Improved Isolation of SlaA and SlaB S-layer proteins in Sulfolobus acidocaldarius.

The Sulfolobus acidocaldarius S-layer is composed of two main proteins: SlaA, which forms the ordered structure of the S-layer matrix, and SlaB, which supports and anchors the S-layer into the tetraether lipid membrane. While SlaA has previously been purified by exploiting its thermotolerance and high resistance to detergents, SlaB has resisted isolation, particularly from the cell membrane. Removal of proteins other than those of the S-layer is especially difficult if large batch-scale culture volumes are unavailable. Here, we describe a benchtop-scale protocol for the purification of SlaA from S. acidocaldarius, enabling isolation of SlaB using size exclusion chromatography (gel filtration). Using this protocol, we were able to identify for the first time tetraether lipids strongly attached to SlaB via heat- and detergent-resistant interactions.

Novel Mechanism for Surface Layer Shedding and Regenerating in Bacteria Exposed to Metal-Contaminated Conditions.

Surface layers (S-layers) are components of the cell walls throughout the Bacteria and the Archaea that provide protection for microorganisms against diverse environmental stresses, including metal stress. We have previously characterized the process by which S-layers serve as a nucleation site for metal mineralization in an archaeon for which the S-layer represents the only cell wall component. Here, we test the hypothesis originally proposed in cyanobacteria that a "shedding" mechanism exists for replacing S-layers that have become mineral-encrusted, using Lysinibacillus sp. TchIII 20n38, metallotolerant gram-positive bacterium, as a model organism. We characterize for the first time a mechanism for resistance to metals through S-layer shedding and regeneration. S-layers nucleate the formation of Fe-mineral on the cell surface, depending on physiological state of the cells and metal exposure times, leading to the encrustation of the S-layer and changes in the cell morphology as observed by scanning electron microscopy. Using Nanoscale Secondary Ion Mass Spectrometry, we show that mineral-encrusted S-layers are shed by the bacterial cells after a period of latency (2 days under the conditions tested) in a heterogeneous fashion likely reflecting natural variations in metal stress resistance. The emerging cells regenerate new S-layers as part of their cell wall structure. Given the wide diversity of S-layer bearing prokaryotes, S-layer shedding may represent an important mechanism for microbial survival in metal-contaminated environments.

Preservation of Archaeal Surface Layer Structure During Mineralization.

Proteinaceous surface layers (S-layers) are highly ordered, crystalline structures commonly found in prokaryotic cell envelopes that augment their structural stability and modify interactions with metals in the environment. While mineral formation associated with S-layers has previously been noted, the mechanisms were unconstrained. Using Sulfolobus acidocaldarius a hyperthermophilic archaeon native to metal-enriched environments and possessing a cell envelope composed only of a S-layer and a lipid cell membrane, we describe a passive process of iron phosphate nucleation and growth within the S-layer of cells and cell-free S-layer "ghosts" during incubation in a Fe-rich medium, independently of metabolic activity. This process followed five steps: (1) initial formation of mineral patches associated with S-layer; (2) patch expansion; (3) patch connection; (4) formation of a continuous mineral encrusted layer at the cell surface; (5) early stages of S-layer fossilization via growth of the extracellular mineralized layer and the mineralization of cytosolic face of the cell membrane. At more advanced stages of encrustation, encrusted outer membrane vesicles are formed, likely in an attempt to remove damaged S-layer proteins. The S-layer structure remains strikingly well preserved even upon the final step of encrustation, offering potential biosignatures to be looked for in the fossil record.

Characterization of leucocin B-KM432Bz from Leuconostoc pseudomesenteroides isolated from boza, and comparison of its efficiency to pediocin PA-1.

