Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity

Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [³H]phosphatidic acid ([³H]PA) and [³H]phosphatidylethanol ([³H]PEth) after labeling the muscle strips with [³H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [³H]PA and [³H]PEth in a concentration-dependent manner (>105–400% vs. controls in the presence of 10^–6 to 10^–4 M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10^–6 M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10^–8 to 10^–6 M) and GFX (10^–8 to 10^–6 M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.     

Pseudomyxoma extraperitonei occurring 35 years after appendicectomy: a case report and review of literature

Background

Pseudomyxoma peritonei is a rare condition consisting of mucinous ascites, most commonly arising from mucinous tumors of the appendix and occasionally from the ovary. Very rarely mucinous implants arise in the retroperitoneum without any intra-peritoneal involvement. This has been termed as pseudomyxoma extraperitonei.

Case presentation

We report a case of a 57 year old man who developed pseudomyxoma extraperitonei, 35 years after undergoing an appendicectomy for a perforated appendix.

Conclusions

Pseudomyxoma extraperitonei has been previously reported, however we report the longest incubation period of 35 years for this condition.

     

Histone h1(0) and its carboxyl-terminal domain bind in the major groove of DNA

The binding of histone H1(0) to T4 bacteriophage DNA was investigated using thermal denaturation of the DNA titrated with varying concentrations of protein. The H1(0) used was expressed in and purified from a strain of E. coli and is therefore homogeneous with respect to H1 subtype and posttranslational modifications. Two types of T4 DNA were used: wild-type, which contains a modification of the cytosine residues that projects into the major groove: and a mutant type, which lacks the modification of the cytosines. Data were compared to simulated thermal denaturation curves to determine estimates for binding affinity and binding site size in base pairs of the protein. Analysis of the data yielded values of 10(8) M(-1) for K, the binding affinity, and 10 base pairs for n, the number of base pairs covered by one protein, for the mutant T4 DNA. Analysis of the wild-type DNA data suggested that the glucose projecting into the major groove of this DNA decreases the number of sites to which the H1(0) protein can bind, indicating that there are interactions between the protein and the major groove of DNA. The binding site size on this DNA is 10 base pairs, the same as on the unmodified DNA. The affinity for wild-type DNA is slightly higher, 10(9) M(-1). Data were collected and analyzed for binding of two domains of the protein as well, the carboxyl-terminal domain and the central globular domain. Binding of the carboxyl-terminal domain was quantitatively and qualitatively similar to that of the full-length protein. In contrast, binding of the globular domain was quite different: it binds much more weakly, with a K of 6 x 10(4) M(-1), and covers fewer base pairs, with an n of 3. Also, there was no evidence that the globular domain interacts with the major groove of DNA.     

Multiple cysteine residues are implicated in Janus kinase 2-mediated catalysis

The redox regulation of Janus kinase 2 (JAK2) is poorly understood, and there are contradictory reports as to whether the enzyme's activity is inhibited or stimulated by oxidizing conditions in the cell. Here we demonstrate that multiple cysteine residues within the JAK2 catalytic domain may be crucial for enzymatic activity. The enzyme is catalytically inactive when oxidized; activity can be restored via reduction to the thiol state. A series of recombinant variants of JAK2 were overproduced using the baculoviral expression vector system. A truncated variant of JAK2, GST/(NDelta661)rJAK2, provided evidence that the amino-terminal autoinhibitory domain was not essential for direct redox regulation and that only nine cysteine residues were potentially involved. The effect of individually and combinatorially altering these nine cysteines was examined via cysteine-to-serine mutagenesis. This identified four cysteine residues in the catalytic domain (Cys866, Cys917, Cys1094, and Cys1105) that cooperatively maintain JAK2's catalytic competency. Our data are consistent with a direct mechanism for redox regulation of JAK2 via oxidation and reduction of critical cysteine residues.     

Support Vector Machine-based method for predicting subcellular localization of mycobacterial proteins using evolutionary information and motifs

Background

Annotations that describe the function of sequences are enormously important to researchers during laboratory investigations and when making computational inferences. However, there has been little investigation into the data quality of sequence function annotations. Here we have developed a new method of estimating the error rate of curated sequence annotations, and applied this to the Gene Ontology (GO) sequence database (GOSeqLite). This method involved artificially adding errors to sequence annotations at known rates, and used regression to model the impact on the precision of annotations based on BLAST matched sequences.

Results

We estimated the error rate of curated GO sequence annotations in the GOSeqLite database (March 2006) at between 28% and 30%. Annotations made without use of sequence similarity based methods (non-ISS) had an estimated error rate of between 13% and 18%. Annotations made with the use of sequence similarity methodology (ISS) had an estimated error rate of 49%.

