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We investigated the roles of the Vibrio cholerae high-molecular-weight bifunctional penicillin binding proteins, PBP1a and PBP1b, in the fitness of this enteric pathogen. Using a screen for synthetic lethality, we found that the V. cholerae PBP1a and PBP1b proteins, like their Escherichia coli homologues, are each essential in the absence of the other and in the absence of the other's putative activator, the outer membrane lipoproteins LpoA and LpoB, respectively. Comparative analyses of V. cholerae mutants suggest that PBP1a/LpoA of V. cholerae play a more prominent role in generating and/or maintaining the pathogen's cell wall than PBP1b/LpoB. V. cholerae lacking PBP1b or LpoB exhibited wild-type growth under all conditions tested. In contrast, V. cholerae lacking PBP1a or LpoA exhibited growth deficiencies in minimal medium, in the presence of deoxycholate and bile, and in competition assays with wild-type cells both in vitro and in the infant mouse small intestine. PBP1a pathway mutants are particularly impaired in stationary phase, which renders them sensitive to a product(s) present in supernatants from stationary-phase wild-type cells. The marked competitive defect of the PBP1a pathway mutants in vivo was largely absent when exponential-phase cells rather than stationary-phase cells were used to inoculate suckling mice. Thus, at least for V. cholerae PBP1a pathway mutants, the growth phase of the inoculum is a key modulator of infectivity.

The peptidoglycan (PG) sacculus once thought to be just a reinforcing, static and uniform structure, is fast becoming recognized as a dynamic cell constituent involved in every aspect of bacterial physiology. Recent advances showed that in addition to 'classical' tasks-as an essential element to define bacterial shape, size, division and resistance to osmotic stress-the sacculus plays very important roles in many other fields. The very few chemical and structural changes that were once considered as bizarre, or maybe exotic exceptions, are now universally accepted as fundamental pieces in bacterial cell wall adaptation to different kinds of environmental stresses; immune response; intra-specific and inter-specific signalling and antibiotics, just to mention a few. Most, if not all, of these implications are a consequence of the enormous adaptability of PG metabolism to cope with changing conditions, a characteristic for which the term plasticity is proposed. Here we overview and comment on a number of recent contributions on the cell wall adaptive responses to environmental challenges that has greatly impacted the already high complexity of the PG biology field. These new evidences have revived the interest in PG plasticity as an exciting and trendy topic in current microbiology which considers this variability as the trustworthy picture of bacterial PG in nature., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan's central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.

A bacterial cell takes on the challenge to preserve and reproduce its shape at every generation against a substantial internal pressure by surrounding itself with a mechanical support, a peptidoglycan cell wall. The enlargement of the cell wall via net incorporation of precursors into the pre-existing wall conditions bacterial growth and morphology. However, generation, reproduction and/or modification of a specific shape requires that the incorporation takes place at precise locations for a defined time period. Much has been learnt in the past few years about the biochemistry of the peptidoglycan synthesis process, but topological approaches to the understanding of shape generation have been hindered by a lack of appropriate techniques. Recent technological advances are paving the way for substantial progress in understanding the mechanisms of bacterial morphogenesis. Here we review the latest developments, focusing on the impact of new techniques on the precise mapping of cell wall growth sites., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The bacterial cell wall consists of peptidoglycan (PG), a sturdy mesh of glycan strands cross-linked by short peptides. This rigid structure constrains cell shape and size, yet is sufficiently dynamic to accommodate insertion of newly synthesized PG, which was long hypothesized, and recently demonstrated, to require cleavage of the covalent peptide cross-links that couple previously inserted material. Here, we identify several genes in Vibrio cholerae that collectively are required for growth - particularly elongation - of this pathogen. V. cholerae encodes three putative periplasmic proteins, here denoted ShyA, ShyB, and ShyC, that contain both PG binding and M23 family peptidase domains. While none is essential individually, the absence of both ShyA and ShyC results in synthetic lethality, while the absence of ShyA and ShyB causes a significant growth deficiency. ShyA is a D,d-endopeptidase able to cleave most peptide chain cross-links in V. cholerae's PG. PG from a ∆shyA mutant has decreased average chain length, suggesting that ShyA may promote removal of short PG strands. Unexpectedly, ShyA has little activity against muropeptides containing pentapeptides, which typically characterize newly synthesized material. ShyA's substrate-dependent activity may contribute to selection of cleavage sites in PG, whose implications for the process of side-wall growth are discussed., (© 2013 John Wiley & Sons Ltd.)

The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell wall synthesis and cell growth. High-performance liquid chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques., (© 2013 John Wiley & Sons Ltd.)

