Effect of reduced pH on physiology and shell integrity of juvenile Haliotis iris (paua) from New Zealand.

The New Zealand paua or black footed abalone, Haliotis iris, is one of many mollusc species at potential risk from ocean acidification and warming. To investigate possible impacts, juvenile paua (~24 mm shell length) were grown for 4 months in seawater pH/pCO2 conditions projected for 2100. End of century seawater projections (pHT 7.66/pCO2 ~1,000 µatm) were contrasted with local ambient conditions (pHT 8.00/pCO2 ~400 µatm) at two typical temperatures (13 and 15 °C). We used a combination of methods (morphometric, scanning electron microscopy, X-ray diffraction) to investigate effects on juvenile survival and growth, as well as shell mineralogy and integrity. Lowered pH did not affect survival, growth rate or condition, but animals grew significantly faster at the higher temperature. Juvenile paua were able to biomineralise their inner nacreous aragonite layer and their outer prismatic calcite layer under end-of-century pH conditions, at both temperatures, and carbonate composition was not affected. There was some thickening of the nacre layer in the newly deposited shell with reduced pH and also at the higher temperature. Most obvious was post-depositional alteration of the shell under lowered pH: the prismatic calcite layer was thinner, and there was greater etching of the external shell surface; this dissolution was greater at the higher temperature. These results demonstrate the importance of even a small (2 °C) difference in temperature on growth and shell characteristics, and on modifying the effects at lowered pH. Projected CO2-related changes may affect shell quality of this iconic New Zealand mollusc through etching (dissolution) and thinning, with potential implications for resilience to physical stresses such as predation and wave action.

Azospirillum brasilense Az39, a model rhizobacterium with AHL quorum-quenching capacity.

AIMS: The aim of this research was to analyse the quorum-sensing (QS) and quorum-quenching (QQ) mechanisms based on N-acyl-l-homoserine lactones (AHLs) in Azospirillum brasilense Az39, a strain with remarkable capacity to benefit a wide range of crops under agronomic conditions. METHODS AND RESULTS: We performed an in silico and in vitro analysis of the quorum mechanisms in A. brasilense Az39. The results obtained in vitro using the reporter strains Chromobacterium violaceum and Agrobacterium tumefaciens and liquid chromatography coupled with mass-mass spectrometry analysis showed that although Az39 does not produce AHL molecules, it is capable of degrading them by at least two hypothetical enzymes identified by bioinformatics approach, associated with the bacterial cell. In Az39 cultures supplemented with 500 nmol l-1 of the C3 unsubstituted AHLs (C4, C6, C8, C10, C12, C14), AHL levels were lower than in noninoculated LB media controls. Similar results were observed upon the addition of AHLs with hydroxy (OH-) and keto (oxo-) substitutions in C3. These results not only demonstrate the ability of Az39 to degrade AHLs. They also show the wide spectrum of molecules that can be degraded by this bacterium. CONCLUSIONS: Although A. brasilense Az39 is a silent bacterium unable to produce AHL signals, it is able to interrupt the communications between other bacteria and/or plants by a QQ activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report confirming by unequivocal methodology the ability of A. brasilense, one of the most agriculturally used benefic bacteria around the world, to degrade AHLs by a QQ mechanism.

The fitness burden imposed by synthesising quorum sensing signals.

It is now well established that bacterial populations utilize cell-to-cell signaling (quorum-sensing, QS) to control the production of public goods and other co-operative behaviours. Evolutionary theory predicts that both the cost of signal production and the response to signals should incur fitness costs for producing cells. Although costs imposed by the downstream consequences of QS have been shown, the cost of QS signal molecule (QSSM) production and its impact on fitness has not been examined. We measured the fitness cost to cells of synthesising QSSMs by quantifying metabolite levels in the presence of QSSM synthases. We found that: (i) bacteria making certain QSSMs have a growth defect that exerts an evolutionary cost, (ii) production of QSSMs negatively correlates with intracellular concentrations of QSSM precursors, (iii) the production of heterologous QSSMs negatively impacts the production of a native QSSM that shares common substrates, and (iv) supplementation with exogenously added metabolites partially rescued growth defects imposed by QSSM synthesis. These data identify the sources of the fitness costs incurred by QSSM producer cells, and indicate that there may be metabolic trade-offs associated with QS signaling that could exert selection on how signaling evolves.

Ocean acidification at high latitudes: potential effects on functioning of the Antarctic bivalve Laternula elliptica.