Conclusion

While the overall error rate is reasonably low, it would be prudent to treat all ISS annotations with caution. Electronic annotators that use ISS annotations as the basis of predictions are likely to have higher false prediction rates, and for this reason designers of these systems should consider avoiding ISS annotations where possible. Electronic annotators that use ISS annotations to make predictions should be viewed sceptically. We recommend that curators thoroughly review ISS annotations before accepting them as valid. Overall, users of curated sequence annotations from the GO database should feel assured that they are using a comparatively high quality source of information.     

Functional significance of dinitrogen fixation in sustaining coral productivity under oligotrophic conditions

Functional traits define species by their ecological role in the ecosystem. Animals themselves are host–microbe ecosystems (holobionts), and the application of ecophysiological approaches can help to understand their functioning. In hard coral holobionts, communities of dinitrogen (N₂)-fixing prokaryotes (diazotrophs) may contribute a functional trait by providing bioavailable nitrogen (N) that could sustain coral productivity under oligotrophic conditions. This study quantified N₂ fixation by diazotrophs associated with four genera of hermatypic corals on a northern Red Sea fringing reef exposed to high seasonality. We found N₂ fixation activity to be 5- to 10-fold higher in summer, when inorganic nutrient concentrations were lowest and water temperature and light availability highest. Concurrently, coral gross primary productivity remained stable despite lower Symbiodinium densities and tissue chlorophyll a contents. In contrast, chlorophyll a content per Symbiodinium cell increased from spring to summer, suggesting that algal cells overcame limitation of N, an essential element for chlorophyll synthesis. In fact, N₂ fixation was positively correlated with coral productivity in summer, when its contribution was estimated to meet 11% of the Symbiodinium N requirements. These results provide evidence of an important functional role of diazotrophs in sustaining coral productivity when alternative external N sources are scarce.     

Complete genome sequence of Mycobacterium phlei type strain RIVM601174

Mycobacterium phlei is a rapidly growing nontuberculous Mycobacterium species that is typically nonpathogenic, with few reported cases of human disease. Here we report the whole genome sequence of M. phlei type strain RIVM601174.     

Sequence preference of mouse H1(0) and H1t.

Histone H1 proteins bind to DNA and are important in formation and maintenance of chromatin structure. Little is known about differences among variant H1 histones in their interactions with DNA. We examined the effects of histones H1(0) and H1t on thermal denaturation of several DNA species. One of the DNA molecules was a 214-base-pair fragment from the plasmid pBR322, which contains an AT-rich and a GC-rich region. Both H1(0) and H1t bound preferentially to one region of the DNA fragment, a region that is relatively GC-rich. This result indicates that histones H1(0) and H1t are not totally nonspecific but rather bind with some sequence preference to DNA. This conclusion was supported by studies of other DNA species, including two 92-base-pair fragments derived from the two regions of the 214-mer, and several synthetic homocopolymers of DNA. Data obtained with the homocopolymers suggested that the binding preference was not simple preference for GC base pairs. The binding of the two H1 variants was not identical: there appear to be differences in binding site sizes, affinities, and sequence selectivities between H1t and H1(0).     

Optimization of citric acid production by Aspergillus niger using two downgraded Algerian date varieties

In the present work, the GHARS and the MECH DEGLA downgraded date varieties were used in a fermentation medium in order to produce citric acid by the Aspergillus niger. The biochemical characteristics of the dates were investigated, along with the chemical and physical characteristics of the solutions of both samples. The analyzed parameters included the moisture and sugar content, the ash residual, the pH values, and the electrical conductivity. The effect of the following fermentation parameters was studied: initial pH, temperature, incubation period, and methanol. For the GHARS and MECH DEGLA date varieties respectively, the ash residual measured at 1.90% and 2.47%. For each date variety, the moisture and total sugars were measured at 11.59% and 85%, for the GHARS, and 12.82% and 80.47% for the MECH DEGLA. Citric acid production using either of the two varieties of dates showed a high yield in a short time.The obtained results showed that the highest production of citric acid by both medium of dates was achieved at the initial pH value of 3.0, temperature 30 °C, and an incubation period of 8 days. Also, the maximum amount of citric acid was produced when both mediums contained 4% of methanol. Both varieties of dates showed a good yield for the citric acid and can be used as a culture medium since they are economic and ensure good growth for the Aspergillus niger.     

The Population Structure of Vibrio cholerae from the Chandigarh Region of Northern India

Background

Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century.

Methodology/Principal Findings

Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII) and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST) distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA) identified 16 distinct clusters.

Conclusions/Significance

The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.