Purpose: Several studies have investigated effectiveness of fibrin spray or bipolar sealer to control peri-operative bleeding and reduce the need for blood transfusion, but a direct comparison between the two methods has not been previously performed. We conducted a prospective randomised trial, with standard electrocautery as a control group., Methods: In our investigation, 95 patients were randomised to one of three parallel groups receiving (1) 10 mL of topical fibrin spray before closure, (2) haemostasis with radiofrequency energy using a bipolar sealer, and (3) standard electrocautery. All patients and staff apart from the surgeons were blinded until data analysis was complete. Peri-operative blood loss has been calculated using a formula described by Ward and Gross (considering estimated patient blood volume, pre- and post-operative haemoglobin and haematocrit levels), with mention of eventual blood re-infusion or transfusion, at given intervals from surgery (6, 24, 48, 72 hours)., Results: Mean blood loss was lower for both methods investigated, compared to the control group at every time interval considered, although differences were stronger for fibrin spray [Quixil]. Mean blood saving at the given intervals from surgery (6-24-48-72 hours) was respectively 96 ml, 129 ml, 296 ml, and 121 ml for bipolar sealer [Aquamantys] and 235 ml, 368 ml, 642 ml, and 490 ml for fibrin spray. These results are statistically significant (p = 0.05) for fibrin spray at every interval compared to control values, while a significance is detectable for bipolar sealer only at 48 hours after surgery., Conclusions: The fibrin spray group had the best performance in terms of blood loss, significantly reduced in comparison with the control group and bipolar sealer group. Blood loss reduction for the bipolar sealer was remarkable only at 48 hours, compared with the control group. Blood loss reduction for fibrin spray was significant at every time interval considered. Differences between the two treatments investigated and the control group narrowed slightly at 72 hours, as an expression of spontaneous homeostasis. Notable is the fact that blood volume saved with fibrin spray at 24 and 48 hours is comparable to the volume of at least one blood unit. A cost-effectiveness analysis should be considered in term of expense, biological risks (related to blood transfusion or human-derived products use) and bleeding-related complications.

Peptidoglycan (PG) is an essential component of the bacterial cell wall. Although experiments with organisms in vitro have yielded a wealth of information on PG synthesis and maturation, it is unclear how these studies translate to bacteria replicating within host cells. We report a chemical approach for probing PG in vivo via metabolic labeling and bioorthogonal chemistry. A wide variety of bacterial species incorporated azide and alkyne-functionalized d-alanine into their cell walls, which we visualized by covalent reaction with click chemistry probes. The d-alanine analogues were specifically incorporated into nascent PG of the intracellular pathogen Listeria monocytogenes both in vitro and during macrophage infection. Metabolic incorporation of d-alanine derivatives and click chemistry detection constitute a facile, modular platform that facilitates unprecedented spatial and temporal resolution of PG dynamics in vivo.

Background: A multicenter retrospective analysis of patients treated for leg fractures was conducted to develop a score that correlates with fracture healing time and to identify the risk gradient for delayed healing., Methods: Fifty-three patients were analyzed and considered healed when full weight bearing was possible. Patients were divided into those who healed within 180 days and those who took longer to heal. Risk factors associated with delayed healing, fracture morphology, and orthopedic treatments were recorded. The available literature was used to weight the relative risk associated with each factor; values were combined into a score evaluating the risk of delayed healing: L-ARRCO (a literature-based score where the risk of delayed bone healing is calculated using a specific algorithm). Other risk factors associated with delayed healing were then considered in order to calculate a new score, ARRCO. Continuous variables were compared between groups using Student's heteroschedastic two-tail t test. Receiver operating characteristic (ROC) curves and the areas under the curves were calculated to determine the ability of this score to discriminate subjects with delayed healing., Results: The mean L-ARRCO scores of the patients who healed within and after 180 days were significantly different (5.78 ± 1.59 and 7.05 ± 2.46, respectively). The mean ARRCO scores of the patients who healed within and after 180 days were also significantly different (5.92 ± 1.78 and 9.03 ± 2.79, respectively). However, the area under the ROC curve was significantly smaller for L-ARRCO than for ARRCO (0.62 ± 0.09 versus 0.82 ± 0.07)., Conclusions: The ARRCO score is significantly associated with fracture healing time and could be used to identify "fractures at risk," allowing early intervention to stimulate osteogenesis.

Tracking a bug's life: Peptidoglycan (PG) of diverse bacteria is labeled by exploiting the tolerance of cells for incorporating different non-natural D-amino acids. These nontoxic D-amino acids preferably label the sites of active PG synthesis, thereby enabling fine spatiotemporal tracking of cell-wall dynamics in phylogenetically and morphologically diverse bacteria. HCC = 7-hydroxycoumarin, NBD = 7-nitrobenzofurazan, TAMRA = carboxytetramethylrhodamine., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Individuals with metabolic syndrome are at high risk of developing chronic kidney disease (CKD) through unclear pathogenic mechanisms. Obesity and diabetes are known to induce glucolipotoxic effects in metabolically relevant organs. However, the pathogenic role of glucolipotoxicity in the aetiology of diabetic nephropathy is debated. We generated a murine model, the POKO mouse, obtained by crossing the peroxisome proliferator-activated receptor gamma 2 (PPARγ2) knockout (KO) mouse into a genetically obese ob/ob background. We have previously shown that the POKO mice showed: hyperphagia, insulin resistance, hyperglycaemia and dyslipidaemia as early as 4 weeks of age, and developed a complete loss of normal β-cell function by 16 weeks of age. Metabolic phenotyping of the POKO model has led to investigation of the structural and functional changes in the kidney and changes in blood pressure in these mice. Here we demonstrate that the POKO mouse is a model of renal disease that is accelerated by high levels of glucose and lipid accumulation. Similar to ob/ob mice, at 4 weeks of age these animals exhibited an increased urinary albumin:creatinine ratio and significantly increased blood pressure, but in contrast showed a significant increase in the renal hypertrophy index and an associated increase in p27(Kip1) expression compared with their obese littermates. Moreover, at 4 weeks of age POKO mice showed insulin resistance, an alteration of lipid metabolism and glomeruli damage associated with increased transforming growth factor beta (TGFβ) and parathyroid hormone-related protein (PTHrP) expression. At this age, levels of proinflammatory molecules, such as monocyte chemoattractant protein-1 (MCP-1), and fibrotic factors were also increased at the glomerular level compared with levels in ob/ob mice. At 12 weeks of age, renal damage was fully established. These data suggest an accelerated lesion through glucolipotoxic effects in the renal pathogenesis in POKO mice.

