Biochemical investigation of multicorer sediment profile PS74/118-2

To detect and track the impact of large-scale environmental changes in a the transition zone between the northern North Atlantic and the central Arctic Ocean, and to determine experimentally the factors controlling deep-sea biodiversity, the Alfred- Wegener-Institute for Polar and Marine Research (AWI) established the deep-sea long-term observatory HAUSGARTEN, which constitutes the first, and until now only open-ocean long-term station in a polar region. Virtually undisturbed sediment samples have been taken using a video-guided multiple corer (MUC) at 13 HAUSGARTEN stations along a bathymetric (1,000 - 4,000 m water depth) and a latitudinal transect in 2,500 m water depth as well as two stations at 230 and 1,200 m water depth within the framework of the KONGHAU project. Various biogenic sediment compounds were analyzed to estimate the input of organic matter from phytodetritus sedimentation, benthic activities (e.g. bacterial exoenzymatic activity), and the total biomass of the smallest sediment-inhabiting organisms (size range: bacteria to meiofauna).

Characterization of Streptococcus bovis from the rumen of the dromedary camel and Rusa deer

Aims: Isolation and characterization of Streptococcus bovis from the dromedary camel and Rusa deer. Methods and Results: Bacteria were isolated from the rumen contents of four camels and two deer fed lucerne hay by culturing on the semi-selective medium MRS agar. Based on Gram morphology and RFLP analysis seven isolates, MPR1, MPR2, MPR3, MPR4, MPR5, RD09 and RD11 were selected and putatively identified as Streptococcus. The identity of these isolates was later confirmed by comparative DNA sequence analysis of the 16S rRNA gene with the homologous sequence from S. bovis strains, JB1, C14b1, NCFB2476, SbR1, SbR7 and Sb5, from cattle and sheep, and the Streptococcus equinus strain NCD01037T. The percentage similarity amongst all strains was >99%, confirming the identification of the camel isolates as S. bovis. The strains were further characterized by their ability to utilize a range of carbohydrates, the production of volatile fatty acids (VFA) and lactate and the determination of the doubling time in basal medium 10 supplemented with glucose. All the isolates produced L-lactate as a major fermentation end product, while four of five camel isolates produced VFA. The range of carbohydrates utilized by all the strains tested, including those from cattle and sheep were identical, except that all camel isolates and the deer isolate RD11 were additionally able to utilize arabinose. Conclusions: Streptococcus bovis was successfully isolated from the rumen of camels and deer, and shown by molecular and biochemical characterization to be almost identical to S. bovis isolates from cattle and sheep. Significance and Impact of the Study: Streptococcus bovis is considered a key lactic acid producing bacterium from the gastrointestinal tract of ruminants, and has been implicated as a causative agent of lactic acidosis. This study is the first report of the isolation and characterization of S. bovis from the dromedary camel and Rusa deer, and suggests a major contributive role of this bacterium to fermentative acidosis.

Characterization of polyamine oxidase from the aquatic nitrogen-fixing fern Azolla imbricata

Biochemical properties of a polyamine oxidase (PAO; EC 1.5.3.3) purified from the aquatic nitrogen-fixing fern Azolla imbricata (Roxb.) Nak. were studied. The native molecular mass of the enzyme estimated by Sephadex G 200 get filtration was 66.2 kDa. SDS-PAGE gave a single protein band corresponding to a molecular mass of 65.5 kDa. The light yellow enzyme had absorption maxima at 278, 372 and 454 nm with 1 mol FAD per mole enzyme molecule as its cofactor. The PAO was active on both the triamine Spd and the tetraamine Spm as substrates. However, it was inactive on the diamines Put and Cad. It had a pH optimum of 6.5 for both Spd and Spm. The K-m(S) for Spd and Spm were 6.71 x 10(-2) and 1.13 x 10(-1) nM, respectively. Pre-incubation with 10 mM of K+ (KCl), Ca2(+) (CaCl2) or Mg2+ (MgCl2) had no effect on PAO activity. However, 10 mM Cu2+ (CuCl2), Mn2+ (MnCl2) and Fe2+ (FeSO4) inhibited enzyme activity by 37%, 43% and 58%, respectively. The metal chelator EDTA (10 mM), the carbonyl reagent hydroxylamine (0.5 mM) and the sulfhydryl reagent p-chloro-mercuribenzoate (0.5 mM) had no effect on PAO activity. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Haematological values of young male rusa deer (Cervus timorensis)

