Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: partial purification and preliminary biochemical characterisation.

Thermus thermophilus HB27 produces important levels of extracellular lipolytic activity when grown for 30 h at 70 degrees C in a complex medium. A method to detect esterase activity in these samples after non-reducing SDS-polyacrylamide electrophoresis was developed. The method, that implies the renaturalisation of the enzymes in the SDS-gels by washing with Triton X-100 at high temperatures, allowed detecting three esterases with different molecular weights (108, 62 and 34 kDa, respectively). The electrophoretic mobility determined under different experimental conditions suggested that the 34- and 108 kDa-esterases might correspond with two oligomeric states of a sole enzyme (monomer and trimer). Dissociation of the trimer into the monomer started when the samples were heated at temperatures higher than 60 degrees C in the presence of sodium dodecyl sulphate. Evidences were found that indicated the independent nature of the 62 kDa-esterase. A method to purify these enzymes from postincubates of T. thermophilus HB27 was developed following three steps: sodium cholate treatment, ethanol/ether precipitation and hydrophobic chromatography. In this way, an enzyme solution was obtained that contained the identified esterases/lipases. The partially purified enzymes showed an optimum of activity for the hydrolysis of p-nitrophenyl laurate at alkaline pH values and 80 degrees C, a high thermal stability and were very stable in the presence of high concentrations of isopropanol.

Involvement of the pancreas in AIDS: a prospective study of 109 post-mortems.

BACKGROUND: Pancreatic involvement in AIDS is rarely mentioned in medical literature. AIMS: To identify the main morphological patterns of the pancreas using optical and electron microscopy in AIDS patients. DESIGN: An open, prospective, and sequential study in a tertiary institutional hospital. METHODS: Consecutive post-mortems of 109 AIDS patients and 38 controls (1995). Baseline characteristics of AIDS patients and controls were evaluated. Morphological analysis consisted of: (i) semi-quantitative score of acinar and parenchymal elements; (ii) qualitative analysis of ducts, vascular components, nerves, and Langerhans' islets; (iii) specific stains and immunohistochemistry for opportunistic agents; (iv) ultrastructural data. RESULTS: The mean age of AIDS patients was 37 years; 80% were male; 60% were white; 21% were alcoholic. All patients with AIDS had normal blood amylase, blood glucose, and pancreatic ultrasound. Histological findings were: acinar atrophy (60%), few zymogen granula in acinar cytoplasm (52%), abnormalities in acinar nucleus (65%), pancreatic steatosis (66%), and focal necrosis (17%). Immunohistochemistry revealed: mycobacteriosis (22%), toxoplasmosis (13%), cytomegalovirus (9%), Pneumocystis carinii (9%), and HIV p24 antigen in macrophage cytoplasm (22%). Ultrastructural examination showed: decreased zymogen granula, enlargement and proliferation of the endoplasmic reticulum and mitochondria, nuclear abnormalities, and increased lipid droplets in acinar cytoplasm. CONCLUSION: Pancreatic involvement in AIDS is very frequent (90%) and is usually asymptomatic. Morphological changes showed three patterns of pancreatic alterations: 'nutritional-like', inflammatory and both of these together. The 'nutritional-like' pattern (atrophy, few zymogen granula and steatosis) may be due to many factors such as nutritional characteristics (Kwashiorkor-like) induced by the HIV infection or related to the HIV virus itself.

A novel chemoenzymatic glycosylation strategy: application to lysozyme modification.

Hen egg lysozyme has been non-specifically glycosylated using a novel two-step strategy. First, a number of sucrose molecules have been chemically bound to the protein surface lysines, then the glycosidic chains have been enzymically lengthened, using a glycosyltransferase. For this task, a fructosyltransferase and a levansucrase have been tested, the latter appearing as the most effective one. In all cases, reactions have been optimised and several degrees of modification have been obtained. Finally, the effects of the modifications on lysozyme hydrophobicity, hydrolytic activity, hydrolysis substrate affinity and thermostability have been assessed.

Influence of surface hydrophilic/hydrophobic balance on enzyme properties.

Two different enzyme surface modifications were carried out in order to alter the protein hydrophilic/hydrophobic balance in opposite directions and to observe the effects induced on enzyme properties. First, a novel chemoenzymatic glycosylation method was applied, which resulted in a higher enzyme surface hydrophilic character. Then, an amphiphilic polymer, PEG, was bound to the enzymes by chemical means, and it brought about an increase in the global hydrophobic character. Two different enzymes, alpha-chymotrypsin and Candida rugosa lipase, were studied, and in all cases, several degrees of modification were obtained. Then, the modified biocatalysts were thoroughly investigated, and the influence of the variation of surface hydrophilic/hydrophobic balance on hydrolytic activity, hydrolysis kinetic parameters, synthetic activity and thermal stability was assessed.

Nonfatal demyelinating encephalomyelitis induced by coronavirus (JHM strain) infection in mice.

