Gastrointestinal pH measurement in rats: influence of the microbial flora, diet and fasting

The pH of the rat intestinal tract was decreased by the presence of a microbial flora, but its influence in the forestomach is less clear. Stomach pH values varied according to the amount of food present at the time of measurement. Fasting increased the pH of the gastrointestinal tract in conventional rats but had little effect in germfree rats. In the conventional rat, feeding a purified diet compared with a commercial diet resulted in a lower pH in the forestomach and a higher pH in the caecal contents. Magnesium trisilicate promoted gastric emptying in conventional rats and its antacid effect was observed only in the caecum and colon.     

Method of sampling chorionic villi in first trimester of pregnancy under guidance of real time ultrasound.

Samples of chorionic villi were obtained in the first trimester by aspiration using a cannula passed transcervically under the guidance of real time ultrasound. In initial studies in 47 anaesthetised patients immediately before therapeutic abortion a method was developed giving a success rate of 89%. In 10 patients successful sampling was performed as an outpatient procedure without anaesthesia. In all, seven diagnostic procedures were undertaken and four of the five unaffected pregnancies continued. The technique of chorionic villous sampling using real time ultrasound is simple to learn and yields material for biochemical analysis and chromosomal study without the need for tissue culture. The exact obstetric risk, however, remains to be defined.     

Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

The self determinants recognized by human virus-immune T cells can be distinguished from the serologically defined HLA antigens

The self specificity of human influenza virus-immune cytotoxic T cells has been analyzed in order to clarify the relationship between the self antigens that they recognize and the serologically defined HLA-A and -B antigens. Virus-immune effectors from HLA-A2-positive donors were tested on panels of virus-infected target cells from donors who were either HLA-mismatched or matched only for HLA-A2. Virus-immune T cells from 11 out of 11 A2-positive donors lysed all A2-matched virus-infected target cells (and no HLA-mismatched targets), except that each of these effector cells consistently failed to lyse virus-infected target cells from one A2-positive donor (designated M7). Although the A2 specificity of donor M7 could also be distinguished from the A2 antigen of other donors by alloimmune cytotoxic T cells, no differences in the A2 antigen of donor M7 could be defined by extensive serologic analyses. These results indicate that there is a strong but incomplete association between a self antigen recognized by virus-immune T cells and the serologically defined HLA-A2 specificity.     

‘Mild mitochondrial uncoupling’ induced protection against neuronal excitotoxicity requires AMPK activity

The preconditioning response conferred by a mild uncoupling of the mitochondrial membrane potential (Δψ_m) has been attributed to altered reactive oxygen species (ROS) production and mitochondrial Ca^2 + uptake within the cells. Here we have explored if altered cellular energetics in response to a mild mitochondrial uncoupling stimulus may also contribute to the protection. The addition of 100 nM FCCP for 30 min to cerebellar granule neurons (CGNs) induced a transient depolarization of the Δψ_m, that was sufficient to significantly reduce CGN vulnerability to the excitotoxic stimulus, glutamate. On investigation, the mild mitochondrial ‘uncoupling’ stimulus resulted in a significant increase in the plasma membrane levels of the glucose transporter isoform 3, with a hyperpolarisation of Δψ_m and increased cellular ATP levels also evident following the washout of FCCP. Furthermore, the phosphorylation state of AMP-activated protein kinase (AMPK) (Thr 172) was increased within 5 min of the uncoupling stimulus and elevated up to 1 h after washout. Significantly, the physiological changes and protection evident after the mild uncoupling stimulus were lost in CGNs when AMPK activity was inhibited. This study identifies an additional mechanism through which protection is mediated upon mild mitochondrial uncoupling: it implicates increased AMPK signalling and an adaptive shift in energy metabolism as mediators of the preconditioning response associated with FCCP-induced mild mitochondrial uncoupling.     

The enzyme that cleaves apolipoprotein A-II upon in vitro incubation of human plasma high-density lipoprotein-3 with blood polymorphonuclear cells is an elastase

The proteolytic activity directed against apolipoprotein A-II (apo-A-II) which is released from human blood polymorphonuclear cells (PMN) when they are incubated with human plasma high-density lipoprotein-3 (HDL3) was studied to assess the properties and site specificity of the enzyme. When 125I-apo-A-II-labeled HDL3 was incubated with the PMN protease at 37 degrees C, a complete cleavage of apo-A-II was observed which paralleled the formation of bands of approximately 11,000 and 7,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 7,000-dalton component had the following N-terminal sequence: NH2-Thr-Asp-Tyr-Gly-Lys-Asp-Leu-Met-Glu-Lys. This corresponds to residues 19 through 28 of the intact apo-A-II monomer. Methoxysuccinyl (MeO-Suc)-Ala-Ala-Pro-Val-chloromethylketone-(CH2Cl) caused a 90% inhibition of apo-A-II hydrolysis at the highest concentration tested (6 X 10(-4)M). Besides apo-A-II, the PMN enzyme also hydrolyzed a synthetic substrate, MeO-Suc-Ala-Ala-Pro-Val-4-nitroanilide and its 4-methylcoumaryl-7-amide analogue. The protease appeared to have a mass of 28,000 daltons as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]diisopropylfluorophosphate-labeled PMN enzyme. That the PMN enzyme which cleaves apo-A-II is an elastase was derived from the following criteria: 1) cleavage at the Val-X bond in apo-A-II and in the two synthetic substrates studied; 2) prevention of the cleavage by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a known specific elastase inhibitor; and 3) a mass comparable to that reported for a pure PMN elastase. These studies establish that apolipoproteins can be suitable substrates for enzymes of the elastase family.     

Analysis and visualization of H7 influenza using genomic,evolutionary and geographic information in a modular web service

We have reported previously on use of a web‐based application, Supramap ( http://supramap.org ) for the study of biogeographic, genotypic, and phenotypic evolution. Using Supramap we have developed maps of the spread of drug‐resistant influenza and host shifts in H1N1 and H5N1 influenza and coronaviruses such as SARS. Here we report on another zoonotic pathogen, H7 influenza, and provide an update on the implementation of Supramap as a web service. We find that the emergence of pathogenic strains of H7 is labile with many transitions from high to low pathogenicity, and from low to high pathogenicity. We use Supramap to put these events in a temporal and geospatial context. We identify several lineages of H7 influenza with biomarkers of high pathogenicity in regions that have not been reported in the scientific literature. The original implementation of Supramap was built with tightly coupled client and server software. Now we have decoupled the components to provide a modular web service for POY ( http://poyws.org ) that can be consumed by a data provider to create a novel application. To demonstrate the web service, we have produced an application, Geogenes ( http://geogenes.org ). Unlike in Supramap, in which the user is required to create and upload data files, in Geogenes the user works from a graphical interface to query an underlying dataset. Geogenes demonstrates how the web service can provide underlying processing for any sequence and metadata database. © The Willi Hennig Society 2012.