It has been generally assumed that the role of D-amino acids in bacterial physiology is rather limited. However, recent new evidence demonstrated that millimolar concentrations of noncanonical D-amino acids are synthesized and released to the environment by bacteria from diverse phyla. These D-amino acids help bacteria adapt to environmental challenges by modulating the structure and composition of the peptidoglycan (PG). This regulation, which appears to be well conserved among bacterial species, occurs principally through the incorporation of the D-amino acids into the terminus of the peptide moiety of muropeptides. These findings revived interest in studies investigating D-amino acids as an exciting and trendy topic in current microbiology, which considers them as fundamental players in different aspects of bacterial physiology. In this article, we provide an overview of the origins of research on the effects of D-amino acids in the biology of bacterial cell walls, including their recent implication as key factors for stress-associated PG remodeling.

Altered microRNA (miRNA) expression may occur early in bladder cancer and may play a role in carcinogenesis and tumor behavior. We evaluated whether alterations in miRNA expression could improve disease stratification and outcome prognosis in bladder tumors and noninvasive diagnosis in urinary samples. miR-143, miR-222, and miR-452 expression levels were analyzed by quantitative RT-PCR (RT-qPCR) in paired urinary and matching tumors and in two independent prospective series of tumors and urinary specimens. Differential expression of miR-143, miR-222, and miR-452 in urine were verified by in situ hybridization in matching tumors. Tumor miRNA expression by RT-qPCR correlated with tumor grade, size, and presence of carcinoma in situ for miR-222, recurrence (miR-222 and miR-143), progression (miR-222 and miR-143), disease-specific survival (miR-222), and overall survival (miR-222). Protein expression patterns of potential miRNA targets, including vascular endothelial growth factor, BCL2, v-erb-b2 erythroblastic leukemia viral oncogene (ERBB) homolog 3, and ERBB4, were evaluated by IHC in tissue arrays containing tumors for which miRNAs were assessed by RT-qPCR. Target expression correlated with expression of their predicted regulatory miRNAs, recurrence (ERBB3), progression (ERBB4), disease-specific survival (ERBB3 and ERBB4), and overall survival (ERBB3 and ERBB4). Furthermore, RT-qPCR of miR-452 (area under the curve, 0.848) and miR-222 (area under the curve, 0.718) in urine provided high accuracies for bladder cancer diagnosis. Thus, bladder tumors were characterized by changes in miRNA expression that could aid in tumor stratification and clinical outcome prognosis, and miRNAs were detected in urinary specimens for noninvasive diagnosis., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Production of non-canonical D-amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non-producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine- and paracrine-like regulators of chemical and physical properties of the cell wall in microbial communities.

The D-enantiomers of amino acids have been thought to have relatively minor functions in biological processes. While L-amino acids clearly predominate in nature, D-amino acids are sometimes found in proteins that are not synthesized by ribosomes, and D-Ala and D-Glu are routinely found in the peptidoglycan cell wall of bacteria. Here, we review recent findings showing that D-amino acids have previously unappreciated regulatory roles in the bacterial kingdom. Many diverse bacterial phyla synthesize and release D-amino acids, including D-Met and D-Leu, which were not previously known to be made. These noncanonical D-amino acids regulate cell wall remodeling in stationary phase and cause biofilm dispersal in aging bacterial communities. Elucidating the mechanisms by which D-amino acids govern cell wall remodeling and biofilm disassembly will undoubtedly reveal new paradigms for understanding how extracytoplasmic processes are regulated as well as lead to development of novel therapeutics.

In all known organisms, amino acids are predominantly thought to be synthesized and used as their L-enantiomers. Here, we found that bacteria produce diverse D-amino acids as well, which accumulate at millimolar concentrations in supernatants of stationary phase cultures. In Vibrio cholerae, a dedicated racemase produced D-Met and D-Leu, whereas Bacillus subtilis generated D-Tyr and D-Phe. These unusual D-amino acids appear to modulate synthesis of peptidoglycan, a strong and elastic polymer that serves as the stress-bearing component of the bacterial cell wall. D-Amino acids influenced peptidoglycan composition, amount, and strength, both by means of their incorporation into the polymer and by regulating enzymes that synthesize and modify it. Thus, synthesis of D-amino acids may be a common strategy for bacteria to adapt to changing environmental conditions.

This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).

Thermus spp is one of the most wide spread genuses of thermophilic bacteria, with isolates found in natural as well as in man-made thermal environments. The high growth rates, cell yields of the cultures, and the constitutive expression of an impressively efficient natural competence apparatus, amongst other properties, make some strains of the genus excellent laboratory models to study the molecular basis of thermophilia. These properties, together with the fact that enzymes and protein complexes from extremophiles are easier to crystallize have led to the development of an ongoing structural biology program dedicated to T. thermophilus HB8, making this organism probably the best so far known from a protein structure point view. Furthermore, the availability of plasmids and up to four thermostable antibiotic selection markers allows its use in physiological studies as a model for ancient bacteria. Regarding biotechnological applications this genus continues to be a source of thermophilic enzymes of great biotechnological interest and, more recently, a tool for the over-expression of thermophilic enzymes or for the selection of thermostable mutants from mesophilic proteins by directed evolution. In this article, we review the properties of this organism as biological model and its biotechnological applications.

