Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus

l-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependend on the extracellular concentration. l-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K _m =1.1 mM), ammonia (K _m =20 mM) and NADH (K _m =0.14 mM) were required for the reductive amination, and l-alanine (K _m =9.1 mM) and NAD (K _m =0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K _i =1.6 mM) and noncompetitively with respect to ammonia (K _i =2.0 mM) and pyruvate (K _i =3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6–7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.Abbreviation Alanine-DH l-alanine:NAD oxidoreductase     

An automated method for the analysis of ‘particulate’ carbon and nitrogen in natural waters

A method is described for the measurement of the carbon and nitrogen content of particulate material in natural waters. Particulate material is separated by filtration through GF/C filters. The dried filter is encapsulated in silver foil using a purpose made press. Analysis is carried out using high temperature combustion with thermal conductivity detection of emission gasses. Analytical performance characteristics obtained with both standards and natural materials are given.     

Comprehensive analysis of the roles of ‘black’ and ‘gray’ clusters in structure and function of rat β-parvalbumin

Recently we found two highly conserved structural motifs in the proteins of the EF-hand calcium binding protein family. These motifs provide a supporting scaffold for the Ca²⁺ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif forms a cluster of three amino acids called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster). Cluster I is much more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II includes a mix of aromatic, hydrophobic, and polar amino acids. The ‘black’ and ‘gray’ clusters in rat β-parvalbumin consist of F48, A100, F103 and G61, L64, M87, respectively. In the present work, we sequentially substituted these amino acids residues by Ala, except Ala100, which was substituted by Val. Physical properties of the mutants were studied by circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probe methods. The Ca²⁺ and Mg²⁺ binding affinities of these mutants were evaluated by intrinsic fluorescence and equilibrium dialysis methods. In spite of a rather complicated pattern of contributions of separate amino acid residues of the ‘black’ and ‘gray’ clusters into maintenance of rat β-parvalbumin structural and functional status, the alanine substitutions in the cluster I cause noticeably more pronounced changes in various structural parameters of proteins, such as hydrodynamic radius of apo-form, thermal stability of Ca²⁺/Mg²⁺-loaded forms, and total energy of Ca²⁺ binding in comparison with the changes caused by amino acid substitutions in the cluster II. These findings were further supported by the outputs of computational analysis of the effects of these mutations on the intrinsic disorder predisposition of rat β-parvalbumin, which also indicated that local intrinsic disorder propensities and the overall levels of predicted disorder were strongly affected by mutations in the cluster I, whereas mutations in cluster II had less pronounced effects. These results demonstrate that amino acids of the cluster I provide more essential contribution to the maintenance of structuraland functional properties of the protein in comparison with the residues of the cluster II.     

Comparison of ‘ignition’ and ‘extraction’ methods for the determination of organic phosphate in rocks and soils

Summary Ignition markedly increased the solubility in dilute acid of iron- and aluminium-bound inorganic phosphate in samples of weathered New Zealand greywacke rock. This observation supports the view that ignition methods may sometimes overestimate the total organic phosphate of soils.     

Exhaled breath analysis: a review of ‘breath-taking’ methods for off-line analysis

Background

The potential of exhaled breath sampling and analysis has long attracted interest in the areas of medical diagnosis and disease monitoring. This interest is attributed to its non-invasive nature, access to an unlimited sample supply (i.e., breath), and the potential to facilitate a rapid at patient diagnosis. However, progress from laboratory setting to routine clinical practice has been slow. Different methodologies of breath sampling, and the consequent difficulty in comparing and combining data, are considered to be a major contributor to this. To fulfil the potential of breath analysis within clinical and pre-clinical medicine, standardisation of some approaches to breath sampling and analysis will be beneficial.

Objectives

The aim of this review is to investigate the heterogeneity of breath sampling methods by performing an in depth bibliometric search to identify the current state of art in the area. In addition, the review will discuss and critique various breath sampling methods for off-line breath analysis.

Methods

Literature search was carried out in databases MEDLINE, BIOSIS, EMBASE, INSPEC, COMPENDEX, PQSCITECH, and SCISEARCH using the STN platform which delivers peer-reviewed articles. Keywords searched for include breath, sampling, collection, pre-concentration, volatile. Forward and reverse search was then performed on initially included articles. The breath collection methodologies of all included articles was subsequently reviewed.

Results

Sampling methods differs between research groups, for example regarding the portion of breath being targeted. Definition of late expiratory breath varies between studies.

Conclusions

Breath analysis is an interdisciplinary field of study using clinical, analytical chemistry, data processing, and metabolomics expertise. A move towards standardisation in breath sampling is currently being promoted within the breath research community with a view to harmonising analysis and thereby increasing robustness and inter-laboratory comparisons.

     

Starter culture for the production of ‘soyiru’

Bacillus subtilis or licheniformis facilitated production of soyiru with the best results being given by using both together. Fermentation employing Streptococcus enterococcus was unsuccessful.H.A. Suberu is with the Department of Biological Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria. J.A. Akinyanju is with the Department of Biological Sciences, University of Ilorin, P.M.B. 1515, Ilorin, Nigeria.     

