermodynamic substrate activity; correlation with the kinetics of enzyme catalyzed reactions. I. Effects of added reagents such as methanol upon alpha-chymotrypsin.

Solvents, detergents, etc., have often been added to the medium to study the kinetics of enzyme action and for binding studies. They have been employed for diverse reasons such as solubilization of substrates or to stabilize an enzyme that was originally membrane bound. Thermodynamic considerations dictate that any added substance, such as methanol, which is present in significant quantity must affect the thermodynamic activities of the enzyme, enzyme-substrate complex, substrate and any other intermediates although cancellation effects may occur in this regard. The influence upon substrate activities is the only one that is easily experimentally accessible. These effects are shown, from the data of Bernard and Laidler, to be large in the case of the alpha-chymotrypsin catalyzed hydrolysis of methylhydrocinnamate. The variation of the Michaelis-Menten constant is quantitatively explainable in terms of the alteration of the thermodynamic activity of the substrate by methanol.     

Deficiency of glucosylsphingosine: beta-glucosidase in Gaucher disease

A deficiency in the activity of glucosylsphingosine: β-glucosidase has been observed in Gaucher's spleen tissue and skin fibroblasts. Preliminary studies indicated the presence of a material similar to glucosylsphingosine in Gaucher's spleen while such a material was not detectable in normal and other pathological control spleen tissue. This is the first report of the natural occurrence of a psychosine-like material in a mammalian tissue.     

Intrinsic binding effects in mixed counterionic polyelectrolyte systems: extension of condensation theory and comparison with voltammetric data

Voltammetric speciation data for the potassium/zinc/polymethacrylate system, recently obtained for various charge densities of the polyelectrolyte (Díaz-Cruz et al., Anal. Chim. Acta, 264 (1992) 163) and for different concentrations of monovalent counterions (van den Hoop and van Leeuwen, Anal. Chim. Acta, 273 (1993) 275), are compared with theoretical predictions computed according to a new thermodynamic model developed by Paoletti et al. (Biophys. Chem., 41 (1991) 73) and recently extended by Benegas and Paoletti (in preparation). The model allows: (i) the simultaneous condensation of both monovalent and divalent counterions and (ii) can account for a certain specific affinity of the polyelectrolyte for one type of the counterion over the other. For various charge densities of the polyelectrolyte, experimentally obtained speciation data for the K/Zn/PMA system agree well with theoretical predictions by considering an extra reduced molar affinity energy of -4RT for the Zn(2+) polyelectrolyte binding. The agreement between experimental and theoretical values for the distribution of Zn(2+) ions over the free and bound state becomes less perfect for relatively high concentrations of monovalent counterions.     

Mechanical properties of single living cells encapsulated in polyelectrolyte matrixes

We have studied the mechanical properties of encapsulated Saccharomyces cerevisiae yeast cells by performing AFM force measurements. Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte layers and mechanically trapped into a porous membrane. Coated and uncoated cells in presence/absence of bud scars, i.e. scars resulting from previous budding events, have been investigated. No significant differences between encapsulated and bare cells could be inferred from AFM topographs. On the other hand, investigation on the system elasticity through the acquisition and analysis of force curves allowed us to put in evidence the differences in the mechanical properties between the hybrid cell/polyelectrolyte system and the uncoated cells. Analysis of the curves contact region indicates that the polyelectrolyte coating increases the system rigidity. Quantitative evaluation of the cell rigidity through the Hertz-Sneddon model showed that coated cells are characterized by a Young's modulus higher than the value obtained for uncoated cells and similar to the value observed on the bud scar region of uncoated cells.     

Mechanical characteristics of synthetic polyelectrolyte gel as a physical model of the cytoskeleton

A physical model of the cytoskeleton based on synthetic polyelectrolyte hydrogel of polymethacrylic acid has been proposed. From the physicochemical point of view, the structures of polyelectrolyte gel and the cytoskeleton show a high degree of similarity. It has been shown that polyelectrolyte gel can shorten and produce mechanical stress in response to changes in the composition of the surrounding solution. The mechanical properties of the model gel have been evaluated: Young modulus (2–6 kPa), stress relaxation time (0.1–1 s), and apparent viscosity (0.3–3 kPa s). The viscoelastic properties of the gel depend on the degree of its swelling. It has been demonstrated that the mechanical properties of gels of polymethacrylic acid are close to those of biological objects.     

