Structural studies of an acidic polysaccharide from the mucin secreted by Drosera capensis

The polysaccharide of the mucin secreted by the leaves of Drosera capensis is composed of l-arabinose, d-xylose, d-galactose, d-mannose, and d-glucuronic acid in the molar ratio of 3.6:1.0:4.9:8.4:8.2. For structural elucidation, methylation analysis using g.l.c. and g.l.c.-m.s. was performed on the native, the carboxyl-reduced, and the degraded polysaccharides. Partial hydrolysis, periodate oxidation, chromium trioxide oxidation, and uronic acid degradation were also performed on the native and carboxyl-reduced polysaccharides. Partial hydrolysis of the native and carboxyl-reduced polysaccharides gave various oligosaccharides that were characterized and suggest a structure containing a d-glucurono-d-mannan backbone having a repeating unit → 4)-β-d-GlcpA-(1 → 2)-α-d-Manp-(1 →. l-Arabinose and d-xylose are present as nonreducing furanosyl and pyranosyl end-groups, respectively, both attached to O-3 of d-glucuronic acid residues of the backbone. d-Galactose is present as non-reducing pyranosyl end-group linked to O-3 of d-mannose residues.     

Aldehyde dehydrogenase (ALDH) isozymes in the gray short-tailed opossum (Monodelphis domestica): Tissue and subcellular distribution and biochemical genetics of ALDH3

Polyacrylamide gel isoelectric focusing (PAGE-IEF), cellulose acetate electrophoresis, and histochemical techniques were used to examine the tissue and subcellular distribution, genetics and biochemical properties of aldehyde dehydrogenase (ALDH) isozymes in a didelphid marsupial, the gray short-tail opossum (Monodelphis domestica). At least 14 zones of activity were resolved by PAGE-IEF and divided into five isozyme groups and three ALDH classes, based upon comparisons with properties previously reported for human, baboon, rat, and mouse ALDHs. Opossum liver ALDHs were distributed among cytosol (ALDHs 1 and 5) and large granular (mitochondrial) fractions (ALDHs 2 and 5). Similarly, kidney ALDHs were distributed between the cytosol (ALDH5) and the mitochondrial fractions (ALDHs 2, 4, and 5), whereas a major isozyme (ALDH3), found in high activity in cornea, esophagus, ear pinna, tail, and stomach extracts, was localized predominantly in the cytosol fraction. Phenotypic variants of the latter enzyme were shown to be inherited in a normal Mendelian fashion, with two alleles at a single locus (ALDH3) showing codominant expression. The data provided evidence for genetic identity of corneal, ear pinna, tail, and stomach ALDH3 and supported biochemical evidence from other mammalian species that this enzyme has a dimeric subunit structure.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

Wavelength-dependent effects of evening light exposure on sleep architecture and sleep EEG power density in men

Light strongly influences the circadian timing system in humans via non-image-forming photoreceptors in the retinal ganglion cells. Their spectral sensitivity is highest in the short-wavelength range of the visible light spectrum as demonstrated by melatonin suppression, circadian phase shifting, acute physiological responses, and subjective alertness. We tested the impact of short wavelength light (460 nm) on sleep EEG power spectra and sleep architecture. We hypothesized that its acute action on sleep is similar in magnitude to reported effects for polychromatic light at higher intensities and stronger than longer wavelength light (550 nm). The sleep EEGs of eight young men were analyzed after 2-h evening exposure to blue (460 nm) and green (550 nm) light of equal photon densities (2.8 x 10(13) photons x cm(-2) x s(-1)) and to dark (0 lux) under constant posture conditions. The time course of EEG slow-wave activity (SWA; 0.75-4.5 Hz) across sleep cycles after blue light at 460 nm was changed such that SWA was slightly reduced in the first and significantly increased during the third sleep cycle in parietal and occipital brain regions. Moreover, blue light significantly shortened rapid eye movement (REM) sleep duration during these two sleep cycles. Thus the light effects on the dynamics of SWA and REM sleep durations were blue shifted relative to the three-cone visual photopic system probably mediated by the circadian, non-image-forming visual system. Our results can be interpreted in terms of an induction of a circadian phase delay and/or repercussions of a stronger alerting effect after blue light, persisting into the sleep episode.     