A bacteriocin-producing bacterium was isolated from boza and identified as Leuconostoc pseudomesenteroides KM432Bz. The antimicrobial peptide was purified and shown to be identical to other class IIa bacteriocins: leucocin A from Leuconostoc gelidum UAL-187 and Leuconostoc pseudomesenteroides QU15 and leucocin B from Leuconostoc carnosum Ta11a. The bacteriocin was named leucocin B-KM432Bz. Leucocin B-KM432Bz gene cluster encodes the bacteriocin precursor (lcnB), the immunity protein (lcnI) and the dedicated export machinery (lcnD and lcnE). A gene of unknown and non-essential function (lcnC), which is interrupted by an insertion sequence of the IS30 family, is localized between lcnB and lcnD. The activity of leucocin B-KM432Bz requires subunit C of the EII(t) Man mannose permease, which is the receptor for entry into target cells. The determination of the minimum inhibitory concentrations revealed the lowest values for leucocin B-KM432Bz over Listeria strains, with 4 to 32 fold better efficiency than pediocin PA-1.

Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury.

Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl2 sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl2 also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.

A randomised, double-blind, placebo-controlled trial of dolasetron, a 5-hydroxytryptamine 3 receptor antagonist, in patients with fibromyalgia.

OBJECTIVE: The purpose of the study was to evaluate the efficacy and safety of dolasetron for symptomatic relief of pain associated with fibromyalgia (FM). METHODS: This prospective, double-blind, placebo-controlled trial randomly assigned 60 patients with FM to receive placebo (n = 31) or dolasetron (n = 29) 12.5mg/d via the intravenous route on 4 days at baseline (M0), 1 month (M1), 2 months (M2) and 3 months (M3) with follow-up to month 12. The primary outcome variable was the reduction in pain intensity measured by visual analogue scale (VAS) between M0 and M3. The secondary outcome variables were patient global impression of change (PGIC), the FM impact questionnaire, assessment of quality of life (SF-36), the hospital anxiety and depression scale, the manual tender point count, and functional symptoms associated with FM. RESULTS: Reduction in pain intensity at M3 was significantly greater in dolasetron-treated patients (p = 0.04, -21.3 on a 0-100 scale) compared with placebo controls (-5.9). More patients in the dolasetron group had ≥ 30% and ≥ 50% improvement in pain (42.5% and 28% respectively in the dolasetron group versus 25% and 16% in the placebo group). The PGIC was significantly greater in the dolasetron group at M3 (p = 0.02). The other secondary outcomes failed to reach statistical significance. The most common adverse events were constipation, nausea, dizziness and headache, with no significant differences between the two groups. CONCLUSION: Intermittent IV dolasetron was safe and efficacious for the reduction of pain intensity associated with FM at 3 months.

Ultrasonography in chondrocalcinosis.

Ultrasonography can visualize calcific deposits within soft tissues. The appearance and location of the deposits distinguishes articular chondrocalcinosis from other crystal deposition diseases. The most common findings are hyperechoic dots or lines running parallel to the joint surface, hyperechoic images within fibrous cartilage (menisci and triangular fibrocartilage complex), and deposits within tendons (Achilles tendon). Studies found that ultrasonography was highly sensitive and specific for detecting calcifications, using calcium pyrophosphate dihydrate crystal detection in joint fluid as the reference standard. Good agreement has been demonstrated between radiographs and ultrasonography for the detection of calcifications. Thus, ultrasonography is valuable for diagnosing articular chondrocalcinosis via the detection of calcifications within the joint cartilage, fibrocartilage, and tendons. In addition, ultrasonography is a noninvasive, widely available, inexpensive investigation that requires no radiation exposure.

Isolation and characterization of two members of the siderophore-microcin family, microcins M and H47.

