Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis.

Based on crystal structure analysis of the Serratia nuclease and a sequence alignment of six related nucleases, conserved amino acid residues that are located in proximity to the previously identified catalytic site residue His89 were selected for a mutagenesis study. Five out of 12 amino acid residues analyzed turned out to be of particular importance for the catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127. Their replacement by alanine, for example, resulted in mutant proteins of very low activity, < 1% of the activity of the wild-type enzyme. Steady-state kinetic analysis of the mutant proteins demonstrates that some of these mutants are predominantly affected in their kcat, others in their Km. These results and the determination of the pH and metal ion dependence of selected mutant proteins were used for a tentative assignment for the function of these amino acid residues in the mechanism of phosphodiester bond cleavage by the Serratia nuclease.     

The curious case of Hermodice carunculata (Annelida: Amphinomidae): evidence for genetic homogeneity throughout the Atlantic Ocean and adjacent basins

Over the last few decades, advances in molecular techniques have led to the detection of strong geographic population structure and cryptic speciation in many benthic marine taxa, even those with long‐lived pelagic larval stages. Polychaete annelids, in particular, generally show a high degree of population divergence, especially in mitochondrial genes. Rarely have molecular studies confirmed the presence of ‘cosmopolitan’ species. The amphinomid polychaete Hermodice carunculata was long considered the sole species within its genus, with a reported distribution throughout the Atlantic and adjacent basins. However, recent studies have indicated morphological differences, primarily in the number of branchial filaments, between the East and West Atlantic populations; these differences were invoked to re‐instate Hermodice nigrolineata, formerly considered a junior synonym of H. carunculata. We utilized sequence data from two mitochondrial (cytochrome c oxidase subunit I, 16S rDNA) markers and one nuclear (internal transcribed spacer) marker to examine the genetic diversity of Hermodice throughout its distribution range in the Atlantic Ocean, including the Mediterranean Sea, the Caribbean Sea, the Gulf of Mexico and the Gulf of Guinea. Our analyses revealed generally low genetic divergences among collecting localities and between the East and West Atlantic, although phylogenetic trees based on mitochondrial data indicate the presence of a private lineage in the Mediterranean Sea. A re‐evaluation of the number of branchial filaments confirmed differences between East and West Atlantic populations; however, the differences were not diagnostic and did not reflect the observed genetic population structure. Rather, we suspect that the number of branchial filaments is a function of oxygen saturation in the environment. Our results do not support the distinction between H. carunculata in the West Atlantic and H. nigrolineata in the East Atlantic. Instead, they re‐affirm the older notion that H. carunculata is a cohesive species with a broad distribution across the Atlantic Ocean.     

Microbial Communities Associated with Zones of Elevated Magnetic Susceptibility in Hydrocarbon-Contaminated Sediments

Recent studies suggest that magnetic susceptibility (MS) measurements can play an important role in identifying zones where microbial-mediated iron mineral transformations are occurring. Here we investigated the microbial community variations within zones of elevated MS in a petroleum hydrocarbon-contaminated aquifer near Bemidji, Minnesota, USA. Our main objective was to 1) identify the key microbial populations that may play a role in hydrocarbon degradation, 2) analyze which microbial populations could be connected to the elevated MS and 3) explore the use of non-destructive geophysical techniques as a tool to guide microbial sampling. Clone libraries based on the 16S rRNA gene revealed the presence of iron-reducing β-Proteobacteria in the vadose zone, whereas the free petroleum phase on the water table was characterized by a methanogenic consortium, in which the syntrophic δ-proteobacterium Smithella and the hydrogenotrophic Methanoregula predominated. Nonmetric multidimensional scaling (NMDS) found a close relationship between elevated MS values and the methanogenic hydrocarbon-degrading consortium. Our results suggest that magnetic susceptibility measurements can guide microbiologists to zones of active microbial biodegradation in aged petroleum spills.     