The bc(1) respiratory complex III constitutes a key energy-conserving respiratory electron transporter between complex I (type I NADH dehydrogenase) and II (succinate dehydrogenase) and the final nitrogen oxide reductases (Nir, Nor and Nos) in most denitrifying bacteria. However, we show that the expression of complex III from Thermus thermophilus is repressed under denitrification, and that its role as electron transporter is replaced by an unusual nitrate reductase (Nar) that contains a periplasmic cytochrome c (NarC). Several lines of evidence support this conclusion: (i) nitrite and NO are as effective signals as nitrate for the induction of Nar; (ii) narC mutants are defective in anaerobic growth with nitrite, NO and N2O; (iii) such mutants present decreased NADH oxidation coupled to these electron acceptors; and (iv) complementation assays of the mutants reveal that the membrane-distal heme c of NarC was necessary for anaerobic growth with nitrite, whereas the membrane-proximal heme c was not. Finally, we show evidence to support that Nrc, the main NADH oxidative activity in denitrification, interacts with Nar through their respective membrane subunits. Thus, we propose the existence of a Nrc-Nar respiratory super-complex that is required for the development of the whole denitrification pathway in T. thermophilus.

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.

Pre-clinical studies have shown that treatment by pulsed electromagnetic fields (PEMFs) can limit the catabolic effects of pro-inflammatory cytokines on articular cartilage and favour the anabolic activity of the chondrocytes. Anterior cruciate ligament (ACL) reconstruction is usually performed by arthroscopic procedure that, even if minimally invasive, may elicit an inflammatory joint reaction detrimental to articular cartilage. In this study the effect of I-ONE PEMFs treatment in patients undergoing ACL reconstruction was investigated. The study end-points were (1) evaluation of patients' functional recovery by International Knee Documentation Committee (IKDC) Form; (2) use of non-steroidal anti-inflammatory drugs (NSAIDs), necessary to control joint pain and inflammation. The study design was prospective, randomized and double blind. Sixty-nine patients were included in the study at baseline. Follow-up visits were scheduled at 30, 60 and 180 days, followed by 2-year follow-up interview. Patients were evaluated by IKDC Form and were asked to report on the use of NSAIDs. Patients were randomized to active or placebo treatments; active device generated a magnetic field of 1.5 mT at 75 Hz. Patients were instructed to use the stimulator (I-ONE) for 4 h per day for 60 days. All patients underwent ACL reconstruction with use of quadruple hamstrings semitendinosus and gracilis technique. At baseline there were no differences in the IKDC scores between the two groups. At follow-up visits the SF-36 Health Survey score showed a statistically significant faster recovery in the group of patients treated with I-ONE stimulator (P < 0.05). NSAIDs use was less frequent among active patients than controls (P < 0.05). Joint swelling resolution and return to normal range of motion occurred faster in the active treated group (P < 0.05) too. The 2-year follow-up did not shown statistically significant difference between the two groups. Furthermore for longitudinal analysis the generalized linear mixed effects model was applied to calculate the group x time interaction coefficient; this interaction showed a significant difference (P < 0.0001) between the active and placebo groups for all investigated variables: SF-36 Health Survey, IKDC Subjective Knee Evaluation and VAS. Twenty-nine patients (15 in the active group; 14 in the placebo group) underwent both ACL reconstruction and meniscectomy; when they were analysed separately the differences in SF-36 Health Survey scores between the two groups were larger then what observed in the whole study group (P < 0.05). The results of this study show that patient's functional recovery occurs earlier in the active group. No side effects were observed and the treatment was well tolerated. The use of I-ONE should always be considered after ACL reconstruction, particularly in professional athletes, to shorten the recovery time, to limit joint inflammatory reaction and its catabolic effects on articular cartilage and ultimately for joint preservation.

Superfolder GFP (sGFP) is a variant of the Green Fluorescent Protein that folds efficiently when fused to poorly folded proteins. In this study, we show that sGFP, but not enhanced GFP, is functional in vivo at 70 degrees C in the extreme thermophile Thermus thermophilus (Tth); thus, permitting the use of sGFP as a localization tag in vivo. We created a suite of plasmids that allow the expression of carboxy-terminal sGFP fusion proteins in both Escherichia coli and Tth. In order to demonstrate the facility of sGFP as an in vivo localization tag in Tth, we tagged GroES (the small subunit of the bacterial GroES/GroEL chaperone), NarC (a membrane component of the nitrate respiration apparatus) and PhoA (a TAT-secreted periplasmic protein), and visualized the distribution of the sGFP fusion proteins using confocal microscopy. Fusions to NarC and PhoA produced enzymatically active proteins that complemented both the narC and the phoA strains respectively. Observation of the distribution of the GroES-sGFP protein by confocal microscopy revealed a homogeneous fluorescence in the cells, which is in full agreement with the cytoplasmic nature of GroES, whereas the NarC-sGFP protein was localized to the membrane. Finally, a combination of confocal microscopy and biochemistry revealed that PhoA is localized in the periplasm. We suggest that sGFP will be broadly applicable in characterizing various extreme thermophile systems.

