Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops.

Protein stability is a widely studied topic, there are still aspects however that need addressing. In this paper we examined the effects of multiple proline substitutions into loop regions of the kinetically stable proteinase K-like serine protease VPR, using the thermostable structural homologue AQUI as a template. Four locations for proline substitutions were chosen to imitate the structure of AQUI. Variants were produced and characterized using differential scanning calorimetry (DSC), circular dichroism (CD), steady state fluorescence, acrylamide fluorescence quenching and thermal inactivation experiments. The final product VPRΔC_N3P/I5P/N238P/T265P was greatly stabilized which was achieved without any noticeable detrimental effects to the catalytic efficiency of the enzyme. This stabilization seems to be derived from the conformation restrictive properties of the proline residue in its ability to act as an anchor point and strengthen pre-existing interactions within the protein and allowing for these interactions to prevail when thermal energy is applied to the system. In addition, the results underline the importance of the synergy between distant local protein motions needed to result in stabilizing effects and thus giving an insight into the nature of the stability of VPR, its unfolding landscape and how proline residues can infer kinetic stability onto protein structures.

Risk factors and outcome in ICU-acquired infections.

BACKGROUND: Nosocomial infections are common in intensive care units (ICU). The objectives of this study were to determine risk factors of ICU-acquired infections, and potential mortality attributable to such infections. METHODS: An observational study was performed in a 10-bed multidisciplinary ICU. For a period of 27 months, all patients admitted for >or=48 h were included. Infections were diagnosed according to Centers for Disease Control and Prevention definitions. Airway colonization was explored by molecular typing. Risk factors for infection were determined by multivariable logistic regression. Survival was analyzed with time-varying proportional hazards regression. RESULTS: Of 278 patients, 81 (29%) were infected: urinary tract infections in 39 patients (14%), primary bloodstream infections in 25 (9%), surgical site infections in 22 (8%) and pneumonia in 21 (8%). Of the total of 147 episodes, Gram-negative bacilli were isolated in 90, Gram-positive cocci in 49 and Candida sp. in 25. Risk factors for pneumonia were mechanical ventilation [odds ratio (OR=7.9, CI 1.8-35), lack of enteral nutriment (OR=8.0, CI 1.4-45) and length of time at risk (OR=1.8, CI 1.2-2.8), while gastric acid inhibitors did not affect the risk (OR=0.99, CI 0.32-3.0). Transmission of bacteria from the stomach to the airway was not confirmed. The risk of death was increased as patients were infected with pneumonia [hazard ratio (HR)=3.6; CI: 1.6-8.1], or primary bloodstream infection (HR=2.5; CI: 1.2-5.4), independent of age and disease severity. CONCLUSIONS: Mortality was increased by ICU-acquired pneumonia and primary bloodstream infections. Our findings did not support the gastro-pulmonary hypothesis of ICU-acquired pneumonia. The proposition that blood transfusions increase the risk of ICU-acquired nosocomial infections was not supported.

Incidence and outcome of Staphylococcus aureus endocarditis--a 10-year single-centre northern European experience.

Staphylococcus aureus is a leading cause of infective endocarditis. Little has been published on the outcome and epidemiology of S. aureus endocarditis (SAE) in the twenty-first century. Our aim was to evaluate the short-term and long-term outcome of SAE in Stockholm, Sweden, and assess its incidence over time. Patients treated for SAE from January 2004 through December 2013 were retrospectively identified at the Karolinska University Hospital. Clinical data were obtained from medical records and the diagnosis was verified according to the modified Duke criteria. Of 245 SAE cases, 152 (62%) were left-sided and 120 (49%) occurred in intravenous drug users. The calculated incidence in Stockholm County was 1.56/100 000 person-years, increasing from 1.28 in 2004-08 to 1.82/100 000 person-years in 2009-13 (p 0.002). In-hospital and 1-year mortality rates were 9.0% (22/245) and 19.5% (46/236), respectively. Age (OR 1.06 per year) and female sex (OR 3.0) were independently associated with in-hospital mortality in multivariate analysis. Involvement of the central nervous system (CNS) was observed in 30 (12%) patients, and valvular surgery was performed during hospitalization in 37 (15%). In left-sided endocarditis the strongest predictors for surgery were severe valvular insufficiency (OR 8.9), lower age (OR 1.07 per year) and no intravenous drug use (OR 10.7), and for CNS involvement lower age (OR 1.04 per year). In conclusion we noted low mortality, low CNS complication rate, and low valvular surgery frequency associated with SAE in our setting. The incidence was high and increased over time. The study provides an update on the outcome and epidemiology of SAE in the twenty-first century.