Targeted resequencing reveals geographical patterns of differentiation for loci implicated in parallel evolution

Parallel divergence and speciation provide evidence for the role of divergent selection in generating biological diversity. Recent studies indicate that parallel phenotypic divergence may not have the same genetic basis in different geographical locations – ‘outlier loci’ (loci potentially affected by divergent selection) are often not shared among parallel instances of phenotypic divergence. However, limited sharing may be due, in part, to technical issues if false‐positive outliers occur. Here, we test this idea in the marine snail Littorina saxatilis, which has evolved two partly isolated ecotypes (adapted to crab predation vs. wave action) in multiple locations independently. We argue that if the low extent of sharing observed in earlier studies in this system is due to sampling effects, we expect outliers not to show elevated F_ST when sequenced in new samples from the original locations and also not to follow predictable geographical patterns of elevated F_ST. Following a hierarchical sampling design (within vs. between country), we applied capture sequencing, targeting outliers from earlier studies and control loci. We found that outliers again showed elevated levels of F_ST in their original location, suggesting they were not generated by sampling effects. Outliers were also likely to show increased F_ST in geographically close locations, which may be explained by higher levels of gene flow or shared ancestral genetic variation compared with more distant locations. However, in contrast to earlier findings, we also found some outlier types to show elevated F_ST in geographically distant locations. We discuss possible explanations for this unexpected result.     

Shedding light on host niches: label‐free in situ detection of Mycobacterium gordonae via carotenoids in macrophages by Raman microspectroscopy

Macrophages are the primary habitat of pathogenic mycobacteria during infections. Current research about the host–pathogen interaction on the cellular level is still going on. The present study proves the potential of Raman microspectroscopy as a label‐free and non‐invasive method to investigate intracellular mycobacteria in situ. Therefore, macrophages were infected with Mycobacterium gordonae, a mycobacterium known to cause inflammation linked to intracellular survival in macrophages. Here, we show that Raman maps provided spatial and spectral information about the position of bacteria within determined cell margins of macrophages in two‐dimensional scans and in three‐dimensional image stacks. Simultaneously, the relative intracellular concentration and distributions of cellular constituents such as DNA, proteins and lipids provided phenotypic information about the infected macrophages. Locations of bacteria outside or close to the outer membrane of the macrophages were notably different in their spectral pattern compared with intracellular once. Furthermore, accumulations of bacteria inside of macrophages exhibit distinct spectral/molecular information because of the chemical composition of the intracellular microenvironment. The data show that the connection of microscopically and chemically gained information provided by Raman microspectroscopy offers a new analytical way to detect and to characterize the mycobacterial infection of macrophages.     

16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys, Antarctica

The prokaryotic diversity of aerobic and anaerobic bacterial isolates and of bacterial and archaeal 16S rDNA clones was determined for a microbial mat sample from the moated region of Lake Fryxell, McMurdo Dry Valleys, Antarctica. Among the anaerobic bacteria, members of Clostridium estertheticum and some other psychrotolerant strains dominated whereas methanogens and other Archaea were lacking. Isolates highly related to Flavobacterium hibernum, Janthiniobacterium lividum, and Arthrobacter flavus were among the aerobic bacteria most frequently isolated. Assessment of more than 350 partial 16S rDNA clone sequences of libraries generated by Bacteria- and Archaea-specific PCR primers revealed a rich spectrum of bacterial diversity but only two different archaeal clone sequences. Among the Bacteria, representative sequences belonged to the class Proteobacteria, order Verrucomicrobiales, class Actinobacteria, Clostridium/Bacillus subphylum of Gram-positives, and the Cytophaga-Flavobacterium-Bacteroides phylum. The clones formed about 70 higher taxonomy groups (<98% sequence similarity) and 133 potential species, i.e., groups of clones sharing greater than 98% similarity. Only rarely were clone sequences found to be highly related to Lake Fryxell isolates and to strains of described species. Subsequent analysis of ten sequencing batches of 36 individual clones indicated that the diversity might be still higher than had been assessed.     

The role of cytosolic free calcium in the generation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate in HL-60 cells. Differential effects of chemotactic peptide receptor stimulation at distinct Ca2+ levels

