A detailed structural description of Escherichia coli succinyl-CoA synthetase

Succinyl-CoA synthetase (SCS) carries out the substrate-level phosphorylation of GDP or ADP in the citric acid cycle. A molecular model of the enzyme from Escherichia coli, crystallized in the presence of CoA, has been refined against data collected to 2.3 A resolution. The crystals are of space group P4322, having unit cell dimensions a=b=98.68 A, c=403.76 A and the data set includes the data measured from 23 crystals. E. coli SCS is an (alphabeta)2-tetramer; there are two copies of each subunit in the asymmetric unit of the crystals. The crystal packing leaves two choices for which pair of alphabeta-dimers form the physiologically relevant tetramer. The copies of the alphabeta-dimer are similar, each having one active site where the phosphorylated histidine residue and the thiol group of CoA are found. CoA is bound in an extended conformation to the nucleotide-binding motif in the N-terminal domain of the alpha-subunit. The phosphoryl group of the phosphorylated histidine residue is positioned at the amino termini of two alpha-helices, one from the C-terminal domain of the alpha-subunit and the other from the C-terminal domain of the beta-subunit. These two domains have similar topologies, despite only 14 % sequence identity. By analogy to other nucleotide-binding proteins, the binding site for the nucleotide may reside in the N-terminal domain of the beta-subunit. If this is so, the catalytic histidine residue would have to move about 35 A to react with the nucleotide.     

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes

This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114+/-18 micromol l-1 and Kd of 0.59+/-0.17 microM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca.     

Effects of test conditions on the outcome of place conditioning with morphine and naltrexone in mice

BACKGROUND: Although it is widely proposed that surgeons, before introducing a novel laparoscopic technique in man, should practice in an appropriate animal model for acquisition of the necessary technical skills, the effectiveness of those hands-on training courses are rarely documented. METHODS: In 1995 we have organized eight hands-on training courses for laparoscopic anterior interbody spine fusion in an in vivo porcine model. A total of 72 colleagues from 50 different centers of 12 countries participated, including orthopedic, trauma, visceral, neuro-, and vascular surgeons. Quality and effectiveness of the course were evaluated by a questionnaire after a 1.5- to 2.5-year period. RESULTS: During this time, 42.2% of the participating centers had applied the new technique successfully in man. Centers which participated in the course with a team that included a skilled laparoscopic surgeon and an orthopedic or trauma surgeon introduced the technique more frequently to clinical practice (57.9%) than those represented by only one participant (30. 8%). Moreover, there was a tendency toward a more frequent introduction of the technique to clinical practice in centers associated with university hospitals (57.1% vs. 29.2%), indicating the requirement of a particular infrastructure for this complex interdisciplinary procedure. Almost all participants (98.3%) agreed that for novel surgical techniques requiring advanced technical skills, there should first be training in a large animal model before the technique is applied in man. CONCLUSIONS: Complex laparoscopic procedures (i.e., laparoscopic spine surgery) can be successfully learned by in vivo hands-on training courses. We propose that for refinements and modifications of the technique (e.g. , the lumboscopic approach), there should also first be training in a large animal model before these are applied in man.     

Functional analysis of human MutSalpha and MutSbeta complexes in yeast

Mismatch repair (MMR) is initiated when a heterodimer of hMSH2*hMSH6 or hMSH2*hMSH3 binds to mismatches. Here we perform functional analyses of these human protein complexes in yeast. We use a sensitive genetic system wherein the rate of single-base deletions in a homopolymeric run in the LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild-type strain. Expression of the human proteins alone or in combination does not reduce the mutation rate of the msh2 strain, and expression of the individual human proteins does not increase the low mutation rate of a wild-type strain. However, co-expression of hMSH2 and hMSH6 in wild-type yeast increases the mutation rate 4000-fold, while co-expression of hMSH2 and hMSH3 elevates the rate 5-fold. Analysis of cell extracts indicates that the proteins are expressed and bind to mismatched DNA. The results suggest that hMutSalpha and hMutSbeta complexes form, bind to and prevent correction of replication slippage errors in yeast. Expression of hMSH6 with hMSH2 containing a proline substituted for a conserved Arg524 eliminates the mutator effect and reduces mismatch binding. The analogous mutation in humans is associated with microsatellite instability, defective MMR and cancer, illustrating the utility of the yeast system for studying human disease alleles.     

Toxin involvement in staphylococcal scalded skin syndrome

The production of staphylococcal exfoliative toxin A (ETA) and toxin B (ETB), toxic shock syndrome toxin (TSST-1), and enterotoxins A-E was analyzed in 60 Staphylococcus aureus strains isolated from children with scalded skin syndrome (15 with generalized exfoliative syndrome, 28 with bullous impetigo, and 17 with staphylococcal scarlet fever). All strains isolated from patients with generalized exfoliative syndrome or bullous impetigo produced ETA and/or ETB and caused a Nikolsky's sign when injected subcutaneously into newborn mice. In contrast, exfoliative toxin was detected in an S. aureus strain from only one of 17 case of staphylococcal scarlet fever; the 16 other S. aureus strains produced TSST-1 and/or an enterotoxin. In conclusion, enterotoxins or TSST-1 are more frequently associated with staphylococcal scarlet fever than are exfoliative toxins. Hence staphylococcal scarlet fever may well represent an abortive form of toxic shock syndrome rather than a milder form of staphylococcal scalded skin syndrome.     