Genotype-phenotype interactions in Wilson's disease: insight from an Icelandic mutation.

Wilson's disease, an autosomal recessive disorder of copper transport, usually presents with symptoms from the liver or central nervous system. Rarely, the initial manifestation is fulminant hepatic failure. The abnormal gene (ATP7B) is located on chromosome 13q and encodes a copper-transporting ATPase. A large number of mutations have been reported. We describe a previously healthy 16-year-old girl who presented with fulminant hepatic failure. The girl died within 24 h of admission to a hospital from refractory shock. Autopsy revealed cirrhosis and widespread necrosis of the liver. The copper content of the liver was markedly increased (975 micrograms/g dry weight), strongly suggesting a diagnosis of Wilson's disease. Genetic studies revealed that the girl was homozygous for the mutation 2007 del7, which is the mutation found in all Wilson's disease patients previously identified in Iceland. This is the first known case of fulminant hepatic failure due to Wilson's disease in Iceland. Despite the same mutation, the clinical picture is vastly different from other Icelandic patients with Wilson's disease, who all presented with relatively late-onset neurological disease. This suggests that factors other than the specific mutation have significant impact on the phenotype of the disease.

Characterization of a collagenolytic serine proteinase from the Atlantic cod (Gadus morhua).

A collagenolytic proteinase was purified from the intestines of Atlantic cod by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The proteinase has an estimated molecular weight of 24.1 (+/- 0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases. The enzyme cleaves native collagen types I, III, IV and V, and also readily hydrolyzes succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as well as succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, a reported elastase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin. The pH optimum of the enzyme was between pH 8.0 and 9.5 and it was unstable at pH values below 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50 degrees C. Calcium binding stabilized the cod collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30 degrees C.

Staphylococcus aureus bacteraemia in Iceland, 1995-2008: changing incidence and mortality.

Little is known about temporal changes in the epidemiology of Staphylococcus aureus bacteraemia. The objective of the present study was to analyse changes in the incidence and mortality of adult S. aureus bacteraemia in Iceland. Individuals 18 years or older with a positive blood culture for S. aureus between 1 January 1995 and 31 December 2008 were identified, with the participation of all clinical microbiological laboratories performing blood cultures in Iceland. Infections were categorized as nosocomial, healthcare-associated or community-acquired. National population statistics and dates of death were retrieved from the National Registry. During the study period, 692 individuals from 19 institutions had 721 distinct episodes of S. aureus bacteraemia. The incidence rose from 22.7 to 28.9 per 100,000 per year during the period (p 0.012). Nosocomial infections comprised 46.3% of cases, 14.6% were healthcare-associated, and 39.1% were community-acquired. The proportion of nosocomial infections decreased during the period (p <0.001), whereas an increase was seen in the proportion of community-acquired infections (p <0.001). All-cause 30-day mortality decreased from 25.0% to 8.1% (p 0.001) and 1-year mortality decreased from 37.0% to 27.9% (p 0.061) between the periods 1995-1996 and 2007-2008. Four cases of bacteraemia caused by methicillin-resistant S. aureus were seen (0.6%), none of which was fatal. In conclusion, there was a significant increase in the incidence of S. aureus bacteraemia in Iceland between 1995 and 2008. Concomitantly, there was a significant reduction in mortality, towards one of the lowest reported. Further studies are needed to understand the basis for these changes.

Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus mykiss).

1. Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). 2. The approximate molecular weights of chymotrypsin I and II were 28,200 (+/- 1200) and 28,800 (+/- 900), respectively, as determined by SDS-PAGE and their isoelectric points were about 5. 3. The pH optima of the enzymes were centered around nine, when assayed for succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-NA) as substrate and both enzymes were unstable at pH values below 5. 4. The amidase activity of both enzymes increased with temperature up to about 55 degrees C. Chymotrypsin I was found to be more heat stable than chymotrypsin II, an effect most likely explained by stronger calcium binding of the former. 5. The trout chymotrypsins were significantly more active than bovine alpha-chymotrypsin when assayed against Suc-AAPF-NA at 25 degrees C and casein at low temperatures (10-20 degrees C), indicating an adaptation of the activities of the trout chymotrypsins to the habitation temperatures of the fish.

