Structural analysis of the GH43 enzyme Xsa43E from Butyrivibrio proteoclasticus.

The rumen of dairy cattle can be thought of as a large, stable fermentation vat and as such it houses a large and diverse community of microorganisms. The bacterium Butyrivibrio proteoclasticus is a representative of a significant component of this microbial community. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fibre-degrading enzymes. This suite of enzymes is essential for the organism to utilize the plant material within the rumen as a fuel source, facilitating its survival in this competitive environment. Xsa43E, a GH43 enzyme from B. proteoclasticus, has been structurally and functionally characterized. Here, the structure of selenomethionine-derived Xsa43E determined to 1.3 Šresolution using single-wavelength anomalous diffraction is reported. Xsa43E possesses the characteristic five-bladed ß-propeller domain seen in all GH43 enzymes. GH43 enzymes can have a range of functions, and the functional characterization of Xsa43E shows it to be an arabinofuranosidase capable of cleaving arabinose side chains from short segments of xylan. Full functional and structural characterization of xylan-degrading enzymes will aid in creating an enzyme cocktail that can be used to completely degrade plant material into simple sugars. These molecules have a range of applications as starting materials for many industrial processes, including renewable alternatives to fossil fuels.

Operative strategy for fistula-in-ano without division of the anal sphincter.

INTRODUCTION: The treatment of perianal fistulas is diverse because no single technique is universally effective. Fistulotomy remains the most effective way of eradicating the pathology but it renders the patient at some risk of faecal incontinence, which many patients are reluctant to take. There are no data in the literature to indicate the healing rate of perianal fistulas when using an operative strategy that routinely avoids division of any part of the anal sphincter. The aim of this paper is to present the long-term results with an operative strategy that aims to avoid division of any part of the anal sphincter complex when treating all types of perianal fistulas, thereby minimising/eliminating the risk of postoperative incontinence. METHODS: We report 54 consecutive cases of anal fistula that presented electively and as an emergency. Patients with known or subsequently diagnosed inflammatory bowel disease or malignancy were excluded from the study. RESULT: Overall, 46 patients (37 male and 9 female) with a median age at presentation of 42 years (range: 19-73 years) were treated by lay-open of the subcutaneous tract of the perianal fistula and insertion of a loose seton for the part of the fistula tract related to the sphincter complex. The types of fistula treated were intersphincteric (89%), transsphincteric (4%) and high suprasphincteric (7%). The median length of time that the seton was left in place was 7 months (range: 1.5-24 months). The healing rate was 86% with a recurrence rate of 19% and a median follow-up duration of 42 months. CONCLUSIONS: Patients who are reluctant to take any risk of faecal incontinence could be treated using an operative strategy that routinely avoids division of any part of the anal sphincter complex as this has a recurrence rate that compares well with other treatment modalities.

The utility of B-type natriuretic peptide in predicting postoperative cardiac events and mortality in patients undergoing major emergency non-cardiac surgery.

B-type natriuretic peptide (BNP) levels predict cardiovascular risk in several settings. We hypothesised that they would identify individuals at increased risk of complications and mortality following major emergency non-cardiac surgery. Forty patients were studied with a primary end-point of a new postoperative cardiac event, and/or development of significant ECG changes, and/or cardiac death. The main secondary outcome was all-cause mortality at 6 months. Pre-operative BNP levels were higher in 11 patients who suffered a new postoperative cardiac event (p = 0.001) and predicted this outcome with an area under the receiver operating characteristic curve of 0.85 (CI = 0.72-0.98, p = 0.001). A pre-operative BNP value > 170 pg x ml(-1) has a sensitivity of 82% and a specificity of 79% for the primary end-point. In this small study, pre-operative BNP levels identify patients undergoing major emergency non-cardiac surgery who are at increased risk of early postoperative cardiac events. Larger studies are required to confirm these data.

Dynamics of leg muscle function in tammar wallabies (M. eugenii) during level versus incline hopping.