Viral phylodynamics and the search for an ‘effective number of infections’

Information on the dynamics of the effective population size over time can be obtained from the analysis of phylogenies, through the application of time-varying coalescent models. This approach has been used to study the dynamics of many different viruses, and has demonstrated a wide variety of patterns, which have been interpreted in the context of changes over time in the ‘effective number of infections’, a quantity proportional to the number of infected individuals. However, for infectious diseases, the rate of coalescence is driven primarily by new transmissions i.e. the incidence, and only indirectly by the number of infected individuals through sampling effects. Using commonly used epidemiological models, we show that the coalescence rate may indeed reflect the number of infected individuals during the initial phase of exponential growth when time is scaled by infectivity, but in general, a single change in time scale cannot be used to estimate the number of infected individuals. This has important implications when integrating phylogenetic data in the context of other epidemiological data.     

Automated analysis of neuromuscular ‘jitter’

This paper describes an interactive program for real time analysis of the neuromuscular jitter. Two implementations, using different hardware for data acquisition, reflect the modular design and flexibility of the software. Details on the time windows are given as well as the available commands. The program features: (1) immediate feedback of acquired data; (2) optional manual editing of data; (3) simple, yet flexible commands; (4) several plot options of raw or analysed data; (5) database interface.     

An analysis of a ‘community-driven’ reconstruction of the human metabolic network

Following a strategy similar to that used in baker’s yeast (Herrgård et al. Nat Biotechnol 26:1155–1160, 2008). A consensus yeast metabolic network obtained from a community approach to systems biology (Herrgård et al. 2008; Dobson et al. BMC Syst Biol 4:145, 2010). Further developments towards a genome-scale metabolic model of yeast (Dobson et al. 2010; Heavner et al. BMC Syst Biol 6:55, 2012). Yeast 5—an expanded reconstruction of the Saccharomyces cerevisiae metabolic network (Heavner et al. 2012) and in Salmonella typhimurium (Thiele et al. BMC Syst Biol 5:8, 2011). A community effort towards a knowledge-base and mathematical model of the human pathogen Salmonella typhimurium LT2 (Thiele et al. 2011), a recent paper (Thiele et al. Nat Biotechnol 31:419–425, 2013). A community-driven global reconstruction of human metabolism (Thiele et al. 2013) described a much improved ‘community consensus’ reconstruction of the human metabolic network, called Recon 2, and the authors (that include the present ones) have made it freely available via a database at http://humanmetabolism.org/ and in SBML format at Biomodels (http://identifiers.org/biomodels.db/MODEL1109130000). This short analysis summarises the main findings, and suggests some approaches that will be able to exploit the availability of this model to advantage.     

Characterisation of a ��-butyrolactone receptor of Streptomyces tacrolimicus: effect on sporulation and tacrolimus biosynthesis

Streptomyces tacrolimicus (ATCC 55098) was reported to produce the immunosuppressant tacrolimus. The wild-type strain sporulates sparsely and produces very low levels of this immunosuppressant. The lack of genetic knowledge of this strain has hampered strain improvement. In this work, we have cloned the gene encoding a γ-butyrolactone receptor protein (Gbr). The gbr gene is linked to two genes encoding two subunits of the dihydroxyacetone kinase, putatively involved in the biosynthesis of the dihydroxyacetone phosphate precursor of γ-butyrolactone but is not flanked by γ-butyrolactone synthetase genes. The Gbr protein was overexpressed in Escherichia coli and purified. Electrophoretic mobility shift assays showed that Gbr binds to a specific autoregulatory element sequence located 338 bp upstream of the gbr gene, indicating that its expression is self-regulated. The deletion mutant Δgbr showed a very early and intense sporulation in two different media. A phenotype similar to that of the wild-type strain was restored by complementation of the Δgbr mutant with a wild-type gbr allele. Duplication of the gbr gene resulted in a slower sporulation. The Δgbr mutant produced much lower amount (32%) of tacrolimus quantified by high performance liquid chromatography. This analysis, using an optimised system, allowed the resolution of tacrolimus from ascomycin and other contaminant metabolites. Our results indicate that the Gbr protein regulates negatively the sporulation and positively the production of tacrolimus.     