Objective To measure haematological values of clinical significance for rusa deer and provide reference data for farmed animals. Design Blood samples were collected regularly from eight male rusa deer from 14 days to 27 months old. Procedure Blood samples, collected by venipuncture, were analysed within 6 hours of collection for red cell count, haemoglobin, packed cell volume, plasma glucose, white cell count and differentials. Results Haemoglobin concentrations appeared to increase with age and ranged from 6.0 to 20.9 g/dL. Packed cell volume and plasma glucose concentration did not appear to vary with age. White cell counts ranged from 6.3 to 7.0 x 10(9)/L and differential counts indicated neutrophils > lymphocytes > monocytes > eosinophils > basophils. In general, the values for packed cell volume, red cell count, mean cell volumes and mean cell haemoglobin concentrations were within ranges previously reported for captive or sedated rusa deer. Conclusions Physical restraint and resultant stress was sufficient to generate some of the effects previously reported for physically immobilised or agitated deer. The values reported here do not differ greatly from those previously reported for rusa deer and can be used as reference values for clinically healthy young farmed male rusa deer.

Characterization of a strong, constitutive mung bean (Vigna radiata L.) promoter with a complex mode of regulation in planta

We report the cloning and characterization in tobacco and Arabidopsis of a Vigna radiata L. (mung bean) promoter that controls the expression of VR-ACS1, an auxin-inducible ACC synthase gene. The VR-ACS1 promoter exhibits a very unusual behavior when studied in plants different from its original host, mung bean. GUS and luciferase in situ assays of transgenic plants containing VR-ACS1 promoter fusions show strong constitutive reporter gene expression throughout tobacco and Arabidopsis development. In vitro quantitative analyses show that transgenic plants harboring VR-ACS1 promoter-reporter constructs have on average 4-6 fold higher protein and activity levels of both reporter genes than plants transformed with comparable CaMV 35S promoter fusions. Similar transcript levels are present in VR-ACS1 and CaMV 35S promoter lines, suggesting that the high levels of gene product observed for the VR-ACS1 promoter are the combined result of transcriptional and translational activation. All tested deletion constructs retaining the core promoter region can drive strong constitutive promoter activity in transgenic plants. This is in contrast to mung bean, where expression of the native VR-ACS1 gene is almost undetectable in plants grown under normal conditions, but is rapidly and highly induced by a variety of stimuli. The constitutive behavior of the VR-ACS1 promoter in heterologous hosts is surprising, suggesting that the control mechanisms active in mung bean are impaired in tobacco and Arabidopsis. The 'aberrant' behavior of the VR-ACS1 promoter is further emphasized by its failure to respond to auxin and cycloheximide in heterologous hosts. VR-ACS1 promoter regulatory mechanisms seem to be different from all previously characterized auxin-inducible promoters.