Ocean acidification is a well recognised threat to marine ecosystems. High latitude regions are predicted to be particularly affected due to cold waters and naturally low carbonate saturation levels. This is of concern for organisms utilising calcium carbonate (CaCO(3)) to generate shells or skeletons. Studies of potential effects of future levels of pCO(2) on high latitude calcifiers are at present limited, and there is little understanding of their potential to acclimate to these changes. We describe a laboratory experiment to compare physiological and metabolic responses of a key benthic bivalve, Laternula elliptica, at pCO(2) levels of their natural environment (430 µatm, pH 7.99; based on field measurements) with those predicted for 2100 (735 µatm, pH 7.78) and glacial levels (187 µatm, pH 8.32). Adult L. elliptica basal metabolism (oxygen consumption rates) and heat shock protein HSP70 gene expression levels increased in response both to lowering and elevation of pH. Expression of chitin synthase (CHS), a key enzyme involved in synthesis of bivalve shells, was significantly up-regulated in individuals at pH 7.78, indicating L. elliptica were working harder to calcify in seawater undersaturated in aragonite (Ω(Ar) = 0.71), the CaCO(3) polymorph of which their shells are comprised. The different response variables were influenced by pH in differing ways, highlighting the importance of assessing a variety of factors to determine the likely impact of pH change. In combination, the results indicate a negative effect of ocean acidification on whole-organism functioning of L. elliptica over relatively short terms (weeks-months) that may be energetically difficult to maintain over longer time periods. Importantly, however, the observed changes in L. elliptica CHS gene expression provides evidence for biological control over the shell formation process, which may enable some degree of adaptation or acclimation to future ocean acidification scenarios.

Quantitative liquid chromatography-tandem mass spectrometry profiling of activated methyl cycle metabolites involved in LuxS-dependent quorum sensing in Escherichia coli.

A rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry assay has been developed and validated for the simultaneous quantification of the metabolites and precursors of the activated methyl cycle, reported in preliminary form by Heurlier et al. (2009) [43]. Analytes were extracted from Escherichia coli MG1655 and chemically derivatized as N(O,S)-iso-butyloxycarbonyl iso-butyl esters using iso-butyl chloroformate in an aqueous iso-butanol/pyridine environment. S-Adenosylmethionine, S-adenosylhomocysteine, S-ribosylhomocysteine, homocysteine, methionine, cystathionine, cysteine, and homoserine were quantified by liquid chromatography-positive ion tandem electrospray ionization mass spectrometry. Internal standards were isotopically labeled [(13)CD(3)]methionine and S-adenosylcysteine. Linearity of the assay was established up to a concentration of 700 microg/g cell dry weight for each analyte. The validated assay was used to quantitatively profile the intracellular activated methyl cycle metabolites as a function of growth in E. coli MG1655 and its derivative Deltapfs and DeltaluxS mutants to determine the metabolic consequences of a disruption to the activated methyl cycle and, hence, LuxS-dependent quorum sensing.

Prone positioning for head and neck reconstructive surgery.

Certain head and neck surgical cases require the patient to be positioned prone. Such positioning carries with it an attendant subset of risks and complications not otherwise encountered in more traditional supine positioning. Gaining awareness of these risks and complications, and developing proactive positioning strategies, will enable the surgical team to position the patient optimally for the procedure and provide for every consideration of patient safety. This article consists of a specific literature review of those issues directly related to the anatomical and physiological concerns arising from prone positioning. Particular attention is paid to the cardiopulmonary, renal, ophthalmologic, and neurological vulnerabilities unique to this position. Proper planning by the surgical team and utilization of the correct equipment are a necessity. A tailored approach to the needs of the individual patient and an intimate awareness of the potential pitfalls will contribute to better outcomes when using the prone position.

Pediatric liver transplant with Campath 1H induction--Preliminary report.

UNLABELLED: Since Campath 1H (C1H) has been successfully used in adult liver transplant recipients since 2001 in our program, we started to use it in children. PATIENTS AND METHODS: C1H induction was employed in 10 children with autoimmune hepatitis (AIH) (n = 6), primary sclerosing cholangitis (PSC) (n = 1), biliary atresia (n = 1), glycogen storage disease (n = 1), and Wilson's disease. Eight were primary transplants, and two retransplants. Patients ages ranged from 5 to 17 years. C1H was administered at a dose of 0.3 mg/kg on days 0, 4, and 7. Tacrolimus level was maintained at 5 to 10 ng/mL. No patient received maintenance steroids posttransplantation except two who were on steroid therapy at the time transplant. They were prescribed small doses of maintenance steroids. Median follow-up of C1H recipients was 679 days (range 115-1143). RESULTS: Postoperative courses were mostly uneventful except for one retransplant recipient who required prolonged hospitalization (40 days) for rehabilitation. Median hospital stay was 12 days (range 7-40 days). All 10 patients in the C1H group are currently alive and well with stable graft function. No opportunistic infection was observed in these patients to date. We compared six patients with AIH who received C1H to the historic control of 10 recipients with AIH who received conventional immunosuppression (tacrolimus + steroid). The patients treated with C1H showed significantly prolonged rejection-free survival. CONCLUSION: In our preliminary experience, C1H induction was well tolerated in pediatric liver recipients. Rejections-free survival was prolonged among recipients with AIH despite a low level of maintenance immunosuppression.

Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivation.

The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 microM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P < 0.001) by thymidine (100 microM) but was reversed (P < 0.001) by the purines, hypoxanthine (Hx; 100 microM) and adenosine (100 microM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 microM) further enhanced (P < 0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 microM) reversed (P < 0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine beta-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 microM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines.

Gelatinase A activation is regulated by the organization of the polymerized actin cytoskeleton.

Gelatinase A (GL-A) is a matrix metalloproteinase (MMP) involved in both connective tissue remodeling and tumor invasion. GL-A activation is mediated by a membrane-type MMP (MT-MMP) that cleaves the GL-A propeptide. In this study, we examined the role of the actin cytoskeleton in regulating GL-A activation and MT-MMP-1 expression. Human palmar fascia fibroblasts and human fetal lung fibroblasts were cultured on a planar substratum or within different types of collagen lattices. Fibroblasts that formed stress fibers, either on a planar substratum or within an attached collagen lattice, showed reduced GL-A activation compared with fibroblasts lacking stress fibers, within either floating or stress-released collagen lattices. To determine whether changes in the organization of the actin cytoskeleton could promote GL-A activation, fibroblasts with stress fibers were treated with cytochalasin D. Within 24 h after treatment, GL-A activation was dramatically increased. Associated with this GL-A activation was an increase in MT-MMP-1 mRNA as determined by Northern blot analysis. Treatment with nocodazole, which induced microtubule depolymerization and cell shape changes without affecting stress fibers, did not promote GL-A activation. These results suggest that the extracellular matrix and the actin cytoskeleton transduce signals that modulate GL-A activation and regulate tissue remodeling.

Mechanical properties of the extracellular matrix influence fibronectin fibril assembly in vitro.

Mechanical properties of the extracellular matrix (ECM) are proposed to influence cell behavior and biological activity. The influence of the mechanical environment on fibronectin fibril assembly was evaluated. Fibroblasts were cultured in hydrated collagen gels with two distinctly different mechanical properties. Cells cultured within a stabilized collagen gel generate stress that is transmitted throughout the matrix (stressed gel). In contrast, cells that are cultured within a collagen gel that is floating freely in media do not generate stress (relaxed gel). Fibroblasts in the stressed collagen gel develop large bundles of actin microfilaments and associated fibronectin fibrils, while fibroblasts within relaxed gels do not form stress fibers or assemble fibronectin into fibrils. In addition, we have evaluated the mechanism of fibronectin fibril assembly employed by fibroblasts cultured within a stressed three-dimensional collagen matrix and the role of fibronectin fibrils in transmission of cell-generated forces to the surrounding matrix. Fibronectin fragments (70-kDa amino terminal fragment, 110-kDa cell-adhesive fragment, and GRGDS peptide) and a monoclonal antibody body blocked fibronectin fibril assembly in stressed three-dimensional collagen gels. These results suggest that the features of fibronectin required for fibronectin fibril assembly by cells in collagen gels is similar to those required by cells cultured on a planar substratum. Although fibronectin fibril assembly was blocked by these inhibiting fragments and antibody, the cells displayed prominent actin bundles and developed isometric tension, indicating that stress fiber formation and contractile force transmission is not dependent on the presence of fibronectin fibrils.

Provision of transplant services.

To a large extent the success of any transplant programme depends ultimately on the expertise, skills and experience of the team of doctors, nurses and other health-care professionals. However, the logistics associated with the service will be the major factors determining the success of the programme. The service should be provided on the basis of epidemiological need, concentrating provision in an optimal number of units so that benefit is gained from the 'experience curve' effect of a relatively high throughput. The availability of the key resource for the programme, namely organs, depends on the willingness of the public to donate, and on the co-operation of other health-care professionals, not directly involved in transplantation, in identifying potential donors, encouraging relatives to consent and ensuring that the donor is maintained in an optimal condition. There are not many medical treatments that require such wide-ranging participation and co-operation as those needed for organ transplantation. All these factors have to be addressed for an organ transplant programme to be successful.

Distribution of ED-A and ED-B containing fibronectin isoforms in Dupuytren's disease.