Neurobiology of tetrahydro-beta-carbolines

Tetrahydro-β-carbolines (THβC's) are tricyclic compounds structurally related to the indoleamines. Recent studies have reported the $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ formation of these compounds in brain and other tissues. This information and other data indicating that THβC's interact relatively specifically with various aspects of the serotonergic neurotransmitter system has suggested that THβC's may serve as endogenous neuromodulators of neurotransmitters. Evidence for the formation of these compounds as well as their neurochemical, neuroendocrinological, and behavioral effects is described in this review.     

Nanomechanical behaviors of microcantilever-based single-stranded DNA chips induced by counterion osmotic effects

Experiments show that deflections of microcantilever-DNA chip can be induced by many factors, such as grafting density, hybridization efficiency, concentration, length and sequence of DNA molecules, buffer salt concentration, time, and temperature variation. However, there are few theoretical works on microcantilever-DNA chips. The present paper is aimed to study the influence of counterion effects of single-stranded DNA (ssDNA) polyelectrolyte solution on the nanomechanical behaviors of microcantilever-based ssDNA chips during packing process. First, the effect of osmotic pressure induced by ingress of counterions into DNA brush structures is studied with Hagan’s model for a cylindrical polyelectrolyte brush system on the basis of Poisson-Boltzmann distribution hypothesis. Second, Zhang’s two-variable method for a laminated cantilever is used to formulate a four-layer energy model for ssDNA chips with weak interactions. Third, the influence of grafting density, ssDNA chain length, and salt concentration on packing deflection is investigated using the principle of minimum energy. The predictive tendency is qualitatively similar to those observed in some related ssDNA chip experiments. The difference between the four-layer model and the simplified two-layer model for ssDNA chips is also discussed.     

Antiangiogenic and antitumor activities of IL-27

IL-27 is a novel IL-6/IL-12 family cytokine playing an important role in the early regulation of Th1 responses. We have recently demonstrated that IL-27 has potent antitumor activity, which is mainly mediated through CD8(+) T cells, against highly immunogenic murine colon carcinoma. In this study, we further evaluated the antitumor and antiangiogenic activities of IL-27, using poorly immunogenic murine melanoma B16F10 tumors, which were engineered to overexpress single-chain IL-27 (B16F10 + IL-27). B16F10 + IL-27 cells exerted antitumor activity against not only s.c. tumor but also experimental pulmonary metastasis. Similar antitumor and antimetastatic activities of IL-27 were also observed in IFN-gamma knockout mice. In NOD-SCID mice, these activities were decreased, but were still fairly well-retained, suggesting that different mechanisms other than the immune response are also involved in the exertion of these activities. Immunohistochemical analyses with Abs against vascular endothelial growth factor and CD31 revealed that B16F10 + IL-27 cells markedly suppressed tumor-induced neovascularization in lung metastases. Moreover, B16F10 + IL-27 cells clearly inhibited angiogenesis by dorsal air sac method, and IL-27 exhibited dose-dependent inhibition of angiogenesis on chick embryo chorioallantoic membrane. IL-27 was revealed to directly act on HUVECs and induce production of the antiangiogenic chemokines, IFN-gamma-inducible protein (IP-10) and monokine induced by IFN-gamma. Finally, augmented mRNA expression of IP-10 and monokine induced by IFN-gamma was detected at the s.c. B16F10 + IL-27 tumor site, and antitumor activity of IL-27 was partially inhibited by the administration of anti-IP-10. These results suggest that IL-27 possesses potent antiangiogenic activity, which plays an important role in its antitumor and antimetastatic activities.     

An electron microscopy study of the structure of polyelectrolyte microcapsules containing protein and containing no protein

Electron micrographs of ultrathin sections of polyelectrolyte microparticles containing protein and free from protein for the formation of which CaCO3 spherulites served as a core basis have been obtained and analyzed. Polyelectrolyte microparticles with the number of alternately layered polyelectrolyte layers of polystyrene sulfonate and polyallylamine from 6 to 11 have been studied. It follows from the data obtained that protein-free polyelectrolyte particles having the dimensions 4.5-5 mm are formations of an intricate internal organization, which consist of a set of threadlike and closed nanoelements of polyelectrolyte nature with a thickness of 20-30 nm. The particles containing six to eight polyelectrolyte layers lack the external envelope; therefore, they were called polyelectrolyte microspherulites. With the number of layers nine and more, when a polyelectrolyte envelope appears on the surface, they transfer into polyelectrolyte microcapsules. It was found that, in a protein-containing polyelectrolyte microcapsule, as distinct from protein-free polyelectrolyte microspherulite and microcapsule, polyelectrolytes are located only in the nearsurface layer, and the external spatially organized envelope restricts the internal volume filled with protein solution. As the number of polyelectrolyte layers increases, the thickness of the envelope increases. The reasons for such substantial differences in the structures of polyelectrolyte microcapsules formed on protein-containing and protein-free CaCO3 spherulite are discussed.     

