Microbial communities colonising nutrient-enriched marine sediment

Sediment cores were set up to study microbial colonisation and interactions on marine sand grains under enrichment conditions. Cores were enriched with photosynthetic media in the light and dark (PL, PD) and heterotrophic media in the light and dark (HL, HD), and were incubated for 25 days. Sediment chlorophylls were then measured by acetone extraction, viable heterotrophic bacteria by plate counts, and numbers of cells mm^–2 sand grain surface by s.e.m. Chlorophyll a occurred in all sediments but was highest in the PL sediment. Bacteriochlorophyll a was only observed in the HL sediment. Heterotrophic viable counts were high in the HL and HD sediments. Dense growth of diatoms and blue-green algae, and a marine fungal Thraustochytrid sp. occurred on PL grains. The blue-green alga Schizothrix was often associated with the diatom Amphora on PL grains. Many different bacteria grew on HL and HD grains and some unusual colony and cell morphologies were recorded (Caulobacter, Flexibacter, polymer strands). Characteristic flakey material sometimes occurred in hollows on grains. The results are discussed in relation to microbial communities in low energy sedimentary environments.     

Membrane receptors for estrogen,progesterone, and testosterone in the rat brain: Fantasy or reality

Summary 1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes.2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17-E-6-[¹²⁵I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimatedK _d of about 3±0.7 nM (X ± SE;n=3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with aK _d of 34±7 nM and, aB _max of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (K _d=1–2 nM) and one low affinity (K _d=62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[¹²⁵I]BSA indicate that the estimatedK _d of 30±2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement withK _d values computed from Scatchard-derived data using the LIGAND algorithm.3. 17-E-6-[¹²⁵I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[¹²⁵I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22.4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase.5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the reality of membrane steroid receptors.     

Calcitonin- and somatostatin-positive cells in thyroid gland of pigs at different ages

Immunohistochemical studies were carried out on calcitonin-, somatostatin- and serotonin-reactive cells in newborn pigs and pigs at 3 weeks and 7 months old. The aim of these studies was to examine if the expression of various bioactive substances by parafollicular cells in the pig thyroid varied during development. The volume density of the follicular epithelium was nearly the same in newborn and 3-week-old piglets and significantly lower in 7-month-old animals. The volume density of calciton-in-positive cells, expressed as a percentage of the follicular epithelium density, was similar in young animals, being 12.10% and 13.03% in newborn and 3-week-old piglets, respectively. A small but significant increase to 14.40% was seen in 7-month-old pigs. Somatostatin-positive cells formed a much smaller population at all time points, but these also showed a significant increase with age (0.13%, 0.17% and 0.52% of follicular epithelium density in newborn, 3-week- and 7-month-old pigs, respectively). However the changes in the volume density of somatostatin-positive cells correlated inversely with thyroid activity, the density being highest when the activation index was lowest, suggesting that thyroid activity may be regulated by an increase in the synthesis of this inhibitory peptide. Serotonin-positive cells were extremely rare at all time points and their volume density was not calculated.     

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

Preparation and structural characterization of large heparin-derived oligosaccharides

Porcine mucosal heparin was partially depolymerized with heparinlyase I and then fractionated into low-molecularweight (<5000)and high-molecular-weight (>5000) oligosaccharides by pressurefiltration. The high-molecular-weight oligosaccharide mixture({small tilde}50 wt% of the starting heparin) also containedintact heparin. This intact polymer complicates oligosacsharidepurification. Thus, the low-molecular-weight fraction was usedto prepare homogeneous oligosaccharides for structural characterization.The low-molecular-weight oligosaccharide mixture was first fractionatedby low pressure gel permeation chromatography into size-uniformmixtures of disaccharides, tetrasaccharides, hexasaccharides,octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharidesand higher oligosaccharides. Each size-fractionated mixturewas then purified on the basis of charge by repetitive semi-preparativestrong-anion-exchange high-performance liquid chromatography.This approach has led to the isolation of 14 homogeneous oligosaccharidesfrom disaccharide to tetradecasaccharide. The purity of theseheparin-derived oligosaccharides was determined by gradientpolyacrylamide gel electrophoresis, analytical strong-anion-exchangehigh-performance liquid chromatography, capillary electrophoresisand one-dimensional nuclear resonance spectroscopy. The structureof these oligosaccharides was established using 600 MHz two-dimensionalnuclear resonance spectroscopy . The spectral methods used includedhomonuclear correlation spectroscopy, nuclear Overhauser effectspectroscopy and heteronuclear multiple quantum coherence spech-clscopy.The ¹H/¹H connectivities of the protons of each sugar residuein an oligosaccharide were established by two-dimensional homonuclearcorrelation spectroscopy, while ¹H/¹³C assignments were madeusing ¹H inverse detection. One- and two-dimensional nuclearresonance spectroscopic analysis of these heparin oligosaccharidesshowed two closely related groups of heparin-oligosaccharidesare afforded by enzymatic depolymerization of heparin. One groupis fully sulphated, having the structures     

Variation in root morphology,enhancement in anti-oxidative enzyme responses and improved arbutin and bergenin levels in Bergenia ciliata (Haw.) Sternb. raised in vitro via EMS and gamma irradiations

Plant Cell, Tissue and Organ Culture (PCTOC) - Plant cells develop defence mechanisms in response to mutagen stress which leads to modulation of certain metabolic and defensive pathways. Owing to...     

