Systematic variation of amino acid substitutions for stringent assessment of pairwise covariation

During protein evolution, amino acids change due to a combination of functional constraints and genetic drift. Proteins frequently contain pairs of amino acids that appear to change together (covariation). Analysis of covariation from naturally occurring sets of orthologs cannot distinguish between residue pairs retained by functional requirements of the protein and those pairs existing due to changes along a common evolutionary path. Here, we have separated the two types of covariation by independently recombining every naturally occurring amino acid variant within a set of 15 subtilisin orthologs. Our analysis shows that in this family of subtilisin orthologs, almost all possible pairwise combinations of amino acids can coexist. This suggests that amino acid covariation found in the subtilisin orthologs is almost entirely due to common ancestral origin of the changes rather than functional constraints. We conclude that naturally occurring sequence diversity can be used to identify positions that can vary independently without destroying protein function.     

Reactivation of formyl peptide receptors triggers the neutrophil NADPH-oxidase but not a transient rise in intracellular calcium

In neutrophils, coupling of chemoattractants to their cell surface receptor at low temperature (相似文献   

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Antisense DNA target sites can be selected by the accessibility of the mRNA target. It remains unknown whether a mRNA site that is accessible to an antisense DNA is also a good candidate target site for a siRNA. Here, we reported a parallel analysis of 12 pairs of antisense DNAs and siRNA duplexes for their potency to inhibit reporter luciferase activity in mammalian cells, both of the antisense DNA and siRNA agents in a pair being directed to same site in the mRNA. Five siRNAs and two antisense DNAs turned out to be effective, but the sites targeted by those effective siRNAs and antisense DNAs did not overlap. Our results indicated that effective antisense DNAs and siRNAs have different preferences for target sites in the mRNA.     

Evidence for a second gene for primary microcephaly at MCPH5 on chromosome 1

Primary microcephaly has been mapped to five loci on different chromosomes. We present here the fine mapping of one of the loci, MCPH5, to a region of only 0.58 Mb located at the 1q31.3-1q32.1 junction. A genome scan was performed on five families from the Netherlands and Jordania, with 14 patients affected by microcephaly. A maximum LOD score of 4.78 was found for marker D1S1660 at the MCPH5 locus. Haplotype analysis suggests that the gene causing microcephaly is located between markers D1S3469 and D1S1660, which excludes the previously reported ASPM gene.     

Trisomy 4q syndrome: presentation of a new case and review of the literature

We describe the 11th case of a de novo partial trisomy of the long arm of chromosome 4, with the extra segment spanning from 4q27 to 4q35. The aberration resulted from an unbalanced translocation of material from 4q to the short arm of chromosome 7, as evident from fluorescent in situ hybridization. Microsatellite analysis revealed the extra material to originate from the father. The karyotype was interpreted as 46,XX,der(7)t(4;7)(q27;p22). The patient is a 13-year-old girl with severe mental retardation, growth retardation, hearing impairment as well as minor foot, thumb and facial anomalies. Although the extent of the aberration varies between the reported patients, there are nevertheless features in common, suggestive of a trisomy 4q syndrome. The clinical findings most frequently reported are: mental retardation, seizures, microcephaly, hearing impairment and growth retardation, as well as epicanthic folds, high/broad/depressed nasal bridge, malformed ears, tooth and thumb anomalies. Almost the entire long arm of chromosome 4, except band q11, has been involved in trisomies/duplications, but 4q27 and 4q31 seem to be preferentially engaged in the trisomy 4q syndrome.     

