Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.     

Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon

We describe here a molecular genetic approach for imaging synaptic inhibition. The thy-1 promoter was used to express high levels of Clomeleon, a ratiometric fluorescent indicator for chloride ions, in discrete populations of neurons in the brains of transgenic mice. Clomeleon was functional after chronic expression and provided non-invasive readouts of intracellular chloride concentration ([Cl(-)](i)) in brain slices, allowing us to quantify age-dependent declines in resting [Cl(-)](i) during neuronal development. Activation of hippocampal interneurons caused [Cl(-)](i) to rise transiently in individual postsynaptic pyramidal neurons. [Cl(-)](i) increased in direct proportion to the amount of inhibitory transmission, with peak changes as large as 4 mM. Integrating responses over populations of pyramidal neurons allowed sensitive detection of synaptic inhibition. Thus, Clomeleon imaging permits non-invasive, spatiotemporally resolved recordings of [Cl(-)](i) in a large variety of neurons, opening up new opportunities for imaging synaptic inhibition and other forms of chloride signaling.     

Genome Sequence of Lactobacillus crispatus ST1

Lactobacillus crispatus is a common member of the beneficial microbiota present in the vertebrate gastrointestinal and human genitourinary tracts. Here, we report the genome sequence of L. crispatus ST1, a chicken isolate displaying strong adherence to vaginal epithelial cells.Lactobacillus crispatus can persist in the vertebrate gastrointestinal tract and is among the most prevalent species of the Lactobacillus-dominated human vaginal microbiota (2, 9, 13, 14). It belongs to the so-called acidophilus group (3), which has attracted interest because some of its species are important factors in the production of fermented foods (12) and some can, at least transiently, colonize the human host (2, 9, 13, 14). Moreover, some specific strains, mainly L. acidophilus NCFM and L. johnsonii NCC 533, have received prominence as intestinal-health-promoting microbes (4). Although the genomes of seven members of the acidophilus complex have been sequenced to date (12), the genome sequences of L. crispatus and other predominant lactobacillar species in the urogenital flora have mostly remained obscure. Vaginal lactobacilli can have an important role in controlling the health of the host (2, 14). They can, for example, positively influence and stabilize the host''s vaginal microbiota via the production of compounds that are acidic or exert a direct inhibiting action toward pathogenic bacteria (2, 14). In addition to the antimicrobial compounds, the competitive exclusion of pathogens is another mechanism by which the host''s microbiota can be balanced (2). L. crispatus ST1 was originally isolated from the crop of a chicken, and PCR profiling of L. crispatus isolates has verified it to be an abundant colonizer of the chicken crop (6, 8). It also displays a strong protein-dependent adhesion to the epithelial cells of the human vagina and has been shown to inhibit the adhesion of avian pathogenic Escherichia coli (6, 7).The genome was sequenced (18× coverage) using a 454 pyrosequencer with GS FLX chemistry (Roche). The contig order was confirmed and gaps were filled by sequencing PCR fragments from the genomic DNA template using ABI 3730 and Big Dye chemistry (Applied Biosystems). Genomic data were processed using the Staden Package (11) and gsAssembler (Roche). Coding sequences (CDSs) were predicted using Glimmer3 (5) followed by manual curation of the start sites. The remaining intergenic regions were reanalyzed for missed CDSs by using BlastX (1). Annotation transfer was performed based on a BlastP search, followed by Blannotator analysis using default settings (http://ekhidna.biocenter.helsinki.fi/poxo/blannotator) and manual verification. Orthologous groups between the different lactobacillar proteomes were identified using OrthoMCL (10).The genome of L. crispatus ST1 consists of a single circular chromosome 2.04 Mbp in size, with an overall G+C content of 37%, without any plasmids. There are 64 tRNA genes, 4 rRNA operons, and 2 CRISPR loci. Out of the 2,024 predicted CDSs, a putative function was assigned to 77%, whereas 10% of the CDSs were annotated as conserved and 13% as novel. Based on the orthologous grouping, 302 (15%) of the CDSs encoded by ST1 have no detectable homologs in any of the Lactobacillus proteomes published to date.     

Bronchoalveolar lavage examined by solid phase microextraction, gas chromatography--mass spectrometry and selected ion flow tube mass spectrometry

Samples (210 in total) of broncholaveolar lavages (BALs), obtained from patients hospitalized with pneumonia in various departments of two hospitals, were analysed using the method of solid phase microextraction-gas chromatography (SPME-GC) with FID detection. Up to 20% (9% unequivocally, 11% probably) of these samples was found to contain volatile fatty acids (VFAs) in the series from acetic acid to heptanoic acid. Importantly, the presence of these acids indicates the presence of fermenting anaerobic bacteria, which were not detected by the conventional microbiological examination. Other compounds, namely the heptanol and cyclohexanone, were also detected by this method in some samples. Cyclohexanone occurred almost exclusively in samples from patients receiving intensive care with mechanical ventilation, and is suspected to originate from plastic parts of ventilators. Selected representative samples were also analysed using further methods, namely gas chromatography-mass spectrometry (GC-MS) of native and silylated samples, and selected ion flow tube mass spectrometry (SIFT-MS). These methods confirmed the identities of above mentioned compounds, and detected numerous other compounds tentatively identified as various alcohols, aldehydes, ketones, esters and hydrogen cyanide, HCN. Most of these compounds occurred in small amounts and their origin and diagnostic significance remains uncertain, except, that is, for the HCN, which indicates the presence of Pseudomonas aeruginosa.     

