Evaluation of hyaluronan from different sources: Streptococcus zooepidemicus, rooster comb, bovine vitreous, and human umbilical cord

Sodium hyaluronate (HA) is widely distributed in extracellular matrixes and can play a role in orchestrating cell function. Consequently, many investigators have looked at the effect of exogenous HA on cell behavior in vitro. HA can be isolated from several sources (e.g., bacterial, rooster comb, umbilical cord) and therefore can possess diverse impurities. This current study compares the measured impurities and the differences in biological activity between HA preparations from these sources. It was demonstrated that nucleic acid and protein content was highest in human umbilical cord and bovine vitreous HA and was low in bacterial and rooster comb HA. Macrophages exposed to human umbilical cord HA produced significantly higher amounts of TNF-alpha relative to control or bacterial-derived HA. These results indicate that the source of HA should be considered due to differences in the amounts and types of contaminants that could lead to widely different behaviors in vitro and in vivo.     

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.     

Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with KS values of approximately 8 and 4mm, respectively, in 0.1 m KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.     

Critical role of RelB serine 368 for dimerization and p100 stabilization

In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory kappaB proteins. In most other cell types inhibitory kappaB proteins prevent nuclear translocation and activation of NFkappaB. We reasoned that this characteristic might be because of post-translational modifications of RelB. In Drosophila, signal-dependent phosphorylation of the Rel homologue Dorsal at serine 317 has been shown to be critical for nuclear import. The evolutionary conservation of this serine prompted us to analyze the function of the corresponding site in RelB. As a model system we used the murine S107 plasmacytoma cell line, which lacks endogenous RelB expression. Analysis of S107 cells expressing wild type RelB and serine 368 mutants reveals that serine 368 is not required for nuclear import but that it is critical for RelB dimerization with other members of the NFkappaB family. Similar effects were obtained when the conserved serine in RelA was mutated. We further demonstrate that expression of functional RelB, but not of serine 368 mutants, severely reduces p52 generation and strongly increases expression of the p52 precursor, p100. Wild type RelB, but not mutant RelB, prolonged p100 half-life. We therefore suggest an inhibitory effect of RelB on p100 processing, which is possibly regulated in a signal-dependent manner.     

Support for association of schizophrenia with genetic variation in the 6p22.3 gene,dysbindin, in sib-pair families with linkage and in an additional sample of triad families

Genetic variants in a gene on 6p22.3, dysbindin, have been shown recently to be associated with schizophrenia (Straub et al. 2002a). There is no doubt that replication in other independent samples would enhance the significance of this finding considerably. Since the gene is located in the center of the linkage peak on chromosome 6p that we reported earlier, we decided to test six of the most positive DNA polymorphisms in a sib-pair sample and in an independently ascertained sample of triads comprising 203 families, including the families for which we detected linkage on chromosome 6p. Evidence for association was observed in the two samples separately as well as in the combined sample (P=.00068 for SNP rs760761). Multilocus haplotype analysis increased the significance further to .00002 for a two-locus haplotype and to .00001 for a three-locus haplotype. Estimation of frequencies for six-locus haplotypes revealed one common haplotype with a frequency of 73.4% in transmitted, and only 57.6% in nontransmitted, parental haplotypes. All other six-locus haplotypes occurring at a frequency of >1% were less often transmitted than nontransmitted. Our results represent a first successful replication of linkage disequilibrium in psychiatric genetics detected in a region with previous evidence of linkage and will encourage the search for causes of schizophrenia by the genetic approach.     

Hydrogen-oxidizing capabilities of Helicobacter hepaticus and in vivo availability of the substrate

Hydrogen-oxidizing hydrogenase activity was detected in Helicobacter hepaticus and compared to the activity in Helicobacter pylori for characteristics associated with hydrogen uptake respiratory hydrogenases. Intact whole cells could couple H(2) oxidation to oxygen uptake, and no H(2) uptake was observed without oxygen available to complete the respiratory pathway. The H. hepaticus enzyme coupled H(2) oxidation to reduction of many positive potential acceptors, and it underwent anaerobic or reductive activation. H. hepaticus had a strong affinity for molecular H(2) (apparent K(m) of 2.5 micro M), and microelectrode measurements on the livers of live mice demonstrated that H(2) is available in the host tissue at levels 20-fold greater than the apparent whole-cell K(m) value.     

LcrV is a channel size-determining component of the Yop effector translocon of Yersinia

Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact-dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127-195 of LcrV and to amino acids 106-173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size-determining structural component of the Yop translocon.     

Inhibition of natural killer cells results in acceptance of cardiac allografts in CD28-/- mice

Successful transplantation of allogeneic organs is an important objective in modern medicine. However, sophisticated immune defense mechanisms, primarily evolved to combat infections, often work against medical transplantation. To investigate the roles of natural and adaptive immune responses in transplant rejection, we functionally inactivated key effector systems of the innate (NK cells) and the adaptive immune system (CD28-mediated costimulation of T cells) in mice. Neither of these interventions alone led to acceptance of allogeneic vascularized cardiac grafts. In contrast, inhibition of NK-receptor-bearing cells combined with CD28-costimulation blockade established long-term graft acceptance. These results indicate a concerted interplay between innate and adaptive immune surveillance for graft rejection. Thus we suggest that inactivation of NK-receptor-bearing cells could be a new strategy for successful survival of solid-organ transplants.