Cloning and expression of the coding regions of the heat shock proteins HSP10 and HSP16 from Piscirickettsia salmonis

The genes encoding the heat shock proteins HSP10 and HSP16 of the salmon pathogen Piscirickettsia salmonis have been isolated and sequenced. The HSP10 coding sequence is located in an open reading frame of 291 base pairs encoding 96 aminoacids. The HSP16 coding region was isolated as a 471 base pair fragment encoding a protein of 156 aminoacids. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from other prokaryotic organisms. Both proteins were expressed in E. coli as fusion proteins with thioredoxin and purified by chromatography on Nicolumn. A rabbit serum against P. salmonis total proteins reacts with the recombinant HSP10 and HSP16 proteins. Similar reactivity was determined by ELISA using serum from salmon infected with P. salmonis. The possibility of formulating a vaccine containing these two proteins is discussed.     

GH values after clonidine stimulation measured by immunofluorometric assay in normal prepubertal children and GH-deficient patients

OBJECTIVE: To establish the cut-off values of GH measured by immunofluorometric assay, a more sensitive and specific assay, in normal prepubertal children and compare their values with those of proven GH-deficient patients. METHODS: 30 normal children (20 males) and 26 patients with known causes of GH deficiency were submitted to the clonidine test and their GH values were compared. A powdered clonidine tablet (0.1 mg/m(2)) was given orally and blood samples for GH measurements were drawn at times -30, 0, 60, 90 and 120 min. RESULTS: GH peak values presented a wide variation ranging from 1.7 to 25 micro g/l (mean +/- SD = 12.87 +/- 5.8 micro g/l) in the normal group. The cut-off values for the 5th and 10th percentiles of the distribution curve were 3.3 and 5.5 micro g/l, respectively. In the GH deficiency group, maximum GH levels after clonidine stimulation ranged from <0.1 to 2.1 micro g/l (0.56 +/- 0.58 micro g/l). CONCLUSIONS: The cut-off values obtained with the immunofluorometric method are lower than the ones obtained by radioimmunoassay. We suggest a cut-off value of 3.3 micro g/l (5th percentile) that ensures 100% of sensitivity along with 93% of specificity to exclude the diagnosis of GH deficiency when using this immunofluorometric method.     

Cathepsin L participates in the production of neuropeptide Y in secretory vesicles, demonstrated by protease gene knockout and expression

Neuropeptide Y (NPY) functions as a peptide neurotransmitter and as a neuroendocrine hormone. The active NPY peptide is generated in secretory vesicles by proteolytic processing of proNPY. Novel findings from this study show that cathepsin L participates as a key proteolytic enzyme for NPY production in secretory vesicles. Notably, NPY levels in cathepsin L knockout (KO) mice were substantially reduced in brain and adrenal medulla by 80% and 90%, respectively. Participation of cathepsin L in producing NPY predicts their colocalization in secretory vesicles, a primary site of NPY production. Indeed, cathepsin L was colocalized with NPY in brain cortical neurons and in chromaffin cells of adrenal medulla, demonstrated by immunofluorescence confocal microscopy. Immunoelectron microscopy confirmed the localization of cathepsin L with NPY in regulated secretory vesicles of chromaffin cells. Functional studies showed that coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY. Furthermore , in vitro processing indicated cathepsin L processing of proNPY at paired basic residues. These findings demonstrate a role for cathepsin L in the production of NPY from its proNPY precursor. These studies illustrate the novel biological role of cathepsin L in the production of NPY, a peptide neurotransmitter, and neuroendocrine hormone.     

Cleavage of the carboxyl tail from the G3 domain of aggrecan but not versican and identification of the amino acids involved in the degradation

Aggrecan, a major structural proteoglycan in cartilage, contains three globular domains, G1, G2, and G3, as well as sequences for glycosaminoglycan modification. A large number of proteases are implicated in aggrecan cleavage in normal metabolism, aging, and arthritis. These proteases are known to cleave at the IGD, KS, and CS domains. Here we report for the first time evidence of cleavage at a novel site, the carboxyl tail of aggrecan. Results from deletion mutants of the tail indicated that the likely cleavage sites were two consensus sequences, RRLXK and RSPR, present in the aggrecan analogs of many species. This was confirmed by site-directed mutagenesis. A construct containing two G3 domains (G3G3) was also found to cleave between the G3 duplicates. When G3 tail was linked to a glycosaminoglycan-modifying sequence, it was protected from cleavage. Furin inhibitor also reduced the levels of tail cleavage. The carboxyl tails of chicken and human versican were not cleaved, despite the presence of the consensus sequence. Our studies indicate that the basic amino acids present in the tail play an important role in cleavage, and this mechanism is specific to aggrecan.     