The goal of our study was to examine whether the in vivo force-length behavior, work and elastic energy savings of distal muscle-tendon units in the legs of tammar wallabies (Macropus eugenii) change during level versus incline hopping. To address this question, we obtained measurements of muscle activation (via electromyography), fascicle strain (via sonomicrometry) and muscle-tendon force (via tendon buckles) from the lateral gastrocnemius (LG) and plantaris (PL) muscles of tammar wallabies trained to hop on a level and an inclined (10 degrees, 17.4% grade) treadmill at two speeds (3.3 m s(-1) and 4.2 m s(-1)). Similar patterns of muscle activation, force and fascicle strain were observed under both level and incline conditions. This also corresponded to similar patterns of limb timing and movement (duty factor, limb contact time and hopping frequency). During both level and incline hopping, the LG and PL exhibited patterns of fascicle stretch and shortening that yielded low levels of net fascicle strain [LG: level, -1.0+/-4.6% (mean +/- S.E.M.) vs incline, 0.6+/-4.5%; PL: level, 0.1+/-1.0% vs incline, 0.4+/-1.6%] and muscle work (LG: level, -8.4+/-8.4 J kg(-1) muscle vs incline, -6.8+/-7.5 J kg(-1) muscle; PL: level, -2.0+/-0.6 J kg(-1) muscle vs incline, -1.4+/-0.7 J kg(-1) muscle). Consequently, neither muscle significantly altered its contractile dynamics to do more work during incline hopping. Whereas electromyographic (EMG) phase, duration and intensity did not differ for the LG, the PL exhibited shorter but more intense periods of activation, together with reduced EMG phase (P<0.01), during incline versus level hopping. Our results indicate that design for spring-like tendon energy savings and economical muscle force generation is key for these two distal muscle-tendon units of the tammar wallaby, and the need to accommodate changes in work associated with level versus incline locomotion is achieved by more proximal muscles of the limb.

Crystal structure of a gamma-herpesvirus cyclin-cdk complex.

Several gamma-herpesviruses encode proteins related to the mammalian cyclins, regulatory subunits of cyclin-dependent kinases (cdks) essential for cell cycle progression. We report a 2.5 A crystal structure of a full-length oncogenic viral cyclin from gamma-herpesvirus 68 complexed with cdk2. The viral cyclin binds cdk2 with an orientation different from cyclin A and makes several novel interactions at the interface, yet it activates cdk2 by triggering conformational changes similar to cyclin A. Sequences within the viral cyclin N-terminus lock part of the cdk2 T-loop within the core of the complex. These sequences and others are conserved amongst the viral and cellular D-type cyclins, suggesting that this structure has wider implications for other cyclin-cdk complexes. The observed resistance of this viral cyclin-cdk complex to inhibition by the p27(KIP:) cdk inhibitor is explained by sequence and conformational variation in the cyclin rendering the p27(KIP:)-binding site on the cyclin subunit non-functional.

Monoclonal antibodies to CD45 modify LPS-induced arachidonic acid metabolism in macrophages.

Signaling by lipopolysaccharide (LPS) through CD14 involves the activation of protein tyrosine kinases of the src family and leads to cytokine production and activation of arachidonic acid metabolism in macrophages. CD45 protein tyrosine phosphatase (PTPase) might play a role in modulating the response through this pathway. Although a critical role in regulation of T-cell signaling for CD45 has been demonstrated, little is known about its role in macrophages. Monoclonal antibodies to CD45 and F(ab')(2) fragments of the monoclonal antibody enhanced the response of differentiated THP-1 monocytic cells to LPS for the release of radiolabeled arachidonic acid metabolites, prostaglandin E(2), and tumor necrosis factor alpha. The enhancing effect of anti-CD45 mAbs was shown to occur primarily through CD14-dependent signaling by performing the experiments under conditions favoring that pathway. Further, LPS may be able to alter the enzymatic activity of CD45, as shown by Western blots of CD45 immunoprecipitates in which LPS caused a transient change in the phosphorylation state of CD45. We conclude that CD45 appears to play a role in LPS-induced responses through the CD14 pathway, possibly through its PTPase activity.

Overcoming inhibitions: subversion of CKI function by viral cyclins.

