Structure of the exceptionally large nonrepetitive carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E-82164.

The structures of the oligosaccharides obtained after acetic acid hydrolysis and alkaline deacylation of the rough-type lipopolysaccharide (LPS) from Pectinatus frisingensis strain VTT E-82164 were analysed using NMR spectroscopy, MS and chemical methods. The LPS contains two major structural variants, differing by a decasaccharide fragment, and some minor variants lacking the terminal glucose residue. The largest structure of the carbohydrate backbone of the LPS that could be deduced from experimental results consists of 25 monosaccharides (including the previously found Ara4NP residue in lipid A) arranged in a well-defined nonrepetitive structure: We presume that the shorter variant with R1 = H represents the core-lipid A part of the LPS, and the additional fragment is present instead of the O-specific polysaccharide. Structures of this type have not been previously described. Analysis of the deacylation products obtained from the LPS of the smooth strain, VTT E-79100T, showed that it contains a very similar core but with one different glycosidic linkage.     

Altered expression and subcellular distribution of GRK subtypes in the dopamine-depleted rat basal ganglia is not normalized by l-DOPA treatment

Dysregulation of dopamine (DA) receptors is believed to underlie Parkinson's disease pathology and l -DOPA-induced motor complications. DA receptors are subject to regulation by G protein-coupled receptor kinases (GRKs) and arrestins. DA lesion with 6-hydroxydopamine caused multiple protein- and brain region-specific changes in the expression of GRKs. In the globus pallidus, all four GRK isoforms (GRK2, 3, 5, 6) were reduced in the lesioned hemisphere. In the caudal caudate-putamen (cCPu) three GRK isoforms (GRK2, 3, 6) were decreased by DA depletion. The decrease in GRK proteins in globus pallidus, but not cCPu, was mirrored by reduction in mRNA. GRK3 protein was reduced in the rostral caudate-putamen (rCPu), whereas other isoforms were either unchanged or up-regulated. GRK6 protein and mRNA were up-regulated in rCPu and nucleus accumbens. l -DOPA (25 mg/kg, twice daily for 10 days) failed to reverse changes caused by DA depletion, whereas D₂/D₃ agonist pergolide (0.25 mg/kg daily for 10 days) restored normal levels of expression of GRK5 and 6. In rCPu, GRK2 protein was increased in most subcellular fractions by l -DOPA but not by DA depletion alone. Similarly, l -DOPA up-regulated arrestin3 in membrane fractions in both regions. GRK5 was down-regulated by l -DOPA in cCPu in the light membrane fraction, where this isoform is the most abundant. The data suggest that alterations in the expression and subcellular distribution of arrestins and GRKs contribute to pathophysiology of Parkinson's disease. Thus, these proteins may be targets for antiparkinsonian therapy.     

lfnA from Pseudomonas aeruginosa O12 and wbuX from Escherichia coli O145 encode membrane-associated proteins and are required for expression of 2,6-dideoxy-2-acetamidino-L-galactose in lipopolysaccharide O antigen

The rare sugar 2,6-dideoxy-2-acetamidino-L-galactose (L-FucNAm) is found only in bacteria and is a component of cell surface glycans in a number of pathogenic species, including the O antigens of Pseudomonas aeruginosa serotype O12 and Escherichia coli O145. P. aeruginosa is an important opportunistic pathogen, and the O12 serotype is associated with multidrug-resistant epidemic outbreaks. O145 is one of the classic non-O157 serotypes associated with Shiga toxin-producing, enterohemorrhagic E. coli. The acetamidino (NAm) moiety of L-FucNAm is of interest, because at neutral pH it contributes a positive charge to the cell surface, and we aimed to characterize the biosynthesis of this functional group. The pathway is not known, but expression of NAm-modified sugars coincides with the presence of a pseA homologue in the relevant biosynthetic locus. PseA is a putative amidotransferase required for synthesis of a NAm-modified sugar in Campylobacter jejuni. In P. aeruginosa O12 and E. coli O145, the pseA homologues are lfnA and wbuX, respectively, and we hypothesized that these genes function in L-FucNAm biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and nuclear magnetic resonance analysis of the lfnA mutant O-antigen structure indicated that the mutant expresses 2,6-dideoxy-2-acetamido-L-galactose (L-FucNAc) in place of L-FucNAm. The mutation could be complemented by expression of either His(6)-tagged lfnA or wbuX in trans, confirming that these genes are functional homologues and that they are required for NAm moiety synthesis. Both proteins retained their activity when fused to a His(6) tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.     