Moraxella catarrhalis bacteraemia. A report on 3 cases and a review of the literature.

Over the last decade, an increase in invasive infections due to Moraxella catarrhalis has been reported. We have analysed 58 cases of bacteraemia due to M. catarrhalis reported in the literature and 3 cases found in Iceland, a total of 61 cases. Patients with bacteraemia could be divided into 3 groups on the basis of host factors. They were either immunocompromised, had underlying respiratory disorders. or were normal hosts. The clinical manifestation of M. catarrhalis bacteraemic infection ranged from a mild febrile illness to a fatal disease. The severity of the clinical picture did not reflect the patients' condition at the time of bacteraemia. The port of entry of the bacteraemia was frequently not elucidated in immunocompromised patients. Patients with a contributory respiratory tract disorder were more likely to develop bacteraemia as a result of a lower respiratory tract infection, whereas bacteraemia in a normal host was more frequently due to an upper respiratory tract infection. The overall prognosis of M. catarrhalis bacteraemia was good, the exception being when it caused endocarditis (5 cases), where mortality rates as high as 80% have been reported.

Alkaline serine proteinase from Thermomonospora fusca YX. Stability to heat and denaturants.

The serine proteinase isolated from Thermomonospora fusca YX shows considerable thermal stability up to 80 degrees C, and progressive inactivation occurs at higher temperatures. Lyotropic salts affected the thermal stability of the enzyme at 85 degrees C, suggesting that disruption of hydrophobic interactions play an important role in the decreased thermal stability of the enzyme above 80 degrees C. Thermal stability is highly pH-dependent; above pH 6.0-6.5 there is a sharp decrease in the stability of the enzyme, reflecting increased autolysis. Although some stabilization occurs upon increasing ionic strength, Ca2+ binding does not appear to play a role in thermal stability. Denaturants, i.e. 8 M-urea, 6 M-guanidinium chloride or 1% SDS, had no significant effect on the activity of the enzyme after 24 h at 25 degrees C.

Heat stable proteinase from Thermomonospora fusca. Characterization as a serine proteinase.

An extracellular proteinase secreted by the thermophilic bacteria Thermomonospora fusca YX (YX-proteinase) is a serine proteinase as shown by its inactivation by the site specific reagents, phenylmethanesulfonyl fluoride, dansyl fluoride, and carbobenzoxy-L-phenylalanine chloromethyl ketone. This conclusion is further supported by the effect of various proteinase inhibitors on its activity. The activity of the proteinase toward small synthetic ester substrates shows that the enzyme has a primary specificity for the aromatic and hydrophobic amino acids. The amino acid composition and NH2-terminal sequence, as well as its size, suggest that the enzyme is related to the chymotrypsin-like microbial proteinase, alpha-lytic protease from Myxobacter 495 and protease A and B from Streptomyces griseus.

Mycobacterium haemophilum infection in immunocompromised patients: case report and review of the literature.

Mycobacterium haemophilum, previously characterized as an unusual pathogen, is found primarily in immunocompromised hosts. This organism has stringent growth characteristics and may not be isolated using routine techniques. M. haemophilum infects the skin and underlying tissues, a circumstance which reflects the organism's propensity for growth in a cooler environment. Infections have been reported in renal transplant recipients, patients with Hodgkin's disease, and, more recently, patients with AIDS. The organism has also been isolated from children with cervical lymphadenitis in the absence of apparent immunodeficiency. Response to therapy has not been uniform, and in some instances improvement in immune status has been associated with regression of lesions. With proliferation of transplantation surgery, chemotherapy, and AIDS, the number of infections due to M. haemophilum is likely to increase.

Polymicrobial and recurrent bacteremia with Shigella in a patient with AIDS.

Shigella gastroenteritis is uncommon among HIV seropositive patients and may be complicated in some patients by bacteremia; S. flexneri being the most frequently detected serogroup. While recurrent Salmonella bacteremia is common among HIV-seropositive patients, recurrent Shigella bacteremia is not. We report here an HIV-seropositive patient with Shigella gastroenteritis, polymicrobial bacteremia due to S. flexneri and S. boydii, and recurrent gastroenteritis and bacteremia with S. boydii. Relapsing infection with the same strain of S. boydii was determined using pulsed field gel electrophoresis. Thus, HIV-seropositive patients who develop Shigella infections may require prolonged treatment and/or suppressive therapy, similar to those infected with Salmonella. Patients who develop recurrent disease should be suspected as having polymicrobial bacteremia since the incidence of this may be underestimated among patients with AIDS, particularly those with concurrent gastroenteritis.