DNA tumour viruses deregulate the mammalian cell cycle to provide a better environment for their replication. Studies of such deregulation have led to the identification of key regulatory steps that normally control the G1-S phase transition of the cell cycle. The balance between the activities of G1-specific cyclin-CDK complexes and their inhibitors is critical. Recent studies suggest that certain herpesviruses disrupt this balance: the viruses encode a cyclin that generates active complexes even in the presence of high inhibitor levels.

A comparison of the effects of lipopolysaccharide and ceramide on arachidonic acid metabolism in THP-1 monocytic cells.

Ceramide has been shown to be an important second messenger for signal transduction in cells of myeloid lineage. Studies have suggested that lipopolysaccharide (LPS) may activate signaling pathways by mimicking the action of ceramide. We explored this hypothesis with THP-1 cells in terms of the effects of LPS, C2 ceramide, and sphingomyelinase on arachidonic acid metabolism as measured by the release of radiolabeled eicosanoids. Arachidonic acid metabolism was activated by both LPS and ceramide. However, the ratio of prostaglandin E2 to leukotriene C4 was 10 times higher in cells treated with LPS than with ceramide. Unlike LPS, prior exposure to ceramide did not desensitize the cells to subsequent challenge with either LPS or ceramide, nor could LPS desensitize the cells to challenge with ceramide. The results suggest that, although LPS and ceramide may share signaling components, the signaling pathways are not identical.

Chromospheric Helium Imaging Photometer (an Instrument for High Time Cadence 1083-nm Wavelength Solar Observations).

The scientific motivation, design criteria, and specifications for a new ground-based instrument to observe the Sun in the He i 1083-nm spectral line is described. The instrument employs a liquid-crystal tunable Lyot-type spectral filter and an array detector that allows the full solar disk to be observed with a time cadence of minutes. We describe the telescope's optical and mechanical features and discuss computer interface and data-reduction procedures employed. Instrument performance during the initial year of operation of the telescope at its high-altitude site is summarized.

A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin.

The hemophilia A mutation database lists more than 160 missense mutations: each represents a molecular defect in the FVIII molecule, resulting in the X-linked bleeding disorder hemophilia A with a clinical presentation varying from mild to severe. Without a three-dimensional FVIII structure it is in most cases impossible to explain biological dysfunction in terms of the underlying molecular pathology. However, recently the crystal structure of the homologous human plasma copper-binding protein ceruloplasmin (hCp) has been solved, and the A domains of FVIII share approximately 34% sequence identity with hCp. This advance has enabled the building of a molecular model of the A domains of FVIII based on the sequence identity between the two proteins. The model allows exploration of predictions regarding the general features of the FVIII molecule, such as the binding-sites for factor IXa and activated protein C; it has also allowed the mapping of more than 30 selected mutations with known phenotype from the database, and the prediction of hypothetical links to dysfunction in all but a few cases. A computer-generated molecular model such as that reported here cannot substitute for a crystal structure. However, until such a structure for FVIII becomes available, the model represents a significant advance in modeling FVIII; it should prove a useful tool for exploiting the increasing amount of information in the hemophilia A mutation database, and for selecting appropriate targets for investigation of the structure-function relationships via mutagenesis and expression in vitro.

Crystallization and preliminary X-ray studies of nitrogenase component 1 (the MoFe protein) from Klebsiella pneumoniae.

Two crystal forms of component 1 (the MoFe protein) of nitrogenase from Klebsiella pneumoniae have been isolated and characterized. The triclinic form has cell dimensions a = 76.0, b = 109.6, c = 144.6 A, alpha = 80.3, beta = 74.9 and gamma = 69.6 degrees, diffracts to around 3.0 A and has two molecules in the asymmetric unit. The monoclinic form belongs to space group P2(1) with a = 76.6, b = 127.8, c = 109.1 A and beta = 104.6 degrees (frozen at 100 K), diffracts to 1.5 A and has one molecule in the asymmetric unit. At this resolution the outstanding questions concerning the structure and the operation of the enzyme, in particular the linkage between the Fe(4)S(4) units in the P clusters, the true geometry of the apparently trigonal Fe atoms in the FeMoco and the reduction site itself, should be answerable.