Hysteresis in systems with a single steady state

It is usually stated that only systems with multiple steady states can exhibit hysteresis. For protein conformations, this would violate the idea that, in fixed environments, primary structure uniquely determines tertiary structure. It is shown that hysteresis-like phenomena can be exhibited by systems possessing only a single steady-state configuration. This property is placed in a more general theoretical setting of recognition and classification systems, and some implications for processes such as memory, learning and pattern generation (morphogenesis) are discussed.     

Culture collections — their services to biotechnology

The need to recruit qualified and experienced employees is a major concern of many organizations engaged in biotechnology. Universities and industry are scoured to find the right people, but where are we to find promising new candidates for the most numerous, and arguably most talented section of the biotechnological workforce — microorganisms and other types of cells? The international network of culture collections offers the best answer, and a proper understanding of their roles and capabilities can save biotechnologists an immense amount of time and money.Among the millions of microbial, plant and animal cells stored in the world's culture collections it is often possible to find one that exactly suits a biotechnologist's particular requirements or one that can be developed to perform a specific task. This article outlines the services offered by culture collections, generally using the UK collections as examples of the main activities throughout the world.     

Cell-Free Degradation of p27kip1, a G1 Cyclin-Dependent Kinase Inhibitor, Is Dependent on CDK2 Activity and the Proteasome

Entry into S phase is dependent on the coordinated activation of CDK4,6 and CDK2 kinases. Once a cell commits to S phase, there must be a mechanism to ensure the irreversibility of this decision. The activity of these kinases is inhibited by their association with p27. In many cells, p27 plays a major role in the withdrawal from the cell cycle in response to environmental cues. Thus, it is likely that p27 is a target of the machinery required to ensure the irreversibility of S-phase entry. We have been interested in understanding the mechanisms regulating p27 at the G₁/S transition. In this report, we define a cell-free degradation system which faithfully recapitulates the cell cycle phase-specific degradation of p27. We show that this reaction is dependent on active CDK2 activity, suggesting that CDK2 activity is directly required for p27 degradation. In addition to CDK2, other S-phase-specific factors are required for p27 degradation. At least some of these factors are ubiquitin and proteasome dependent. We discuss the relationships between CDK2 activity, ubiquitin-dependent, and possibly ubiquitin-independent proteasomal activities in S-phase extracts as related to p27.     

Increase in rat striatal acetylcholine content by bromocriptine: Evidence for an indirect dopaminergic action

Bromocriptine, at the optimal dose and time of 4 mg/kg, 90 min, increased the content of acetylcholine in the rat striatum by about 30% without affecting the acetylcholine content in other brain regions. Striatal choline acetyltransferase and acetylcholinesterase activities and sodium-dependent high affinity choline uptake were not affected by the $\text{in vivo}$ administration or the $\text{in vitro}$ incubation with even high amounts of the drug. The increase in striatal acetylcholine by bromocriptine was mediated through the dopaminergic system since pretreatment with pimozide or penfluridol, powerful dopamine receptor antagonists, completely prevented the effect while parachlorophenylaline and phenoxybenzene pretreatment were ineffective. The action of bromocriptine, differently from that of apomorphine, was also blocked upon inhibition of tyrosine hydroxylase by alphamethylparatyrosine, suggesting that intact catecholamine synthesis is necessary for the drug to act. The requirement of dopamine by bromocriptine was further indicated when no potentiation of the cholinergic response to bromocriptine occurred following induction of dopamine receptor supersensitivity by long-term 6-hydroxydopamine lesion of the nigroneostriatal pathway. On the other hand, evidence is presented to show that bromocriptine acts in synergism with dopamine as the latency period for the onset of bromocriptine's cholinergic action was significantly decreased when it was administered in combination with a subthreshold dose of L-dopa, the dopamine precursor. There also was no summation of bromocriptine's increase with apomorphine's increase in striatal acetylcholine content at supramaximal doses possibly indicating that the same population of intrastriatal cholinergic neurons is the common target of both drugs.It is proposed that bromocriptine exerts an inhibitory effect on the striatal cholinergic neurons through a stimulation of the dopaminergic system but, differently from apomorphine, it requires the presence of endogenous dopamine for its action.