The nitrate conjugative element (NCE) encodes the ability to respire nitrate in the facultative Thermus thermophilus NAR1 strain. This process is carried out by two heterotetrameric enzymes that catalyse the oxidation of NADH (Nrc) and the reduction of nitrate (Nar), whose expression is activated by the NCE-encoded transcription factors DnrS and DnrT. We report the presence of NCE in other facultative strains of T. thermophilus and analyse its role in subsequent steps of the denitrification pathway. We encountered that nrc mutants of denitrifying strains show a decrease in anaerobic growth rates not only with nitrate, but also with nitrite, NO and N(2)O, which is concomitant to their lower NADH oxidation activities in vitro. We show that nitrate, nitrite and NO are activating signals for transcription of nrc in these strains. Finally, we demonstrate that DnrS and DnrT are required for anaerobic growth not only with nitrate, but also with nitrite, NO and N(2)O. These data allow us to conclude that: (i) Nrc constitutes the main electron donor for the four reductases of the denitrification pathway, and (ii) the NCE controls the expression of the whole denitrification pathway and the repression of the aerobic respiration through the transcription factors DnrS and DnrT.

Background: Cinacalcet has improved the management of hyperparathiroidism (HPTH) in hemodialysis. To our knowledge there are no specific studies on peritoneal dialysis (PD)., Aim: The aim of the present study was to evaluate the efficacy of Cinacalcet on the achievement of optimal and suboptimal targets on treatment of hyperparathiroidism (HPTH) in PD patients. As secondary objectives we have studied the safety of treatment and estimate the mean time to reach these targets, and evaluate economic cost., Methods: Eighteen patients undergoing more than 4 months on PD with a severe HPTH (PTH > 500 pg/ml) resistant to conventional treatment with diet, chelants and vitamin D were included in this prospective open-label study. We have used the targets of K/DOQITM-clinical guidelines as optimal target. We have selected as suboptimal targets: PTH < 350 pg/ml, phosphorus < 6 mg/dl and calcium < 10.4 mg/dl (only when simultaneous CaxP was under 55 mg2/dl2). Oral Cinacalcet was given with main meal in a single daily start dose of 30 mg and titrated thereafter monthly. We considered the first value on target as an event and used a Kaplan-Meyer survival analysis to estimate mean time to reach target., Results: On inclusion all patients have at least two previous PTH values over 500 pg/ml, PTH mean 695,3 (SD 96) and they were on PD with an appropriate efficacy during a mean of 15.56 months (SD 0.78). Mean follow-up time under Cinacalcet treatment was 12 months. The percentage of patients with a PTH under 350 pg/ml was 66,7% on month 3, 60% on month 6 and 100% after 1 year. The percentage of patients that reach an aggregate of all suboptimal targets (PTH< 350 pg/ml and calcium < 10.4 mg/dl and phosphorus< 6 mg/dl and CaxP < 55 mg2/dl2) was 33.3% on month 6 and 66.7% after 1 year. The mean time to reach PTH target was 2.33 months with a 95% confident interval [1.35-3.32] and to reach the aggregate of all target was 16.94 months [11.38-22.5]. Cinacalcet has been well tolerated, we reduced the dose in a single patient due to secondary effects, but treatment was not discontinued in any case., Conclusion: In summary the addition of Cinacalcet to conventional treatment in PD patients with resistant HPTH has improved the achievement of targets, and has been reasonably safe in our patients.

We describe an activity-independent method for the selection of thermostable mutants of any protein. It is based on a fusion construct comprising the protein of interest and a thermostable antibiotic resistance reporter, in such a way that thermostable mutants provide increased resistance in a thermophile. We isolated thermostable mutants of three human interferons and of two enzymes to demonstrate the applicability of the system.

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.

Quantitative data on the components of biological variation (BV) are used for several purposes, including calculating the reference change value (RCV) required for the assessment of the significance of changes in serial results in an individual. Pathology may modify the set point in diseased patients and, more importantly, the variation around that set-point. Our aim was to collate all published BV data in situations other than health. We report the within-subject coefficient of variation (CV(I)) for 66 quantities in 34 disease states. We compared the results with the CV(I) determined in healthy individuals and examined whether the data derived in specific diseases could be useful for clinical applications. For the majority of quantities studied, CV(I) values are of the same order in disease and health: thus the use of RCV derived from healthy subjects for monitoring patients would be reasonable. However, for a small number of quantities considered to be disease specific markers, the CV(I) differed from those in health. This could mean that RCV derived from healthy CV(I) may be inappropriate for monitoring patients in certain diseases. Hence, disease-specific RCVs may be clinically useful.

The strains of Thermus thermophilus that contain the nitrate respiration conjugative element (NCE) replace their aerobic respiratory chain by an anaerobic counterpart made of the Nrc-NADH dehydrogenase and the Nar-nitrate reductase in response to nitrate and oxygen depletion. This replacement depends on DnrS and DnrT, two homologues to sensory transcription factors encoded in a bicistronic operon by the NCE. DnrS is an oxygen-sensitive protein required in vivo to activate transcription on its own dnr promoter and on that of the nar operon, but not required for the expression of the nrc operon. In contrast, DnrT is required for the transcription of these three operons and also for the repression of nqo, the operon that encodes the major respiratory NADH dehydrogenase expressed during aerobic growth. Thermophilic in vitro assays revealed that low DnrT concentrations allows the recruitment of the T. thermophilus RNA polymerase sigma(A) holoenzyme to the nrc promoter and its transcription, whereas higher DnrT concentrations are required to repress transcription on the nqo promoter. In conclusion, our data show a complex autoinducible mechanism by which DnrT functions as the transcriptional switch that allows the NCE to take the control of the respiratory metabolism of its host during adaptation to anaerobic growth.

Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N-terminal coiled-coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C-terminal domain (CTD). We show that depending on pH, Gfh1-CTD exists in two alternative orientations. At pH above 7, it assumes an inactive 'flipped' orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1-CTD switches to an active 'Gre-like' orientation, which enables Gfh1 to bind to and inhibit RNAP.

The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.

The expression of an antisense RNA revealed that an Mn-catalase was required in Thermus thermophilus for aerobic but not for anaerobic growth. The antisense system is based on the constitutive expression of a "bicistronic" transcript consisting of the kanamycin resistance gene mRNA followed by the antisense RNA against the selected target.

A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor. Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfur-containing subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs). The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank. Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants. A fusion to a thermophilic reporter gene was used for the first time in Thermus spp. to show that expression of nrc required the presence of nitrate and anoxic conditions. Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced. Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen. The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system. Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins. Interactions were also detected between NrcN and NrcF. Inactivation of nrcF produced solubilization of NrcN, but not of NrcD. These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH.

Background: [corrected] Data on within- and between-subject biological variation are available for around 250 analytes commonly used in medical laboratories., Methods: Integration of this data into the quality system occurs at all three levels of laboratory activity: (a) Preanalytic process: biological variation provides the basis for selecting the most appropriate specimen for analysis, for defining sample stability and for deciding suitable timing between samplings; (b) analytic process: biological variation-derived goals are fundamental for designing internal quality control procedures, and for evaluating laboratory performance; and (c) postanalytic process: delta checks based on within-subject biological variation values are used for validating results and for interpreting serial results from a patient., Conclusion: The biological variation is a pillar for managing quality in laboratory medicine.

Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H(2)O(2)-detoxifying systems. We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a Thermus-specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T. thermophilus under the control of the Pnar promoter. This promoter belongs to a respiratory nitrate reductase from of T. thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in T. thermophilus HB8, a 20- to 30-fold increase in catalase specific activity was observed, whereas a 90- to 110-fold increase was detected when the laboratory strain T. thermophilus HB27::nar was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of T. thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.

Electron microscopy of isolated cell walls of the ancient bacterium Thermus thermophilus revealed that most of the peptidoglycan (PG) surface, apart from the septal region, was shielded against specific alphaPG antibodies. On the other hand, an antiserum raised against S-layer-attached cell wall fragments (alphaSAC) bound to most of the surface except for the septal regions. Treatments with alpha-amylase and pronase E made the entire cell wall surface uniformly accessible to alphaPG and severely decreased the binding of alphaSAC. We concluded that a layer of strongly bound secondary cell wall polymers (SCWPs) covers most of the cell wall surface in this ancient bacterium. A preliminary analysis revealed that such SCWPs constitute 14% of the cell wall and are essentially composed of sugars. Enzyme treatments of the cell walls revealed that SCWP was required in vitro for the binding of the S-layer protein through the S-layer homology (SLH) motif. The csaB gene was necessary for the attachment of the S-layer-outer membrane (OM) complex to the cell wall in growing cells of T. thermophilus. In vitro experiments confirmed that cell walls from a csaB mutant bound to the S-layer with a much lower affinity ( approximately 1/10) than that of the wild type. CsaB was found to be required for pyruvylation of components of the SCWP and for immunodetection with alpha-SAC antiserum. Therefore, the S-layer-OM complex of T. thermophilus binds to the cell wall through the SLH motif of the S-layer protein via a strong interaction with a highly immunogenic pyruvylated component of the SCWP. Immuno-cross-reactive compounds were detected with alphaSAC on cell walls of other Thermus spp. and in the phylogenetically related microorganism Deinococcus radiodurans. These results imply that the interaction between the SLH motif and pyruvylated components of the cell wall arose early during bacterial evolution as an ancestral mechanism for anchoring proteins and outer membranes to the cell walls of primitive bacteria.

Bacterial single-stranded DNA-binding proteins (SSBs) are required for DNA replication and repair. We have over-expressed and purified the native form and two His-tagged fusions of the SSB from Thermus thermophilus (TthSSB). The three proteins were found as dimers in solution. They bound in vitro to single-stranded DNA specifically over a temperature range of 4-80 degrees C, and the wild-type protein could withstand incubation at 94 degrees C for 2 min. Addition of TthSSB to PCR halved the elongation time required for the DNA polymerases of T.thermophilus (Tth) and Pyrococcus furiosus (Pfu) to synthesise DNA fragments in PCRs. The presence of TthSSB increased the fidelity of the proof- reading-free DNA polymerase of T.thermophilus. TthSSB was also able to bind single-stranded RNA, allowing a dramatic enhancement of the reverse transcription activity of its cognate Tth DNA polymerase during cDNA synthesis.

A specific expression system for Thermus spp. is described. Plasmid pMKE1 contains replicative origins for Escherichia coli and Thermus spp., a selection gene encoding a thermostable resistance to kanamycin, and a 720 bp DNA region containing the promoter (Pnar), and the regulatory sequences of the respiratory nitrate reductase operon of Thermus thermophilus HB8. Two genes, encoding a thermophilic beta-galactosidase and an alkaline phosphatase were cloned in pMKE1 as cytoplasmic and periplasmic reporters, respectively. The expression of the reporters was specifically induced by the combined action of nitrate and anoxia in facultative anaerobic derivatives of T. thermophilus HB27 to which the gene cluster for nitrate respiration was transferred by conjugation. Overexpressions in the range of approximately 200-fold were obtained for the cytoplasmic reporter, whereas that of the periplasmic reporter was limited to approximately 20-fold, with respect to their intrinsic respective activities., (Copyright 2002 Elsevier Science (USA))

A cytochrome c (NarC) is encoded as the first gene of the operon for nitrate respiration in Thermus thermophilus. NarC is required for anaerobic growth and for the synthesis of active nitrate reductase (NR). The alpha and delta subunits (NarG, NarJ) of the NR were constitutively expressed in narC::kat mutants, but NarG appeared in the soluble fraction instead of associated with the membranes. Our data demonstrate for NarC an essential role in the synthesis of active enzyme and for the attachment to the membrane of the respiratory NR from T. thermophilus.