Crystallization and preliminary X-ray crystallographic analysis of the putative [6Fe-6S] prismane protein from Desulfovibrio vulgaris (Hildenborough).

Crystals of the prismane protein from Desulfovibrio vulgaris (Hildenborough) containing a putative [6Fe-6S] cluster have been obtained and X-ray data collected to a resolution of 1.7 A using synchrotron radiation. The unit cell is orthorhombic with a = 64.1, b = 65.1 and c = 154.1 A, space group P2(1)2(1)2(1) (No. 19). The unit cell will readily accommodate four molecules of molecular weight 60 kDa with a corresponding solvent content of approximately 48%.

Activation of arachidonic acid metabolism in mouse macrophages by bacterial amphiphiles.

The relative activities of lipoteichoic acid (LTA) from four Gram-positive bacteria were compared to different lipopolysaccharide (LPS) preparations for activation of arachidonic acid metabolism in mouse peritoneal macrophages. Total eicosanoid was determined in cultures labeled with [3H]-arachidonic acid. Prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) were determined by EIA analysis. The relative potencies of the different preparations were: smooth LPS from Salmonella abortus > or = Re-LPS from Salmonella minnesota (R-595) > or = LTA from Streptococcus pyogenes approximately Streptococcus faecalis approximately Staphylococcus aureus > or = monophosphoryl lipid A derived from the Re-LPS >> LTA from Bacillus subtilis. Activation of eicosanoid release was inhibited by staurosporin for all of the amphiphiles tested. Treatment of the macrophage cultures with LTA from S. pyogenes, S. faecalis, and S. aureus, either in the presence or absence of indomethacin, desensitized the cells to eicosanoid release on subsequent challenge with LPS. The desensitized cells remained responsive to the phorbol ester phorbol myristate acetate. LPS from Gram-negative bacteria has immunostimulatory and endotoxic activities which result, in part, from the release of eicosanoids and other mediators from activated macrophages. The similarities in the patterns of cell activation by LPS and LTA suggest that lipoteichoic acids might contribute to the pathogenicities of Gram-positive bacteria.

Loneliness, depression, and social support of patients with COPD and their spouses.

Thirty clients with chronic obstructive pulmonary disease (COPD) and their spouses were interviewed to examine differences in the relationships among loneliness, depression, and social support. Data were collected during structured in-home interviews using the UCLA loneliness scale, the Center for Epidemiological Studies depression scale, and the social support questionnaire. The clients and spouses did not differ significantly on measures of loneliness and depression, with mean scores for both groups higher than those in other comparable groups. Spouses, however, tended to be a little lonelier than clients, and clients tended to be a little more depressed than spouses. The two groups were also similar with respect to the number of people in their social networks but different as to network composition. Spouses were less satisfied with their networks than clients. Social support satisfaction was linked to loneliness and depression for clients but not for spouses. Results of the study suggest that community nurses working in home settings must be sensitive to clients' and spouses' psychologic reactions to COPD, which may be expressed in feelings of loneliness and depression.

Carbonate inhibition of leukotriene D4-dipeptidase in human serum.

Human serum contains an inhibitor of leukotriene D4 (LTD4) dipeptidase which was separated from the enzyme by ultrafiltration (Amicon, YM-10). Removal of the inhibitor resulted in a 3- to 5-fold increase in total LTD4-dipeptidase activity in the material retained by the filter. Inhibitor activity (which was assayed with a partially purified LTD4-dipeptidase) was recovered in the filtrate. Ultrafiltration of serum using YM-3, YM-1, and YC-05 membranes suggested an inhibitor molecular weight of less than 500. Elution of inhibitor activity from a Bio Gel P2 gel filtration column was identical to the elution pattern of pure carbonate. The inhibitor was heat stable (95 degrees C, 30 min), stable in 0.1 N NaOH, but rapidly inactivated by 0.1 N HCl at both 4 degrees C and 30 degrees C. Partially purified LTD4-dipeptidase was inhibited by carbonate and phosphate but not by nitrate, sulfate, or chloride. Based on these observations it was concluded that the inhibitor of LTD4-dipeptidase in human serum either was carbonate or required carbonate. The relative concentrations of LTC4, LTD4, and LTE4 appear to be important parameters in determining the duration and intensity of LT mediated reactions. The relative concentration of carbonate in serum or extracellular fluids might, therefore, be a factor in modulating localized LT mediated responses.