Bacterial community structure within an activated sludge reactor added with phenolic compounds

Biodegradation of phenolic compounds in bioreactors is well documented, but the changes in the bacterial populations dynamics during degradation were not that often. A glass bubble column used as reactor was inoculated with activated sludge, spiked with 2-chlorophenol, phenol and m-cresol after 28 days and maintained for an additional 56 days, while the 16S rRNA gene from metagenomic DNA was monitored. Proteobacteria (68.1%) dominated the inoculum, but the bacterial composition changed rapidly. The relative abundance of Bacteroidetes and Firmicutes decreased from 4.8 and 9.4 to <0.1 and 0.2% respectively, while that of Actinobacteria and TM7 increased from 4.8 and 2.0 to 19.2 and 16.1% respectively. Phenol application increased the relative abundance of Proteobacteria to 94.2% (mostly Brevundimonas 17.6%), while that of Bacteroidetes remained low (1.2%) until day 42. It then increased to 47.3% (mostly Leadbetterella 46.9%) at day 84. It was found that addition of phenolic compounds did not affect the relative abundance of the Alphaproteobacteria initially, but it decreased slowly while that of the Bacteroidetes increased towards the end.

     

Hyperadrenocorticism of calorie restriction contributes to its anti‐inflammatory action in mice

Calorie restriction (CR), which lengthens lifespan in many species, is associated with moderate hyperadrenocorticism and attenuated inflammation. Given the anti‐inflammatory action of glucocorticoids, we tested the hypothesis that the hyperadrenocorticism of CR contributes to its attenuated inflammatory response. We used a corticotropin‐releasing‐hormone knockout (CRHKO) mouse, which is glucocorticoid insufficient. There were four controls groups: CRHKO mice and wild‐type (WT) littermates fed either ad libitum (AL) or CR (60% of AL food intake), and three experimental groups: (a) AL‐fed CRHKO mice given corticosterone (CORT) in their drinking water titrated to match the integrated 24‐hr plasma CORT levels of AL‐fed WT mice, (b) CR‐fed CRHKO mice given CORT to match the 24‐hr CORT levels of AL‐fed WT mice, and (c) CR‐fed CHRKO mice given CORT to match the 24‐hr CORT levels of CR‐fed WT mice. Inflammation was measured volumetrically as footpad edema induced by carrageenan injection. As previously observed, CR attenuated footpad edema in WT mice. This attenuation was significantly blocked in CORT‐deficient CR‐fed CRHKO mice. Replacement of CORT in CR‐fed CRHKO mice to the elevated levels observed in CR‐fed WT mice, but not to the levels observed in AL‐fed WT mice, restored the anti‐inflammatory effect of CR. These results indicate that the hyperadrenocorticism of CR contributes to the anti‐inflammatory action of CR, which may in turn contribute to its life‐extending actions.     

Screening for a Potent Antibacterial Peptide to Treat Mupirocin-Resistant MRSA Skin Infections

Mupirocin is the first-line topical antibacterial drug for treating skin infections caused primarily by meticillin-resistant Staphylococcus aureus (MRSA). Its widespread use since its introduction more than 30 years ago has resulted in the global emergence of mupirocin-resistant strains of MRSA. Antimicrobial peptides (AMPs) are a promising class of antibacterial compounds that can potentially be developed to replace mupirocin due to their rapid membrane-targeting bactericidal mode of action and predicted low propensity for resistance development. Herein, we conducted and compared the antibacterial activities of 61 AMPs between 3 and 11 residues in length reported in the literature over the past decade against mupirocin-resistant MRSA. The most potent AMP, 11-residue peptide 50, was selected and tested against a panel of clinical isolates followed by a time-kill and a human dermal keratinocyte cytotoxicity assay. Lastly, peptide 50 was formulated into a topical spray which showed strong in vitro bactericidal effects against mupirocin-resistant MRSA. Our results strongly suggest that peptide 50 has the potential to be further developed into a new class of topical antibacterial agent for treating drug-resistant MRSA skin infections.