HLA class II genes in chronic hepatitis C virus-infection and associated immunological disorders

To investigate the factors that may confer susceptibility or protection to hepatitis C virus (HCV) infection and to HCV-associated immunological disorders, we designed two studies on 420 Sardinian transfusion-dependent thalassemia patients followed in our department in Cagliari since 1974. The first one was an epidemiological survey aimed to evaluate the prevalence of HCV infection and HCV-associated immunological disorders. In the second study, the distribution of different HLA class II genes was examined by DNA analysis in 116 HCV positive patients, 30 HCV negative patients, and 606 healthy controls. Three hundred fourteen patients became infected with HCV (74.7%) after 5.6 +/- 2.8 years of regular transfusion program. Mixed cryoglobulinemia, purpura, arthritis, proteinuria, decreased complement levels, rheumatoid factor and anti-GOR, smooth muscle antibody (SMA), anti-nuclear antibody (ANA), and liver, kidney microsome (LKM) autoantibodies were significantly more represented in HCV positive patients than in negative ones (P < .05). A significant increase of HLA class II DR2 subtype (DRB1*1601,DQB1*0502) was observed in a group of 30 HCV negative patients who despite 10.3 +/- 2.2 years in a regular blood transfusion program did not show any evidence of HCV infection (Pc < .0092). Our results represent clear evidence for a relationship between HCV infection and immune extrahepatic abnormalities. A gene(s) located in the human major histocompatibility complex (MHC) region may play an important role in conferring protection against HCV infection.     

Characterization of graf, the GTPase-activating protein for rho associated with focal adhesion kinase. Phosphorylation and possible regulation by mitogen-activated protein kinase

Graf is a GTPase-activating protein for Rho that interacts with focal adhesion kinase and co-localizes with the actin cytoskeleton (Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (1996) Mol. Cell. Biol. 16, 3169-3178). We examined the expression and regulation of Graf as a prelude to understanding the role of Graf in mediating signal transduction in vivo. We demonstrated that Graf is a ubiquitously expressed 95-kDa protein with high levels observed in heart and brain and cells derived from these tissues. Stimulation of PC12 cells with epidermal growth factor or nerve growth factor induced a phosphatase-reversible mobility shift upon gel electrophoresis, indicative of phosphorylation. In vitro, purified mitogen-activated protein (MAP) kinase catalyzed the phosphorylation of Graf on serine 510, suggesting that Graf phosphorylation may be mediated through MAP kinase signaling. In addition, the mutation of serine 510 to alanine inhibited the epidermal growth factor-induced mobility shift of mutant Graf protein in vivo, consistent with serine 510 being the site of in vivo phosphorylation. Based on these data we suggest that phosphorylation of Graf by MAP kinase or related kinases may be a mechanism by which growth factor signaling modulates Rho-mediated cytoskeletal changes in PC12 and perhaps other cells.     

Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins

Vaccinia virus vectors were used to express the major (L1) and minor (L2) capsid proteins of human papillomavirus type 1 (HPV-1) with the vaccinia virus early (p7.5K) or late (pSynth, p11K) promoters. All constructs expressed the appropriate-sized HPV proteins, and both L1 and L2, singly or in combination, localized to the nucleus. Capsids were purified by cesium chloride density gradient centrifugation from nuclei of cells infected with a vaccinia virus-L1 (vac-L1) recombinant or a vac-L1-L2 recombinant but not from vac-L2-infected cells. Electron microscopy showed that the particles were 55 nm in diameter and had icosahedral symmetry. Immunogold-labeled antibodies confirmed the presence of the L1 and L2 proteins in the HPV-1 capsids. Capsids containing L1 alone were fewer and more variable in size and shape than capsids containing the L1 and L2 proteins. The L1-plus-L2 capsids were indistinguishable in appearance from HPV-1 virions obtained from plantar warts. The ability to produce HPV capsids in vitro will be useful in many studies of HPV pathogenicity.     

Does the preoperative administration of ketorolac improve postoperative analgesia

AIM OF THE STUDY: 1) To verify the usefulness of ketorolac administration (30 mg i.v.) before a surgical operation in terms of postoperative analgesia improvement; 2) To evaluate the impact of preoperative ketorolac administration on perioperative renal function and on intraoperative water balance; 3) to evaluate the presence of adverse effect due to preoperative NSAID use. DESIGN: Prospective randomized trial. SETTING: University surgical department. PATIENTS AND METHODS: Forty adult patients undergoing major abdominal surgery, randomized in 2 groups: in group 1 ketorolac (30 mg i.v.) was administered immediately after the induction and, for postoperative analgesia, ketorolac (30 mg i.v.) was administered beginning at the time of skin closure; in group 2 no ketorolac was administered before the operation and postoperative treatment was the same. Buprenorphine (0.3 mg i.m.) was administered in case of unsatisfactory analgesia. Fluids infused and diuresis were measured intraoperatively. One, 6 and 24 hours after the end of operation pain was evaluated using pain intensity score and VAS. The day after the operation serum creatinine and urea were measured. RESULTS: No statistically significant differences were found between groups regarding fluids infused, intraoperative diuresis, postoperative pain, adverse effects and number of bleeding episodes. More than 50% of patients, in either groups, required opioids administration. CONCLUSIONS: Ketorolac (30 mg i.v.) administration before a major abdominal operation does not improve postoperative analgesia nor determines significant alterations in renal function or increase in the frequency of abnormal bleedings. Opiate administration is necessary in more than 50% of the patients to achieve adequate analgesia.