Eutrophication-induced changes in benthic algae affect the behaviour and fitness of the marine amphipod Gammarus locusta

This study, conducted in mesocosms, natural field sites, and in laboratory aquaria, showed that eutrophication altered the nutrient status and dominance patterns among marine macroalgae, which in turn, stimulated gammaridean density. Gammaridean abundance correlated positively with both nutrient addition and the amount of green algae (also stimulated by nutrient enrichment). Path analysis indicated that the direct effect of nutrients on gammaridean density was of less importance than the indirect effect through increased production of green algae. In cage colonisation experiments, either in the field or in a control mesocosm kept under ambient nutrient conditions, more gammarids colonised nutrient enriched algae (E-algae) than algae with ambient nutrient levels (A-algae). Gammarus locusta generally grew faster on nutrient enriched algal specimens and when reared on green rather than on brown algae (fucoids). The nutrient status of periphytic algae did not affect gammaridean growth significantly, but the number of egg-carrying females (and thus egg production) was significantly higher among gammarids reared on E-periphyton. The gammaridean habitat preference order (red > green > brown > periphyton) was almost the reverse of their growth rate in feeding assays (periphyton > green > brown). This implies that macroalgae may be more important as a habitat than as a food source for these animals, which then have to become mobile in search of optimal food items. In this process, algal nutrient content was important as the gammarids in our study actively chose high quality nutrient-rich food, which, in addition, increased their fitness. Stimulated growth rates and egg production may ultimately lead to population increase, which, combined with the preference for high nutrient food items may dampen the initial effect of nutrient enrichment (i.e. blooms of green macroalgae) in shallow coastal waters.     

NADPH oxidase inhibition reduces tubular sodium transport and improves kidney oxygenation in diabetes

Sustained hyperglycemia is associated with increased oxidative stress resulting in decreased intrarenal oxygen tension (Po(2)) due to increased oxygen consumption (Qo(2)). Chronic blockade of the main superoxide radicals producing system, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalizes Qo(2) by isolated proximal tubular cells (PTC) and reduces proteinuria in diabetes. The aim was to investigate the effects of acute NADPH oxidase inhibition on tubular Na(+) transport and kidney Po(2) in vivo. Glomerular filtration rate (GFR), renal blood flow (RBF), filtration fraction (FF), Na(+) excretion, fractional Li(+) excretion, and intrarenal Po(2) was measured in control and streptozotocin-diabetic rats during baseline and after acute NADPH oxidase inhibition using apocynin. The effects on tubular transporters were investigated using freshly isolated PTC. GFR was increased in diabetics compared with controls (2.2 ± 0.3 vs. 1.4 ± 0.1 ml·min(-1)·kidney(-1)). RBF was similar in both groups, resulting in increased FF in diabetics. Po(2) was reduced in cortex and medulla in diabetic kidneys compared with controls (34.4 ± 0.7 vs. 42.5 ± 1.2 mmHg and 15.7 ± 1.2 vs. 25.5 ± 2.3 mmHg, respectively). Na(+) excretion was increased in diabetics compared with controls (24.0 ± 4.7 vs. 9.0 ± 2.0 μm·min(-1)·kidney(-1)). In controls, all parameters were unaffected. However, apocynin increased Na(+) excretion (+112%) and decreased fractional lithium reabsorption (-10%) in diabetics, resulting in improved cortical (+14%) and medullary (+28%) Po(2). Qo(2) was higher in PTC isolated from diabetic rats compared with control. Apocynin, dimethylamiloride, and ouabain reduced Qo(2), but the effects of combining apocynin with either dimethylamiloride or ouabain were not additive. In conclusion, NADPH oxidase inhibition reduces tubular Na(+) transport and improves intrarenal Po(2) in diabetes.     

Regulation of p97 in the ubiquitin-proteasome system by the UBX protein-family

The AAA protein p97 is a central component in the ubiquitin-proteasome system, in which it is thought to act as a molecular chaperone, guiding protein substrates to the 26S proteasome for degradation. This function is dependent on association with cofactors that are specific to the different biological pathways p97 participates in. The UBX-protein family (ubiquitin regulatory X) constitutes the largest known group of p97 cofactors. We propose that the regulation of p97 by UBX-proteins utilizes conserved structural features of this family. Firstly, they act as scaffolding subunits in p97-containing multiprotein complexes, by providing additional interaction motifs. Secondly, they provide regulation of multiprotein complex assembly and we suggest two possible models for p97 substrate recruitment in the UPS pathway. Lastly, they impose constraints on p97 and its interaction with substrates and further cofactors. These features allow the regulation, within the UPS, of the competitive interactions on p97, a regulation that is crucial to allow the diverse functionality of p97.