The generation of the two inositol trisphosphate (IP3) isomers, 1,4,5-IP3 and 1,3,4-IP3, and its relation to changes in the cytosolic free calcium concentration, [Ca2+]i, in response to the chemotactic peptide fMet-Leu-Phe was studied in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Stimulation by fMet-Leu-Phe within seconds transiently elevates 1,4,5-IP3 to peak values averaging 8-fold basal levels, and leads to a concomitant rise in [Ca2+]i and to degranulation. These responses are followed by a slower and more sustained rise in 1,3,4-IP3. Alterations in [Ca2+]i modulate differentially the generation of the two IP3 isomers. At [Ca2+]i lower than 30 nM, no IP3 is generated upon fMet-Leu-Phe stimulation. Working at normal resting [Ca2+]i, but preventing the fMet-Leu-Phe induced transient rise in [Ca2+]i (by prior depletion of intracellular Ca2+ stores and working in calcium-free medium) the fMet-Leu-Phe stimulation of 1,3,4-IP3 levels is attenuated, whereas the response of 1,4,5-IP3 is not significantly altered. Maintained elevation of [Ca2+]i to micromolar levels with the Ca2+ ionophore ionomycin generates enhanced 1,3,4-IP3 levels in the absence of fMet-Leu-Phe, whereas the fMet-Leu-Phe stimulation of 1,4,5-IP3 generation is markedly inhibited. Pertussis toxin selectively abolishes the fMet-Leu-Phe-induced IP3 production, whereas ionomycin stimulation of 1,3,4-IP3 generation is unaffected. These findings indicate that in intact cells: receptor-triggered phosphatidylinositol bisphosphate phosphodiesterase activation has a minimal Ca2+ requirement, but does not depend on a previous or concomitant rise in [Ca2+]i; Ca2+ elevations above micromolar levels decrease the fMet-Leu-Phe-induced generation of 1,4,5-IP3; and 1,3,4-IP3 generation is not directly linked to receptor activation and appears to result both from increased [Ca2+]i and 1,4,5-IP3 levels.     

Characterization of hereditary inclusion body myopathy myoblasts: possible primary impairment of apoptotic events

Hereditary inclusion body myopathy (HIBM) is a unique muscular disorder caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. GNE encodes a bi-functional enzyme acting in the biosynthetic pathway of sialic acid. Since the underlying myopathological mechanism leading to the disease phenotype is poorly understood, we have established human myoblasts cultures, derived from HIBM satellite cells carrying the homozygous M712T mutation, and identified cellular and molecular characteristics of these cells. HIBM and control myoblasts showed similar heterogeneous patterns of proliferation and differentiation. Upon apoptosis induction, phosphatidylserine externalization was similar in HIBM and controls. In contrast, the active forms of caspase-3 and -9 were strongly enhanced in most HIBM cultures compared to controls, while pAkt, downregulated in controls, remained high in HIBM cells. These results could indicate impaired apoptotic signaling in HIBM cells. Since satellite cells enable partial regeneration of the post-mitotic muscle tissue, these altered processes could contribute to the muscle mass loss seen in patients. The identification of survival defects in HIBM affected muscle cells could disclose new functions for GNE in muscle cells.     

Two novel proteins activate superoxide generation by the NADPH oxidase NOX1

NOX1, an NADPH oxidase expressed predominantly in colon epithelium, shows a high degree of similarity to the phagocyte NADPH oxidase. However, superoxide generation by NOX1 has been difficult to demonstrate. Here we show that NOX1 generates superoxide when co-expressed with the p47(phox) and p67(phox) subunits of the phagocyte NADPH oxidase but not when expressed by itself. Since p47(phox) and p67(phox) are restricted mainly to myeloid cells, we searched for their homologues and identified two novel cDNAs. The mRNAs of both homologues were found predominantly in colon epithelium. Differences between the homologues and the phagocyte NADPH oxidase subunits included the lack of the autoinhibitory domain and the protein kinase C phosphorylation sites in the p47(phox) homologue as well as the absence of the first Src homology 3 domain and the presence of a hydrophobic stretch in the p67(phox) homologue. Co-expression of NOX1 with the two novel proteins led to stimulus-independent high level superoxide generation. Stimulus dependence of NOX1 was restored when p47(phox) was used to replace its homologue. In conclusion, NOX1 is a superoxide-generating enzyme that is activated by two novel proteins, which we propose to name NOXO1 (NOX organizer 1) and NOXA1 (NOX activator 1).     

Oligo-riboprobes

Summary In situ hybridisation detection of mRNAs using riboprobes has become a widely used technique. However, the identification of cells producing closely-related yet distinct mRNAs is difficult with the usual size probes. More-over, it is not always easy to obtain the required cDNA essential for cRNA probe synthesis. To avoid these problems, we have used synthetic oligodeoxynucleotides to generate short, single stranded RNA probes (oligo-riboprobes). These probes can be labelled to very high (10⁹ cpm/g) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from preprotachykinin cDNA.