1-Deoxyglycitolation of protein amino groups and their regeneration by periodate oxidation.

Reductive alkylation of lysyl epsilon-amino groups with sugars (1-deoxyglycitolation) using pyridine borane as the reducing agent has been recently described [Wong, W.S.D., Osuga, D.T. & Feeney, R.E. (1984) Anal. Biochem. 139, 58-67]. The regeneration of the lysines has now been achieved by oxidation with approximately 10 mM periodate. In experiments with glycitolated bovine serum albumin, reactions using 5, 20, and 80 mM periodate were essentially complete in the first 10 min. Oxidations of methionine to the sulfoxide were evident even at this lower concentration of periodate, while higher concentrations and prolonged reaction times only caused unnecessary destruction of amino acids. The removal was shown to occur in a wide pH range with little variation in the recovery of the lysines. The degree of glycitolation, or the nature of the attached carbohydrate residues, had no effect on the yield of products. Reductive 1-deoxygalactitolation of approximately 55% of the lysyl epsilon-amino groups of lysozyme caused no loss in enzymatic activity; when 5 mM periodate was used to remove the 1-deoxygalactitol moiety, approximately 95% of the amino groups were regenerated, concomitant with destruction of approximately 16% of the activity. On reductive 1-deoxygalactitolation of the amino groups of turkey ovomucoid, 65% of the lysyl epsilon-amino groups were modified with 70% loss of the inhibitory activity against trypsin. On treatment with 5 mM periodate, approximately 80% of the amino groups were regained with a similar recovery of the inhibitory activity.

Pneumonia caused by penicillin-non-susceptible and penicillin-susceptible pneumococci in adults: a case-control study.

The objective of this study was to investigate the observation that patients with pneumonia due to penicillin-non-susceptible pneumococci (PNSP) in many instances present with milder disease than patients with pneumonia caused by penicillin-susceptible pneumococci (PSP) and to compare the cost of treatment. The clinical data, APACHE II score and laboratory features of hospitalized adults with pneumonia caused by PNSP or PSP were compared, along with antibiotic and hospital costs. Each patient with PNSP pneumonia (n = 36) was matched to a control with PSP pneumonia of the same age and gender. There was no difference in smoking history, but PNSP pneumonia patients had received prior antibiotics more frequently (p < 0.007). The mean APACHE II score was not different, but when broken down into acute vs. chronic scores those with PSP pneumonia had a significantly higher acute score (p = 0.005). Bacteraemia was present in 9 of 31 (29%) patients with PSP compared to 2 of 25 (8%) with the PNSP pneumonia (p = 0.09). The majority of isolates in the PNSP group were of serotype 6B (minimum inhibitory concentration range 0.125-2.0 mg/l), whereas serotypes 7, 9, 14, 18 and 19 were noted among the 9 PSP isolates. Compared with the control group, patients with the PNSP strains had a significantly longer hospital stay, 26.8 vs. 11.5 days (p = 0.001) and higher average antibiotic cost, $736 vs. $213 (p < 0.0001). In conclusion, pneumonia in adults caused by PNSP is associated with a milder clinical presentation than infection caused by PSP, suggesting either that resistance carries a price or that the serotypes of PNSP are less virulent. Pneumonia due to PNSP resulted in increased cost because of prolonged hospitalization and the use of more expensive antibiotics.

Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus.

Small-colony variants (SCVs) of Staphylococcus aureus were cultured from five patients with persistent and relapsing infections. All five SCV strains were nonhemolytic and nonpigmented and grew very slowly on routine culture media in an ambient atmosphere. In several instances, these phenotypic characteristics led to the initial misidentification of the organisms in the clinical microbiology laboratory. All four strains available for further analysis were shown to be auxotrophs that reverted to normal growth and morphology in the presence of menadione, hemin, and/or a CO2 supplement. Similarly, these isolates were resistant to aminoglycosides under routine conditions but susceptible in the presence of the metabolic supplements. For two patients, the large and small colony forms isolated concurrently were indistinguishable when analyzed by pulsed field gel electrophoresis and thus represented phenotypic variants within individual clones. We propose a model relating the phenotypic characteristics of S. aureus SCVs with the clinical pattern of persistent and relapsing infection.

Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species comparison with proteinase K and aqualysin I.