Indinavir and nelfinavir inhibit proximal insulin receptor signaling and salicylate abrogates inhibition: Potential role of the NFkappa B pathway

The molecular basis of insulin resistance induced by HIV protease inhibitors (HPIs) remains unclear. In this study, Chinese hamster ovary cells transfected with high levels of human insulin receptor (CHO‐IR) and 3T3‐L1 adipocytes were used to elucidate the mechanism of this side effect. Indinavir and nelfinavir induced a significant decrease in tyrosine phosphorylation of the insulin receptor β‐subunit. Indinavir caused a significant increase in the phosphorylation of insulin receptor substrate‐1 (IRS‐1) on serine 307 (S307) in both CHO‐IR cells and 3T3‐L1 adipocytes. Nelfinavir also inhibited phosphorylation of Map/ERK kinase without affecting insulin‐stimulated Akt phosphorylation. Concomitantly, levels of protein tyrosine phosphatase 1B (PTP1B), suppressor of cytokines signaling‐1 and ‐3 (SOCS‐1 and ‐3), Src homology 2B (SH2B) and adapter protein with a pleckstrin homology domain and an SH2 domain (APS) were not altered significantly. When CHO‐IR cells were pre‐treated with sodium salicylate (NaSal), the effects of indinavir on tyrosine phosphorylation of the IR β‐subunit and phosphorylation of IRS‐1 at S307 were abrogated. These data suggest a potential role for the NFκB pathway in insulin resistance induced by HPIs. J. Cell. Biochem. 114: 1729–1737, 2013. © 2013 Wiley Periodicals, Inc.     

Neutralizing Nanobodies Targeting Diverse Chemokines Effectively Inhibit Chemokine Function

Chemokine receptors and their ligands play a prominent role in immune regulation but many have also been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, allograft rejection after transplantation, and also in cancer metastasis. Most approaches to therapeutically target the chemokine system involve targeting of chemokine receptors with low molecular weight antagonists. Here we describe the selection and characterization of an unprecedented large and diverse panel of neutralizing Nanobodies (single domain camelid antibodies fragment) directed against several chemokines. We show that the Nanobodies directed against CCL2 (MCP-1), CCL5 (RANTES), CXCL11 (I-TAC), and CXCL12 (SDF-1α) bind the chemokines with high affinity (at nanomolar concentration), thereby blocking receptor binding, inhibiting chemokine-induced receptor activation as well as chemotaxis. Together, we show that neutralizing Nanobodies can be selected efficiently for effective and specific therapeutic treatment against a wide range of immune and inflammatory diseases.     

THE IMPORTANCE OF MOSQUITO BEHAVIOURAL ADAPTATIONS TO MALARIA CONTROL IN AFRICA

Over the past decade the use of long‐lasting insecticidal nets (LLINs), in combination with improved drug therapies, indoor residual spraying (IRS), and better health infrastructure, has helped reduce malaria in many African countries for the first time in a generation. However, insecticide resistance in the vector is an evolving threat to these gains. We review emerging and historical data on behavioral resistance in response to LLINs and IRS. Overall the current literature suggests behavioral and species changes may be emerging, but the data are sparse and, at times unconvincing. However, preliminary modeling has demonstrated that behavioral resistance could have significant impacts on the effectiveness of malaria control. We propose seven recommendations to improve understanding of resistance in malaria vectors. Determining the public health impact of physiological and behavioral insecticide resistance is an urgent priority if we are to maintain the significant gains made in reducing malaria morbidity and mortality.     

A Lightweight Radio Propagation Model for Vehicular Communication in Road Tunnels

Radio propagation models (RPMs) are generally employed in Vehicular Ad Hoc Networks (VANETs) to predict path loss in multiple operating environments (e.g. modern road infrastructure such as flyovers, underpasses and road tunnels). For example, different RPMs have been developed to predict propagation behaviour in road tunnels. However, most existing RPMs for road tunnels are computationally complex and are based on field measurements in frequency band not suitable for VANET deployment. Furthermore, in tunnel applications, consequences of moving radio obstacles, such as large buses and delivery trucks, are generally not considered in existing RPMs. This paper proposes a computationally inexpensive RPM with minimal set of parameters to predict path loss in an acceptable range for road tunnels. The proposed RPM utilizes geometric properties of the tunnel, such as height and width along with the distance between sender and receiver, to predict the path loss. The proposed RPM also considers the additional attenuation caused by the moving radio obstacles in road tunnels, while requiring a negligible overhead in terms of computational complexity. To demonstrate the utility of our proposed RPM, we conduct a comparative summary and evaluate its performance. Specifically, an extensive data gathering campaign is carried out in order to evaluate the proposed RPM. The field measurements use the 5 GHz frequency band, which is suitable for vehicular communication. The results demonstrate that a close match exists between the predicted values and measured values of path loss. In particular, an average accuracy of 94% is found with R² = 0.86.