The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed in a mutant (HB8(Delta)UTR1) defective in the regulation of its S-layer (surface crystalline layer). This mutant forms round multicellular bodies (MBs) surrounded by a common envelope as the culture approaches the stationary phase. Confocal microscopy revealed that the origin of the MBs is the progressive detachment of the external layers coupled with the accumulation of NH(2)-containing material between the external envelopes and the peptidoglycan. A specific pattern of proteins was found as soluble components of the intercellular space of the MBs by a single fractionation procedure, suggesting that they are periplasmic-like components. To demonstrate this, we cloned a gene ( phoA) from T. thermophilus HB8 encoding a signal peptide-wearing alkaline phosphatase (AP), and engineered it to be overexpressed in the mutant from a shuttle vector. Most of the AP activity (>80%) was found as a soluble component of the MBs' intercellular fraction. All these data indicate that Thermus thermophilus actually has a periplasmic space which is functionally similar to that of Proteobacteria. The potential application of the HB8(Delta)UTR1 mutant for the overexpression of periplasmic thermophilic proteins is discussed.

The biological variation of anti-TPO and anti-Tg autoantibodies was studied in 17 clinically and biochemically stable female patients with autoimmune thyroid disease (AITD), at regular monthly intervals over a period of 6 consecutive months. The mean and standard deviation (SD), within-subject coefficient of variation (CV), between-subject CV, index of individuality, reliability coefficient, and critical differences were as follows: for anti-TPO 238 (197) U/ml, 9.2%, 81.4%, 0.11, 0.96, and 27.6%; and for anti-Tg 1,785 (3,170) U/ml, 6.9%, 174%, 0.04, 0.99, and 22.3%. The data indicate a low within-subject CV, and a high between-subject CV that is particularly pronounced for anti-Tg. The high individuality of both autoantibodies indicates that an isolated result compared to conventional population-based reference intervals is of very little value for diagnosis. Furthermore, the near to 1 reliability coefficient for both autoantibodies correctly classifies the patient with respect to his or her homeostatic mean antibody concentration in a 6-month period of clinical and biochemical stability of thyroid disease. Imprecision goals for anti-TPO and anti-Tg antibodies are attainable with current methodology., (Copyright 2002 Wiley-Liss, Inc.)

Components of biological variation can be used to assess the usefulness of reference values, to evaluate the significance of changes in serial results from an individual and to define objective analytical goals. The aim of the study was to assess, in 15 healthy subjects studied at regular monthly intervals over a period of 6 consecutive months, the biological variation of interleukin-1beta (IL-1beta), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha). Biological variation data (within-subject and between-subject coefficient of variation (CV)) were determined using a simple nested analysis of variance. Derived parameters (index of individuality, reliability coefficient and critical diferences) were calculated from within-subject and between-subject CV. The mean and standard deviation (SD), within-subject CV, between-subject CV, index of individuality and reliability coefficient were as follows: for IL-1beta, 0.67 (0.32) pg/ml, 30%, 36%, 0.85, and 0.76; for IL-8, 3.68 (1.45) pg/ml, 24%, 31%, 0.85 and 0.75; and for TNF-alpha, 3.14 (1.87) pg/ml, 43%, 29%, 1.56 and 0.50, respectively. We conclude that between-subject variation and within-subject variation are quite similar for IL-1beta and IL-8 and are relatively high for the three cytokines studied. Index of individuality is less than 1.4 for IL-1beta and IL-8, and thus reference intervals based on population studies are of limited value. On the contrary, the index of individuality for TNF-alpha is greater than 1.4 and reference values can be used for diagnosis. Quality goals for imprecision are easily achieved for the three cytokines with current methodology.

Objective: To assess the influence that self-measurement of capillary blood glucose (SMBG) has on glucaemia control in patients with diabetes mellitus., Design: Quasi-experimental before and after study., Setting: Semi-urban health centre., Patients: All diabetic patients, with at least two years of evolution, who used reactive strips for SMBG in 1996., Measurements and Main Results: Mean values of base glucaemia in the SMBG study year were compared with those of the year before in 85 patients, 33 men and 52 women, with average age 62.38. Thirteen were type 1 and 72 type 2 diabetics, with a mean 15.61 years of evolution of the disease. A drop of -11.47 mg/dl (SD 44.32) was observed, which was significant (p < 0.05, CI 95%) in the overall results. In all the subgroups, except those treated with non-pharmacological measures, there were drops, ranging between -2.17 mg/dl and -17.01 mg/dl, which were significant in women, in patients with type 2 diabetes, in those who had received health education and in those treated with non-pharmacological measures plus insulin., Conclusions: Despite the limitations of this kind of study, our findings point towards a slightly better control of glucaemia levels in diabetic patients after SMBG. It is very doubtful whether it signifies any real improvement in managing the illness.