Activation of the neutrophil nicotinamide adenine dinucleotide phosphate oxidase by galectin-1

Galectins are a group of lactose-binding proteins widely distributed in nature. Twelve mammalian galectins have so far been identified, but their functions are to a large extent unknown. In this work we study galectin-1 in its interaction with human neutrophils, with regard to both cell surface binding and activation of the superoxide-producing NADPH-oxidase. We show that galectin-1 is able to activate the neutrophil NADPH-oxidase, provided that the cells have been primed by extravasation from the blood into the tissue, an activation pattern that is similar to that of galectin-3. Using in vitro priming protocols, the galectin-1 responsiveness was found to correlate to granule mobilization and galectin-1 binding to the cells, suggesting the presence of granule-localized receptors that are up-regulated to the cell surface upon priming. By galectin-1 overlay of fractionated neutrophils we identified potential galectin-1 receptor candidates localized in the membranes of the secretory vesicle and gelatinase granules. The binding of galectin-1 and galectin-3 to neutrophil proteins was compared, as were the dose dependencies for activation by the two lectins. The results suggest that, although similarities are found between the two galectins, they appear to activate the NADPH-oxidase using different receptors. In conclusion, galectin-1 appears to have proinflammatory functions, mediated through activation of the neutrophil respiratory burst.     

Outer membrane protein A deficient Escherichia coli activates neutrophils to produce superoxide and shows increased susceptibility to antibacterial peptides

The outer membrane protein A (OmpA) of Gram-negative bacteria has been ascribed multiple functions including maintenance of structural membrane integrity and porin activity. OmpA has also been implicated in various host defense processes in that it contributes to bacterial serum resistance and activates certain immune cells. Recently, OmpA was shown to be the molecular target for neutrophil elastase (NE), and Escherichia coli mutants lacking OmpA were resistant to the bactericidal effects of NE. In addition to NE, neutrophils use a variety of other antibacterial effector molecules such as oxygen radicals and bactericidal peptides or proteins. The aim of this study was to investigate the role of E. coli OmpA regarding susceptibility to other neutrophil-derived defense systems. We found that OmpA-deficient (OmpA(-)), but not wild-type isogenic, E. coli activated human neutrophils to produce oxygen radicals intracellularly. This activation was found to require an intact neutrophil cytoskeleton but was independent of bacterial phagocytosis. Furthermore, we found that the OmpA(-) strain was more susceptible to membrane-acting bactericidal peptides than the wild-type strain, although the susceptibility to different oxygen radicals was independent of the presence of OmpA. Taken together, these data suggest an important role for OmpA in the context of bacteria vs. host interactions.     

Clinical relevance of matrix metalloproteinase-13 determined with a new highly specific and sensitive ELISA in ascitic fluid of advanced ovarian carcinoma patients

Matrix metalloproteinases (MMPs) are involved in many physiological and pathophysiological processes, including tumor cell invasion and metastasis. For one member of this family, MMP-13 (collagenase-3), a new, highly specific ELISA with a sensitivity of 0.5 ng MMP-13/ml was established. The protein levels of MMP-13 in ascitic fluids of 30 patients with advanced ovarian cancer FIGO stage III (n = 19) and IV (n = 11) were measured with this ELISA. Using a cut-off value of 0.5 ng MMP-13/mg total protein, two patient subpopulations with short (median 16 months) and long (median 36 months) overall survival were identified. Together with other prognostic markers, determination of MMP-13 in ascitic fluid may help to identify patients at risk for early death and help to individualize adjuvant therapy.     

Specific regulation of lipocalin-type prostaglandin D synthase in mouse heart by estrogen receptor beta

Estrogens have important physiological roles in the cardiovascular system. We use DNA microarray technology to study the molecular mechanism of estrogen action in the heart and to identify novel estrogen-regulated genes. In this investigation we identify genes that are regulated by chronic estrogen treatment of mouse heart. We present our detailed characterization of one of these genes, lipocalin-type prostaglandin D synthase (L-PGDS). Northern and Western blot analysis revealed that L-PGDS was induced both by acute and chronic estrogen treatment. Northern blot analysis, using estrogen receptor (ER)-disrupted mice, suggests that L-PGDS is specifically induced by ERbeta in vivo. In further support of ERbeta-selective regulation, we identify a functional estrogen-responsive element in the L-PGDS promoter, the activity of which is up-regulated by ERbeta, but not by ERalpha. We demonstrate that a one-nucleotide change (A to C) in the L-PGDS estrogen-responsive element affects receptor selectivity.