Phylogeny and taxonomy of the ‘manna lichens’

We present ataxonomic revision of the ‘manna lichens’ based on morphological, chemical, ecological and molecular data. A large number of herbarium specimens and fresh collections were examined. Phylogenetic analyses were performed using nuclear ribosomal (nrITS, nrLSU) and mitochondrial small subunit (mtSSU) sequences. Some notable phenotypic characters were plotted on the phylogenetic tree, and the analysis reveals that some of these characters are useful for genus and species level distinction of certain ‘manna lichens.’ Phylogeny of the Megasporaceae was revised using a combined data set of nrLSU and mtSSU and performing parsimony and Bayesian analyses. Five genera (Aspicilia, Circinaria, Lobothallia, Megaspora and Sagedia) are recognized. Further, the relationships of five presumably closely related genera of ‘manna lichens’, namely Agrestia (vagrant), Aspicilia (crustose) Circinaria (crustose), Chlorangium (vagrant) and Sphaerothallia (vagrant) with different growth forms were analysed. The analyses revealed that ‘manna lichens’ do not form a monophyletic group but occur in different clades within the genus Circinaria. The genera Agrestia, Chlorangium and Sphaerothallia are assigned as new synonyms under the genus Circinaria and no vagrant or erratic species remain in the genus Aspicilia. The analyses also show that five new erratic, vagrant and crustose species can be recognized. In this study two ‘manna lichens’, viz. Circinaria rostamii sp. nov. (Azerbaijan, Iran and Turkey), and Circinaria gyrosa sp. nov. (Armenia, Azerbaijan, Iran, Turkey, Turkmenistan and Spain) are described as new to science. Three potentially new species with crustose and erratic forms need additional study. Aspicilia fruticolosofoliacea is reduced to synonymy under C. alpicola, and a lectotype is designated for C. aspera. Thirteen new combinations in Circinaria are presented. The phenomenon of vagrancy is briefly discussed, and the biogeography of the ‘manna lichens’ is outlined. Illustrations, distribution maps, and an identification key to the species are provided.     

‘Gender’ and the Concerns of Carnal Self-Consumption

This article analyses the co-occurrence of two concerns often expressed by indigenous leaders involved with reparations for victims of paramilitaries in Colombia. These concerns revolve around discomfort with the intense use of hegemonic ‘gender’ discourses in the context of the reparations, and a feeling that leaders' involvement in the latter brings about a betrayal of the family and of themselves. By engaging with the local category of eirruku (flesh), I argue that ways of engaging women in reparations entail the commoditisation of caring relationships. This article, based on 13 months of fieldwork with an indigenous organisation and the communities integrated into it between 2007 and 2008, contributes to the understanding of the articulation of gender and ethnic/racial identification in the context of contemporary forms of governance of indigenous peoples, while delving into ‘carnality’ as a concept to understand gendered forms of reproduction, reparation and selfhood.     

The ‘fluid mosaic model’ and the ‘lattice model’ as two nonequilibrium states of the same membrane and the significance for biochemical control

Summary Biochemical control involves steep and hysteretic response. But the law of mass action does not allow for cooperativity. Therefore resort is classically made to concerted conformational change of protomers. This explanation of steepness and hysteresis by cooperativity is supported by regular surface patterns often observed by electron microscopy. But at other times the lattice appearance which gave rise to the lattice model is not observed. By contrast, a random appearance is observed and the fluid mosaic model of the membrane is assumed. So we are faced with the choice between the fluid mosaic model and the lattice model. Recently the fluid mosaic model is favoured but unlike the lattice model it does not explain the steep hyteretic response.It is suggested that the lattice model and the fluid mosaic model are in fact expression of two states of the very same membrane. The random state corresponds to a resting state. The lattice state corresponds to an active or inhibited state. Thus the transition from random distribution to hexagonal distribution provides simultaneously for triggering and hysteretic cycle with respect to both chemical production and transport across the membrane. This is a universal mechanism for rapid responsiveness and cyclic activity which is largely independent of the chemical mechanism assumed. It is based on the law of mass action supplemented by lateral diffusion. Conformational change and cooperativity are not invoked at all.     

‘Placebos’ and the logic of placebo comparison

Robin Nunn has argued that we should stop using the terms ‘placebo’ and ‘placebo effect’. I argue in support of Nunn’s position by considering the logic of why we perform placebo comparisons. Like all comparisons, placebo comparison is just a case of comparing one thing with another, but it is a mistake, I argue, to think of placebo comparison as a case where something is compared to ‘a placebo’. Rather, placebo comparison should be understood as a situation which sets-up the treatment and control groups in a particular way; not as a case involving objects or procedures called ‘placebos’ employed in order to control for ‘placebo effects’.     

Making sense of ‘mixture’: states and the classification of ‘mixed’ people

Caribbean diaspora intellectuals have contributed significantly to African-American political struggles over the years, despite arguments by some African-American intellectuals about what they discern as West Indian negativism, extremism, and divisiveness in American political life. This essay examines the legacy of a representative sample of diasporic Caribbean intellectuals since the time of the Harlem Renaissance in the 1920s and culminating in the 1970s, notably, Garvey, members of the African Blood Brotherhood [ABB], C.L.R. James, and Walter Rodney, to demonstrate the phenomenal ideological vision, organizational capability, and political activism they have brought into fostering solidarity with African Americans in the struggle for fundamental change. The focus here is on their respective contributions to the debate on the relationship between class and race in America and the black diaspora generally, which has helped to shape the form, and orient the direction of African-American and Caribbean diasporic struggles for the better part of the twentieth century.