In this paper we provide the first biochemical evidence of the existence of a family of structure-related antimicrobial peptides, the siderophore-microcins, in the Enterobacteriaceae family. We isolated and characterized two novel siderophore-microcins, MccM and MccH47, previously characterized through genetic studies. MccM and MccH47 were expressed from several Escherichia coli strains containing the microcin gene clusters. The spectra of their bactericidal activities were found to be restricted to some species of the Enterobacteriaceae. MccM and MccH47 were unable to inhibit the growth of strains carrying mutations in the fepA, cir, and fiu genes, which showed the requirement of the iron-catecholate receptors for their recognition. The MccM and MccH47 peptide moieties contain 77 and 60 residues, respectively, and are derived from the microcin precursors McmA and MchB, respectively. In addition, both peptides carried a C-terminal posttranslational modification containing a salmochelin-like siderophore moiety also found in MccE492 (X. Thomas et al., J. Biol. Chem., 279:28233-28242, 2004). Interestingly, when MccM was isolated from E. coli Nissle 1917, which lacks the two genes necessary for modification biosynthesis, it was devoid of posttranslational modification. Those two genes could be complemented by their homologues from the MccH47 gene cluster, thereby showing their functional interchangeability between at least two members of the siderophore-microcin family. Finally, from the sequence analysis of the MccE492 gene cluster, we hypothesized the existence of an additional member of the siderophore-microcin family. Therefore, we propose that the siderophore-microcin family contains five representatives.

[Contribution of sacroiliac magnetic resonance imaging (MRI) to the evaluation of infliximab efficacy in spondyloarthropathy (a prospective study of 34 patients)]. / Contribution de l'IRM des sacro-iliaques dans l'evaluation des spondylarthropathies sous infliximab (etude prospective sur 34 patients).

Thirty-four patients who met ESSG criteria for spondylarthropathy and were eligible for anti-TNFalpha treatment (infliximab) were enrolled in this open-label study lasting 14 weeks. The aims were to evaluate the progression of sacroiliitis by means of MRI, and to determine the positive predictive value of this exam for the treatment response. Patients underwent MRI of the sacroiliac region at baseline (W0), and also at 14 weeks if the baseline MRI showed sacroiliitis. Two blinded readers reviewed all imaging studies. The patients also had a physical examination and ESR and CRP assays at W0 and W14. Sacroiliitis was found in 22 patients (65%) at W0, but only 18 of these patients had a second MRI at W14, for technical reasons. After 14 weeks of therapy, MRI signs of sacroiliac inflammation diminished by 77.7% on average. Clinical and biological parameters also improved. However, MRI was not predictive of the treatment response. Sacroiliac MRI seems to be interesting for objective therapeutic evaluation and monitoring of patients with spondyloarthropathy.

[What are the cardiovascular complications of the analgesics and glucocorticoids?]. / Quelles sont les complications cardiovasculaires des antalgiques et des glucocorticoides?

When prescribing a non-steroidal anti-inflammatory treatment but also an analgesic or a glucocorticoid, the cardiovascular risk of the patient should be assessed. The analgesics have few cardiovascular side effects and the main complications observed are linked essentially to the vagal action of the opioids. Acetaminophen is considered by several scientific societies to be the first line analgesic treatment, particularly in case of cardiovascular risk but with caution since cardiovascular toxicity of acetaminophen cannot be totally excluded. An overdose of dextropropoxyphene can result in cardiotoxicity. On the other hand, the glucocorticoids need to be prescribed cautiously, at the lowest possible dose and for the shortest possible duration due to the non-negligible cardiovascular risk, hypertension, dyslipidemia, hypokaliemia.

Assays of matrix metalloproteinases (MMPs) activities: a review.

Measurement of matrix metalloproteinase (MMP) activity often remains a challenge, mainly in complex media. Two sets of methods are currently used. The first one measures the hydrolysis of natural protein substrates (labeled or not) and includes the popular zymography. These techniques which are quite sensitive, cannot generally be carried out on a continuous basis. The second one takes mainly advantage of the increase of fluorescence, which is associated to the hydrolysis of initially quenched fluorogenic peptide substrates. Quite recently, another group, which is a compromise between the other two, has been developed. It measures the hydrolysis of synthetic triple-helical peptide substrates. These different methods are described and discussed.