Characterization of heat-treated porous carbons using argon adsorption

The microstructural variation of Norit RI Extra activated carbon, progressively heated at 1373 K, was explored in terms of pore size and pore wall thickness distributions, for various periods of heating time, determined by argon adsorption at 87 K, both using an infinite as well as and finite wall thickness model. The latter approach has recently been developed in our laboratory and has been applied to several virgin carbons. The current results show significant variations in small pore size regions (< 7 angstrom) in association with strong growth of thick walls having at least three carbon sheets, as a result of heat treatment. In particular, shrinkage of the smallest pores due to strong interaction between their opposite walls as well as smoothening of carbon wall surfaces due to an increase in graphitization degree under thermal treatment have been found. Further, the results of pore wall thickness distribution are well corroborated by X-ray diffraction. The results of pore size and pore wall thickness distributions are also shown to be consistent with transmission electron microscopy analyses. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular dynamics characterization of n-octyl-beta-D-glucopyranoside micelle structure in aqueous solution

n-Octyl-beta-D-glueopyranoside (OG) is a non-ionic glycolipid, which is used widely in biotechnical and biochemical applications. All-atom molecular dynamics simulations from two different initial coordinates and velocities in explicit solvent have been performed to characterize the structural behaviour of an OG aggregate at equilibrium conditions. Geometric packing properties determined from the simulations and small angle neutron scattering experiment state that OG micelles are more likely to exist in a non-spherical shape, even at the concentration range near to the critical micelle concentration (0.025 M). Despite few large deviations in the principal moment of inertia ratios, the average micelle shape calculated from both simulations is a prolate ellipsoid. The deviations at these time scales are presumably the temporary shape change of a micelle. However, the size of the micelle and the accessible surface areas were constant during the simulations with the micelle surface being rough and partially elongated. Radial distribution functions computed for the hydroxyl oxygen atoms of an OG show sharper peaks at a minimum van der Waals contact distance than the acetal oxygen, ring oxygen, and anomeric carbon atoms. This result indicates that these atoms are pointed outwards at the hydrophilic/hydrophobic interface, form hydrogen bonds with the water molecules, and thus hydrate the micelle surface effectively. (c) 2005 Elsevier Inc. All rights reserved.

Preliminary crystallographic characterization of an RNA helicase from Kunjin virus

Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C-terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3' nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid-binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti-flaviviral compounds.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Cloning and characterization of natriuretic peptides from the venom glands of Australian elapids

The venom from Australian elapid snakes contains a complex mixture of polypeptide toxins that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Included in these toxin families are the recently described venom natriuretic peptides, which display similar structure and vasoactive functions to mammalian natriuretic peptides. This paper describes the identification and detailed comparative analysis of the cDNA transcripts coding for the mature natriuretic peptide from a total of nine Australian elapid snake species. Multiple isoforms were identified in a number of species and represent the first description of a natriuretic peptide from the venom gland for most of these snakes. Two distinct natriuretic peptide isoforms were selected from the common brown snake (Pseudonaja textilis), PtNP-a, and the mulga (Pseudechis australis), PaNP-c, for recombinant protein expression and functional analysis. Only one of these peptides, PtNP-a, displayed cGMP stimulation indicative of normal natriuretic peptide activity. Interestingly, both recombinant peptides demonstrated a dose-dependent inhibition of angiotensin converting enzyme (ACE) activity, which is predictive of the vasoactive effects of the toxin. The natriuretic peptides, however, did not possess any coagulopathic activity, nor did they inhibit or potentiate thrombin, adenosine diphosphate or arachidonic acid induced platelet aggregation. The data presented in this study represent a significant resource for understanding the role of various natriuretic peptides isoforms during the envenomation process by Australian elapid snakes. (c) 2006 Published by Elsevier Masson SAS.

Preparation and characterization of mesoporous Ni/Zr-laponite for the catalytic deoxygenation of vegetable oils into liquid hydrocarbons

The deoxygenation of vegetable oils was studied over a Ni/Zr-laponite catalyst in various nickel loading composition. Stearic acid has been used as a model compounds. The liquid and gas products were analyzed using gas chromatograph equipped with thermal conductivity detector and flame ionization detector. From product distribution it was determined the effect of catalyst composition on reaction conditions

Molecular characterization of three gonad cell lines

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce Sox9 transcription upon expression of Sry, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate Sox9 expression upon transfection of Sry-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of Sox9 expression during testis determination. Copyright © 2003 S. Karger AG, Basel

Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology."