An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases. The enzyme is secreted as a 47-kDa protein, but under mild heat treatment (30 min at 40 degrees C) undergoes autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47-kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. The catalytic efficiency (kcat/Km) for the amidase activity of the cold-adapted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I. One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant. The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H2O2-oxidation, as a function of temperature. The order of reactivity observed in these reactions most likely reflects the accessibility of the reactive cystine or methionine side chains present in the three related proteinases, and hence a difference in the compactness of their protein structures.

[Measurements of plasma HIV-1 RNA in Iceland.].

OBJECTIVE: First to measure plasma HIV-1 RNA in Icelandic HIV infected individuals and second to evaluate the initial effects of new combination regimens on viral load and CD4+ cell counts in HIV infected patients in Iceland. MATERIAL AND METHODS: The cohort studied consis notted of all HIV infected individuals we received samples from during the period September 1995 to November 1996. HIV-1 RNA and CD4+ cells were measured initially and subsequently every three to six months except when a change was made in the antiretroviral regimen, when samples were measured before the change, three to four weeks later and then every three to six months. The quantitative measurement of viral RNA was performed using the Amplicor HIV Monitor Test (Roche Diagnostic Sys nottems). CD4+ cell counts were measured by flow cytometry. RESULTS: A total of 44 patients were evaluated. The initial RNA ranged from %lt; 2.6 logio to 6.13 logio with a mean of 5.02 log. CD4+ cell counts ranged from 2 to 641 per mm3 (mean 230 cells/mm3). Eleven patients had never been treated with antiretroviral drugs and had greater than 10 000 viral copies per mL of plasma. Twenty five of the patients were evaluated following a change in or initiation of a new treatment. The initial change in treatment led to a +0.7 to -2.88 log change in plasma RNA (mean -0.9 log) and a mean of 6.9 cells per mm3 increase in CD4+ cells. Saquinavir was added to two reverse transcriptase (RT) inhibitors in 11 patients with a resulting mean of 0.23 log fall in RNA levels (range +0.70 log to -0.78 log). Saquinavir plus one RT inhibitor were added to one RT inhibitor in six patients with a subsequent mean of 0.65 log reduction in viral load (range +0.24 to -2.26 log). Saquinavir plus two RT inhibitors were given to four antiretroviral naive patients with a resulting mean of 2.37 log reduction in viral load (range -1.8 log to -2.67 log). CONCLUSIONS: 1. In a mixed cohort of RT inhibitor naive and treated patients, the viral RNA ranged throughout the range of the RNA assay. 2. Changes in viral load following changes in treatment were quite variable. 3. Saquinavir alone added to two RT inhibitors did not lead to a significant reduction in viral load. 4. In antiretroviral naive patients the viral load was reduced 100 fold following treatment with saquinavir and two RT inhibitors.

Comparison of restriction endonuclease analysis, ribotyping, and pulsed-field gel electrophoresis for molecular differentiation of Clostridium difficile strains.

A combined clinical and molecular epidemiologic analysis of 46 strains of Clostridium difficile, including 16 nosocomial isolates from one ward (outbreak ward) plus 17 other nosocomial isolates and 13 community-acquired isolates, was performed. HindIII digests of total cellular DNA were analyzed by restriction enzyme analysis (REA) and ribotyping; SmaI digests were analyzed by pulsed-field gel electrophoresis (PFGE). Isolates were assigned to typing groups on the basis of the profiles detected; isolates with closely related profiles were assigned to subgroups. The 16 isolates from the outbreak ward were resolved by both REA and PFGE into five distinct groups; 13 isolates represented two REA groups and three PFGE groups and two isolates were resolved as distinct groups by both techniques. DNA obtained from one isolate was persistently partially degraded, precluding analysis by PFGE. Seventeen sporadic nosocomial isolates were resolved by REA and PFGE into comparable numbers of groups (i.e., nine groups) and subgroups (i.e., 15 and 14 subgroups, respectively), with two isolates not evaluable by PFGE. The 13 epidemiologically unrelated community-acquired isolates were assigned to 11 groups by REA and to 12 groups by PFGE. Overall, ribotyping identified only nine groups among the 46 isolates. We conclude that REA and PFGE have comparable discriminatory powers for epidemiologic typing of C. difficile isolates and that ribotyping is appreciably less discriminatory. For a few isolates, partial DNA degradation prevented analysis by PFGE but not by REA or ribotyping; the cause of the degradation is unknown.