Role of anionic lipid in bacterial membranes.

The major phospholipids of Bacillus stearothermophilus are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Under the growth conditions used in this study the concentration of anionic lipid (PG + CL) was determined by the pH of the culture medium. Cells grown in a complex medium at pH 5.8, 7.0, and 8.0 contained 17, 29 and 36 nmol of anionic (PG + CL) lipid/mg cell (dry weight). The concentration of the zwitterionic lipid phosphatidylethanolamine (PE) was 17-20 nmol/mg cell (dry weight) under all conditions. Analysis of isolated membrane preparations suggested that the amount of anionic lipid per unit area of membrane increased as the pH of the growth medium was increased. Membranes from cells grown at pH 5.8 and 8.0 contained 130 and 320 nmol anionic lipid/mg membrane protein, respectively. Phosphatidylethanolamine appeared to be localized on the inner membrane surface in cells grown under all conditions. Increasing the ionic strength of the culture medium by the addition of NaCl or KCl had little effect on growth at pH 5.8 but inhibited growth at pH 7 and 8. It was concluded that anionic phospholipid plays an important physiological role in maintaining an acidic pH at the outer membrane surface.

Leukotriene metabolism and hypersensitivity in man and mouse.

The relative rates of cysteinyl-leukotriene metabolism were analyzed in fresh human and mouse serum. Human serum contained higher gamma-glutamyl-transpeptidase activity than mouse serum, and a higher percentage of the metabolized leukotriene C4 was recovered as leukotriene D4 in the human serum than in the mouse serum. The results suggest that the patterns of metabolism of the cysteinyl-leukotrienes could be an important factor in determining the relative sensitivity of an animal to the development of hypersensitivity reactions.

Products of phospholipid metabolism in Bacillus stearothermophilus.

The products of phospholipid turnover in Bacillus stearothermophilus were determined in cultures labeled to equilibrium and with short pulses of [32P]phosphate and [2-3H]glycerol. Label lost from the cellular lipid pool was recovered in three fractions: low-molecular-weight extracellular products, extracellular lipid, and lipoteichoic acid (LTA). The low-molecular-weight turnover products were released from the cells during the first 10 to 20 min of a 60-min chase period and appeared to be derived primarily from phosphatidylglycerol turnover. Phosphatidylethanolamine, which appeared to be synthesized in part from the phosphatidyl group of phosphatidylglycerol, was released from the cell but was not degraded. The major product of phospholipid turnover was LTA. Essentially all of the label lost from the lipid pool during the final 40 min of the chase period was recovered as extracellular LTA. The LTA appeared to be derived primarily from the turnover of cardiolipin and the phosphatidyl group of phosphatidylglycerol. Three types of LTA were isolated; an extracellular LTA was recovered from the culture medium, and two types of LTA were extracted from membrane preparations or whole-cell lysates by the hot phenol-water procedure. Cells contained 1.5 to 2.5 mg of cellular LTA per g of cells (dry weight), over 50% of which remained associated with the membrane when cells were fractionated. Over 75% of the 3H label incorporated into the cellular LTA pool during a 90-min labeling period was released from the cells during the first cell doubling after the chase. Label lost from the lipid pool was incorporated into cellular LTA which was then modified and released into the culture medium.

The relationships between growth temperature, fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica.

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37 degrees C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37 degrees C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73--76%), phosphatidylglycerol (9--11%) and cardiolipin (11--13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37 degrees C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at --13, --9 and 1 degree C for membranes from cells grown at 5, 22 and 37 degrees C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5 degrees C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.

Protein-associated lipid of Bacillus stearothermophilus.

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.