Different fibronectin (FN) isoforms arise via alternate splicing of a single gene transcript in a cell- and tissue-specific manner. Antibodies were used to evaluate the presence and distribution of FN and its isoforms in Dupuytren's diseased and normal palmar fascia. Immunolocalization studies show extracellular FN fibrils, including FN isoforms containing extra domains A (A-FN) and B (B-FN), in proliferative and involutional stage Dupuytren's diseased tissue. However, B-FN appears less abundant and more restricted in its distribution as compared to A-FN or total FN. Total FN and A-FN are significantly reduced in residual tissue, while B-FN is not present. A-FN and B-FN are not present in normal palmar fascia, while total FN staining is slight and restricted to the loose connective tissue surrounding the large, parallel bundles of collagen fibers. The presence of A-FN and B-FN in Dupuytren's diseased palmar fascia represents a disease-induced appearance of these FN isoforms and further evidence of an association between Dupuytren's disease and wound healing.

The pharmacodynamics of rocuronium in pediatric patients anesthetized with halothane.

The aim of this study was to determine the neuromuscular blocking potency of rocuronium (ORG 9426) in 4-to 14-year old children anesthetized with halothane. After induction of anesthesia, the ulnar nerve was stimulated with electrical impulses of 0.2 ms duration every 12 s and the force of contraction of the thumb (P) was continuously recorded. Doses of 0.12, 0.16, 0.20, and 0.24 mg·kg(-1) rocuronium were administered, in a randomized fashion, to 4 groups of 12 patients each. The ED50, ED90, and ED95 of rocuronium determined from the log dose-probit regression lines were 0.18, 0.34, and 0.40 mg·kg(-1), respectively. To facilitate tracheal intubation, after the development of the maximal effect of the first dose, a variable second dose of rocuronium was administered to increase the total dose to 0.3 mg·kg(-1). If after the second dose P was greater than 10% of control, additional 0.025-0.1 mg·kg(-1) increments of rocuronium were administered until P became less than 10% of control. At this time the trachea was intubated. Muscular relaxation was maintained with 0.075, 0.1, or 0.125 mg·kg(-1) rocuronium, administered whenever P recovered to 25% of control. The clinical duration of these doses was 6.9±2.8, 6.1±0.4, and 8.1±0.6 min, respectively. On repeated administration of three 0.1 or 0.125 mg·kg(-1) doses, rocuronium showed little cumulative tendency. Time for spontaneous recovery of P from 25% to 75%, 8.4±0.39 min and from 10% to 90%, 16.19±0.15 min, of control, were relatively short. When at termination of anesthesia T4/T1 ratios were lower than 0.75, the residual neuromuscular block could be antagonized with 0.5 mg·kg(-1) edrophonium in 2 min. Rocuronium, 0.3 mg·kg(-1) caused a 13.5% increase of heart rate but had no effect on blood pressure. In conclusion, in 4 to 14-year-old children, rocuronium appears to have a more rapid onset and shorter duration of action than other steroid-type muscle relaxants.

I.v. temazepam: theoretical and clinical considerations.

Two injectable forms of temazepam, in 90% propylene glycol or 40% salicylic acid, were studied in volunteers, and before surgery in healthy patients. The volunteers also received two forms (capsule and elixir) by mouth. The salicylate preparation was painful on injection and both i.v. formulations caused an unacceptably high incidence of venous thrombosis. Temazepam was detected in plasma earlier following the elixir preparation than the capsule. Plasma concentrations were similar following both injectable preparations. The potency of i.v. temazepam in inducing drowsiness in patients was much less than expected and doses greater than 0.6 mg kg-1 were required to produce adequate sedation. There was a significant reduction in thiopentone induction dose in patients receiving temazepam i.v.

Aspirin and probenecid pretreatment influences the potency of thiopentone and the onset of action of midazolam.

The effects of pretreatment with aspirin and probenecid, two drugs which are highly bound to albumin, on an induction dose of thiopentone and time to onset of action of a fixed dose of midazolam were investigated. Both drugs significantly potentiate thiopentone and shorten the induction time with midazolam. The importance of individual variations in plasma proteins in influencing the action of highly bound drugs is stressed.

Pretreatment with opioids. The effect on thiopentone induction requirements and on the onset of action of midazolam.

Pretreatment with small doses of fentanyl (100 micrograms) or alfentanil (300 micrograms) was found significantly to reduce the induction dose of thiopentone. Fentanyl 50 micrograms and alfentanil 150 micrograms also significantly reduced the onset time and increased the consistency of action of midazolam. Respiratory depression was not a problem when 50 micrograms fentanyl or 150 micrograms alfentanil were used.