Thermostable RNase P RNAs lacking P18 identified in the Aquificales

The RNase P RNA (rnpB) and protein (rnpA) genes were identified in the two Aquificales Sulfurihydrogenibium azorense and Persephonella marina. In contrast, neither of the two genes has been found in the sequenced genome of their close relative, Aquifex aeolicus. As in most bacteria, the rnpA genes of S. azorense and P. marina are preceded by the rpmH gene coding for ribosomal protein L34. This genetic region, including several genes up- and downstream of rpmH, is uniquely conserved among all three Aquificales strains, except that rnpA is missing in A. aeolicus. The RNase P RNAs (P RNAs) of S. azorense and P. marina are active catalysts that can be activated by heterologous bacterial P proteins at low salt. Although the two P RNAs lack helix P18 and thus one of the three major interdomain tertiary contacts, they are more thermostable than Escherichia coli P RNA and require higher temperatures for proper folding. Related to their thermostability, both RNAs include a subset of structural idiosyncrasies in their S domains, which were recently demonstrated to determine the folding properties of the thermostable S domain of Thermus thermophilus P RNA. Unlike 16S rRNA phylogeny that has placed the Aquificales as the deepest lineage of the bacterial phylogenetic tree, RNase P RNA-based phylogeny groups S. azorense and P. marina with the green sulfur, cyanobacterial, and delta/epsilon proteobacterial branches.     

Inflammatory cytokines induce protein tyrosine nitration in rat astrocytes

Protein tyrosine nitration may be relevant for the pathogenesis of hepatic encephalopathy (HE). Infections, sepsis, and trauma precipitate HE episodes. Recently, serum levels of tumor necrosis factor (TNF)-alpha were shown to correlate with severity of HE in chronic liver failure. Here the effects of inflammatory cytokines on protein tyrosine nitration in cultured rat astrocytes and rat brain in vivo were studied. In cultured rat astrocytes TNF-alpha (50 pg/ml-10 ng/ml) within 6h increased protein tyrosine nitration. TNF-alpha-induced tyrosine nitration was related to an increased formation of reactive oxygen and nitrogen intermediates, which was downstream from a NMDA-receptor-dependent increase of intracellular [Ca(2+)](i) and nNOS-catalyzed NO production. Astroglial tyrosine nitration was also elevated in brains of rats receiving a non-lethal injection of lipopolysaccharide, as indicated by colocalization of nitrotyrosine immunoreactivity with glial fibrillary acidic protein and glutamine synthetase, and by identification of the glutamine synthetase among the tyrosine-nitrated proteins. It is concluded that reactive oxygen and nitrogen intermediates as well as protein tyrosine nitration by inflammatory cytokines may alter astrocyte function in an NMDA-receptor-, Ca(2+)-, and NOS-dependent fashion. This may be relevant for the pathogenesis of HE and other conditions involving cytokine exposure the brain.     

Continuous Enzymatic Regeneration of Electron Acceptors Used by Flavoenzymes: Cellobiose Dehydrogenase-Catalyzed Production of Lactobionic Acid as an Example

An efficient enzymatic bioprocess is described in which lactose, an abundant renewable resource produced by the dairy industry, is completely and efficiently converted with a specific productivity of up to 32 g (kU h)^-1 into lactobionic acid, without the formation of any by-products. The key biocatalyst of this new process is the fungal enzyme cellobiose dehydrogenase which oxidizes several β-1,4-linked disaccharides including lactose specifically at position C-1 of the reducing sugar moiety to the corresponding lactones. The electron acceptor employed in this reaction is continuously regenerated with the help of laccase, a H₂O-producing, copper-containing oxidase, and therefore has to be added in low, catalytic amounts only. Redox mediators that were successfully employed in this novel process and hence are compatible with the laccase regeneration system include benzoquinone, ABTS, ferricyanide, or ferrocene, amongst others. Factors affecting operational stability of the biocatalysts employed in this process include the redox mediator used, the temperature, and importantly the volumetric gas flow necessary for maintaining the dissolved oxygen tension. Lactobionic acid is a mild and sweet tasting acid with excellent chelating properties. These useful characteristics have lead to a growing number of patents for diverse applications in the food, pharmaceutical and detergent industries.     

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.