Characterization of tumour necrosis factor alpha release by human granulocytes in response to procainamide challenge

The role of human granulocytes in the promotion of procainamide (PA) toxicity in vitro has been studied and one of the agents responsible for DNA strand scission and cell death in human target cells has been characterized. Crude peripheral blood mononuclear cells (cPBMNs) isolated by density centrifugation, and the lymphocyte cell lines--CCRF-HSB2 and WIL-2NS--were exposed to PA, and DNA strand breaks were quantified by fluorescent analysis of DNA unwinding. Therapeutic plasma concentrations of PA (0-50 microM) caused dose-dependent cytotoxicity, determined by dye exclusion, and strand breaks in cPBMNs incubated for 3 and 1.5 hr at 37 degrees, respectively. Using 50 microM PA a five-fold increase in DNA strand breaks was observed after 1.5 hr, with significant induction of strand breaks also being observed for 10 and 25 microM concentrations. Toxicity was much reduced in lymphocyte cell lines (maximal killing = 3.0% at 50 microM PA compared with 13.2% in cPBMNs). A similar decrease in toxicity was observed where N-acetyl procainamide (NAPA) was substituted for PA (less than 50% of strand breaks at all concentrations). Further investigations showed that the presence of a contaminating granulocyte population in the cPBMN fraction was responsible for the induction of PA toxicity. Incubation of a highly enriched granulocyte population with PA for 1 hr prior to exposure to purified peripheral blood mononuclear cells (pPBMNs) led to the complete restoration of the toxic effects. The resulting cyto- and genotoxicity were not significantly different to levels observed in cPBMNs. Significantly, incubation of granulocytes with NAPA did not induce toxicity in target pPBMNs. Ultrafiltration of granulocyte supernatants led to the identification of two toxic fractions of < 3000 and > 30,000 Da. Temporal studies showed that the toxicity associated with the < 3000 Da fraction appeared during the first 10-15 min incubation with PA whereas the > 30,000 Da fraction did not display significant toxicity until the 40-60 min period. Further assessment of the nature of these agents indicated that the 30,000 Da fraction was a protein. SDS-PAGE analysis showed an inducible 17,800 Da species appearing in granulocyte supernatants after 40 min incubation with PA. Dot blot analysis indicated that tumour necrosis factor alpha (TNF alpha) was present in the > 30,000 Da fraction. Evidence that TNF alpha was the high-molecular weight species responsible for PA-induced toxicity was obtained from neutralization assays employing an anti-TNF alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical and physical characterization of clay bodies

Suitable methods for the assessment of the effect of freeze-thaw action upon ceramic tiles have been determined. The results obtained have been shown to be reproducible with some work in this area still warranted. The analysis of Whichford Potteries clays via a variety of analytical techniques has shown them to be a complex mix of both clay and non-clay minerals. 57Fe Mössbauer spectroscopy has highlighted the presence of both small and large particleα-Fe203, removable via acid washing. 19F MAS NMR has demonstrated that the raw Whichford Pottery clays examined have negligible fluorine content. This is unlikely to be detrimental to ceramic wares during the heating process. A unique technique was used for the identification of fluorine in solid-state systems. The exchange of various cations into Wyoming Bentonite clay by microwave methodology did not show the appearance of five co-ordinate aluminium when examined by 27Al MAS NMR. The appearance of Qo silicate was linked to an increase in the amount of tetrahedrally bound aluminium in the silicate framework. This is formed as a result of the heating process. The analysis of two Chinese clays and two Chinese clay raw materials has highlighted a possible link between the two. These have also been shown to be a mix of both clay and non-clay minerals. Layered double hydroxides formed by conventional and microwave methods exhibited interesting characteristics. The main differences between the samples examined were not found to be solely attributable to the differences between microwave and conventional methods but more attributable to different experimental conditions used.