The elucidation of the structure of the core part of the LPS from Plesiomonas shigelloides serotype O17 expressing O-polysaccharide chain identical to the Shigella sonnei O-chain

Plesiomonas shigelloides O17 LPS contains the same O-antigenic polysaccharide chain as a causative agent of dysentery, Shigella sonnei. This polysaccharide can be used as a component of a vaccine against dysentery. Core part of the P. shigelloides O17 LPS was studied using NMR and mass spectrometry and the following structure was proposed: Significant similarity of the P. shigelloides O17 LPS core with the structure of the P. shigelloides O54 core was observed.     

The structure of the carbohydrate backbone of the LPS from Shewanella spp. MR-4

The rough type lipopolysaccharide isolated from Shewanella spp. strain MR-4 was analyzed using NMR, mass spectroscopy, and chemical methods. Two structural variants have been found, both contained 8-amino-3,8-dideoxy-d-manno-octulosonic acid and lacked l-glycero-d-manno-heptose. A minor variant of the LPS contained phosphoramide substituent.     

Characteristics of Resonance in Heart Rate Variability Stimulated by Biofeedback

As we previously reported, resonant frequency heart rate variability biofeedback increases baroreflex gain and peak expiratory flow in healthy individuals and has positive effects in treatment of asthma patients. Biofeedback readily produces large oscillations in heart rate, blood pressure, vascular tone, and pulse amplitude via paced breathing at the specific natural resonant frequency of the cardiovascular system for each individual. This paper describes how resonance properties of the cardiovascular system mediate the effects of heart rate variability biofeedback. There is evidence that resonant oscillations can train autonomic reflexes to provide therapeutic effect. The paper is based on studies described in previous papers. Here, we discuss the origin of the resonance phenomenon, describe our procedure for determining an individual's resonant frequency, and report data from 32 adult asthma patients and 24 healthy adult subjects, showing a negative relationship between resonant frequency and height, and a lower resonant frequency in men than women, but no relationship between resonant frequency and age, weight, or presence of asthma. Resonant frequency remains constant across 10 sessions of biofeedback training. It appears to be related to blood volume.     

Organization of core spliceosomal components U5 snRNA loop I and U4/U6 Di-snRNP within U4/U6.U5 Tri-snRNP as revealed by electron cryomicroscopy

In eukaryotes, pre-mRNA exons are interrupted by large noncoding introns. Alternative selection of exons and nucleotide-exact removal of introns are performed by the spliceosome, a highly dynamic macromolecular machine. U4/U6.U5 tri-snRNP is the largest and most conserved building block of the spliceosome. By 3D electron cryomicroscopy and labeling, the exon-aligning U5 snRNA loop I is localized at the center of the tetrahedrally shaped tri-snRNP reconstructed to approximately 2.1 nm resolution in vitrified ice. Independent 3D reconstructions of its subunits, U4/U6 and U5 snRNPs, show how U4/U6 and U5 combine to form tri-snRNP and, together with labeling experiments, indicate a close proximity of the spliceosomal core components U5 snRNA loop I and U4/U6 at the center of tri-snRNP. We suggest that this central tri-snRNP region may be the site to which the prespliceosomal U2 snRNA has to approach closely during formation of the catalytic core of the spliceosome.     

Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant <Emphasis Type="Italic">E. coli</Emphasis> strains with predesigned genome structure

Background  

The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on in vitro and in vivo recombination processes have been developed.