Acid-base disturbances were examined during experimentally induced infection in the rabbit with Eimeria stiedai, a parasite that profoundly modifies liver morphology and physiology, resulting in anatomical and functional alterations similar to those appearing in different human hepatic diseases. Over 28 days of infection, bicarbonate and lactate concentrations, partial pressures of O2 and CO2, and pH values were determined in the blood and bile of infected animals and compared with the values obtained in noninfected rabbits. The plasma activity of several liver-indicator enzymes was also evaluated. Under our experimental conditions we observed an uncompensated metabolic acidosis that developed with elevated levels of lactate and reduced concentrations of bicarbonate in blood and bile and tended to be compensated by respiratory and biliary mechanisms.

We investigated the effect of cyclosporine A (CyA) administered as a single i.v. dose of 20 and 40 mg/kg body wt, on biliary secretion of cholesterol, phospholipid, bile acid, and lysosomal marker and canalicular plasma membrane marker enzymes in anaesthetized Wistar rats. CyA reduced the concentration and biliary secretion of cholesterol, phospholipid and bile acid to a considerable extent; the inhibitory effect of CyA on the biliary secretion of phospholipid and bile acid was greater than that on cholesterol. The biliary outputs of acid phosphatase (AcP) and gamma-glutamyltransferase (gamma-GT) were also diminished by the drug, all these effects being dose-dependent. Maximum decreases in bile acid secretion were observed 10 min after administration, whereas those of cholesterol and phospholipid were delayed. Bile acid concentrations and secretion returned to pretest values at 30-50 min after CyA injection whereas those of cholesterol and phospholipid remained significantly reduced at this time point. The greater inhibitory effect of CyA on the biliary outputs of phospholipid and bile acid relative to cholesterol secretion together with the asynchronous fall and recovery of bile acid, cholesterol and phospholipid concentrations and secretion alter the cholesterol/bile acid, phospholipid/bile acid and cholesterol/phospholipid molar ratios as well as the lithogenic index, thus suggesting that CyA would uncouple biliary lipid secretion from bile acid secretion. Since under physiological conditions biliary lipid and gamma-GT secretion is related to and dependent upon bile acid secretion, we propose that the CyA-induced inhibition on lipid and gamma-GT secretion is, at least partly, secondary to the fall in bile acid output caused by the drug. However, since CyA inhibits secretory processes independent of the hepatobiliary flux of bile acid, such as the exocytic discharge of AcP, and because it also uncouples biliary lipid from bile acid secretion, other mechanisms and factors involved in lipid and protein secretion (such as intracellular transport, canalicular membrane fluidity and/or intracanalicular events) might also be altered by this drug.

Carbohydrate antigen 15-3 (CA 15-3) and mucinous carbohydrate antigen (MCA) immunoreactivities were detected in human seminal plasma. Mean values for CA 15-3 (8.2 +/- 3.7 U/ml, range 2.6 - 18.4 U/ml) and MCA (13.8 +/- 8.2 U/ml, range 2.1-31.9 U/ml) in the seminal plasma were of the same magnitude as that found in serum. No correlation was obtained between seminal plasma, either CA 15-3 or MCA immunoreactivities, and volume of seminal plasma, sperm count or percent of motile spermatozoa. Seminal plasma CA 15-3 and MCA levels were significantly (p less than 0.001) correlated (r = 0.55). The nature and origin of CA 15-3 and MCA immunoreactivities in human plasma are unknown.

We have studied vitamin B6 status in 26 uremic patients, 18 on maintenance hemodialysis and 8 nonhemodialyzed. The vitamin B6 status was estimated by an assay of erythrocyte aspartate aminotransferase and coenzyme stimulation. Hemodialyzed uremic patients were found to have vitamin B6 deficiency. Patients were treated with 150 mg pyridoxine hydrochloride daily for 4 weeks. Erythrocyte aspartate aminotransferase increased significantly in both groups of uremic patients, the increase being greater in hemodialyzed patients. In vitro pyridoxal phosphate stimulation produces an erythrocyte aspartate aminotransferase activity greater than that obtained before pyridoxine hydrochloride administration. After cessation of pyridoxine hydrochloride treatment, erythrocyte aspartate aminotransferase decreases in hemodialyzed patients, while it remains elevated in nonhemodialyzed patients. The data obtained appear to indicate that vitamin B6 administration to patients with chronic renal insufficiency must be appraised not only for correcting the deficit but also for increasing the intracellular pyridoxal phosphate concentration, which could modify the possible functional impairment at the level of apoenzymes that use pyridoxal phosphate.

Although there is general agreement on the hydrocholeretic properties of the so-called "synthetic choleretics" on biliary secretion, related simply to the kinetics of excretion, recent studies suggest that some of these drugs also have a pharmacodynamic effect; mainly, stimulation of bile acid secretion. In the present work, we studied the biliary response to different doses of cyclobutyrol (CB) in order to determine whether this agent stimulates the secretion of bile acids and to establish the relationships between dose and the choleretic effects in anaesthetized rats. Biliary bile flow, sodium, potassium, chloride and bicarbonate outputs were found to be increased and bile acid concentrations reduced in a dose-dependent fashion after 0.40, 0.54, 0.80, 1.08 and 2.16 mmol/kg b.wt. of CB administration. All assayed doses had no effect on the bile acids secretion rate. These findings suggest that a) CB-induced choleresis is unrelated to bile acids; b) CB and bile acids do not compete for the hepatobiliar transport mechanisms, despite the anionic character of both compounds, and c) in the rat the active mechanisms involved in the biliary elimination of CB are not saturated even at the large doses employed.