C3H mice infected intravenously with the JHM strain of coronavirus showed high incidence of demyelination (44.8%) and low incidence of encephalitis-induced mortality (6.9%). High titers of virus were detectable in the brain and liver of mice only during the first 3 to 12 days of infection (10(3) and 10(4) PFU/g, respectively). Most of the animals recovered from the first phase of disease and some (11.1%) came down with paralysis 6 to 7 weeks after the infection, with no histological changes or virus detectable in their tissues.

Comparison of Bacillus subtilis and Serratia marcescens as protease producers under different operating conditions.

Two microorganisms, Bacillus subtilis and Serratia marcescens, have been selected in order to assess their ability to produce proteases. First, their performances have been studied using three media providing different levels of the main nutrients (complex, semi-defined and defined). The optimal absolute production was obtained in the complex media, while maximum protease activity per cell weight appeared in defined medium for Bacillus and in semi-defined and complex media for Serratia. Then, the effects of applying different environmental conditions to the cells were studied. The two microorganisms were immobilised in calcium alginate beads, protease production by these systems was assessed in the previously tested culture media, and the performances in free and immobilized operating conditions were compared. The richest medium seemed to be the best one in terms of absolute protease production, although the use of semi-defined or defined media could be considered more appropriate, in order to minimise growth of cells leaked from the support, and therefore downstream processing cost. Productivity was slightly higher in free cultures than in immobilized cultures, and retention of enzyme within the alginate beads was detected in the latter, indicating the occurrence of diffusional limitations. In all the cases studied, Serratia marcescens appeared as a better protease producer than B. subtilis, in terms of absolute production. This fact could be related to the different cell growth levels observed for the two microorganisms.

Diffusion of proteases in calcium alginate beads.

Diffusion of proteases from Bacillus subtilis and Serratia marcescens within calcium alginate beads has been assayed, and the experimental data fitted into a mathematical model for diffusion into a finite volume liquid medium. Values of effective diffusivity were calculated for the studied proteases and compared with the available data in the literature for molecules of lower molecular weight.

Pancreatic involvement in co-infection visceral leishmaniasis and HIV: histological and ultrastructural aspects.

The involvement of the gastrointestinal tract in the co-infection of HIV and Leishmania is rarely reported. We report the case of an HIV-infected adult man co-infected with a disseminated form of leishmaniasis involving the liver, lymph nodes, spleen and, as a feature reported for the first time in the English literature, the pancreas. Light microscopy showed amastigote forms of Leishmania in pancreatic macrophages and immunohistochemical staining revealed antigens for Leishmania and also for HIV p24. Microscopic and ultrastructural analysis revealed severe acinar atrophy, decreased zymogen granules in the acinar cytoplasm and also nuclear abnormalities such as pyknosis, hyperchromatism and thickened chromatin. These findings might correspond to the histologic pattern of protein-energy malnutrition in the pancreas as shown in our previous study in pancreas with AIDS and no Leishmania. In this particular case, the protein-energy malnutrition may be due to cirrhosis, or, Leishmania or HIV infection or all mixed. We believe that this case represents the morphologic substratum of the protein energy malnutrition in pancreas induced by the HIV infection. Further studies are needed to elucidate these issues.

[Tumors of the brain stem: anatomo-pathologic study of 35 necropsy cases]. / Tumores do tronco cerebral: estudo anatomopatológico em 35 casos de necrópsia.

Brain stem tumors are rare and are usually treated without histological diagnosis. Its incidence varies in literature between 1.09% and 17.5% of all cerebral tumors. The objective of our study is to present a casuistic of these tumors in 28500 necropsies performed from 1952 till 1985 at the Department of Pathology, Santa Casa Hospital, São Paulo. We emphasize neuropathologic aspects, and compare our cases with those of other series. Also, we point out those tumors which are most frequent in this region, with the aim of possibly contributing to a better therapeutic approach. We used some clinical data and pathological diagnosis, and localized the tumor in the brain stem. Its microscopic diagnosis was determined according to the World Health Organization criterion. Among 428 intracranial tumors observed, 35 were localized in the brain stem. Intrinsic tumors and metastasis were included, and excluded those tumors which infiltrated the brain stem. Greatest incidence occurred in the first decade and cerebral edema was the predominant cause of death. Preferential topography was in the pons and the most frequent tumor was glioblastoma multiformis (19 cases). Metastasis ranked second (9 cases), being mostly from lungs. Some authors oppose tumor biopsy in this region because of high surgical risk. We do not agree with this opinion. We believe that the diagnosis of glioma is important to rule out the characteristics of malignancy. We can not leave without considering the diagnosis of other pathologies which can cause expansion in the brain stem. Therapeutical approach can be more adequate with the histological diagnosis of glioma or of other possibilities as shown here.

Technoeconomic assessment of phenanthrene degradation by Pseudomonas stutzeri CECT 930 in a batch bioreactor.

Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent pollutants that accumulate in natural environment mainly as a result of anthropogenic activities. Therefore, the improvement of the available bank of microbial resources and information is crucial to the proper management of PAHs-polluted sites and effluents. In this work, Pseudomonas stutzeri CECT 930 was selected for aerobically degrading an aqueous effluent containing phenanthrene (PHE). Maximum PHE degradation of 90% was obtained both at flask and stirred tank bioreactor scale. All the experimental data were fitted to logistic and Luedeking and Piret models, and licensed to quantitatively ascertain a stronger dependence on the biomass of the metabolites triggering the bioremediation process. In addition, PHE degradation via protocatechuate pathway was elucidated through GC-MS data. Finally, based on the promising results of biodegradation, a preliminary economic evaluation of this process at industrial scale was approached by means of simulation data obtained with SuperPro Designer.

Assessment of a process to degrade metal working fluids using Pseudomonas stutzeri CECT 930 and indigenous microbial consortia.

The development of a novel biological process to treat metal working fluids (MWFs)-containing effluents at bioreactor scale was pursued in this work. The bacteria Pseudomonas stutzeri CECT 930 was investigated for the first time as an alternative agent for MWF degradation. An adequate medium design and mixing and aeration system, as well as an appropriate microorganism proved to be crucial for reaching high levels of degradation by P. stutzeri and by an indigenous consortium (about 70% and 50% of reduction in total petroleum hydrocarbon content in less than 2 wk, respectively). Additionally, as there is no information in literature trying to kinetically characterize an MWF-polluted effluent degradation process, all the experimental data were fitted to logistic and Luedeking and Piret models, that allowed to elucidate the growth-associated character of the biodegradation process.

Decolorization of dye Reactive Black 5 by newly isolated thermophilic microorganisms from geothermal sites in Galicia (Spain).

In this study, thermophilic microbial strains from thermal spots in northwestern Spain displaying excellent decolorization capability were isolated. The research work tackled: (i) the ability of consortia to degrade a model di-azo dye Reactive Black at different pHs in flask cultures, obtaining that just neutral pHs licensed degradation levels near to 70%, (ii) the isolation of tree of the bacteria, which rendered possible reaching high levels of decolorization (80%) after just 24 h in aerobic conditions, and which were identified through 16S rRNA sequencing to possess high homology (99%) with Anoxybacillus pushchinoensis, Anoxybacillus kamchatkensis and Anoxybacillus flavithermus, and (iii) the cultivation of the isolates in a bench-scale bioreactor, which led to a decolorization rate two-fold higher than that obtained in flask cultures. Therefore, this work makes up the first time that a decolorization process of an azo dye by thermophilic microorganisms in aerobic conditions is investigated.

Influence of operational parameters on electro-Fenton degradation of organic pollutants from soil.

The combination of the Fenton's reagent with electrochemistry (the electro-Fenton process) represents an efficient method for wastewater treatment. This study describes the use of this process to clean soil or clay contaminated by organic compounds. Model soil of kaolinite clay polluted with the dye Lissamine Green B (LGB) was used to evaluate the capability of the electro-Fenton process. The effects of operating parameters such as electrode material and dye concentration were investigated. Operating in an electrochemical cell under optimized conditions while using electrodes of graphite, a constant potential difference of 5 V, pH 3, 0.2 mM FeSO(4). 7H(2)O, and electrolyte 0.1 M Na(2)SO(4), around 80% of the LGB dye on kaolinite clay was decolorized after 3 hours with an electric power consumption around 0.15 W h g(-1). Furthermore, the efficiency of this process for the remediation of a real soil polluted with phenanthrene, a typical polycyclic aromatic hydrocarbon, has been demonstrated.

Production of a thermostable extracellular lipase by Kluyveromyces marxianus.

Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids). The highest extracellular lipolytic enzyme production (about 80 U ml(-1) in 3 d) was obtained when the medium was supplemented with 2 g urea l(-1) plus 5 g tributyrin l(-1). Addition of surfactants (1 g l(-1)) did not improve production. The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 degrees C, 16 min half-life time at 100 degrees C). It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).

A novel application of solid state culture: production of lipases by Yarrowia lipolytica.

An extracellular lipase was produced in solid state cultures of Yarrowia lipolytica CECT 1240 using nylon sponge and several food and agroindustrial wastes (barley bran and triturated nut) as, respectively, inert support and support substrate. The highest activity was obtained with triturated nut (23 kU l(-1)) followed by sunflower oil-soaked barley bran (21 kU l(-1)). The activities were 5 fold greater those obtained in the control cultures with just inert support.

Quantification of intra- and extra-cellular thermophilic lipase/esterase production by Thermus sp.

Four Thermus strains produced lipolytic activity when grown in liquid medium for 30 h at 70 degrees C. The highest total lipase/esterase activity (57 U l(-1)) was in Thermus aquaticus YT-1, followed by Thermus thermophilus HB27 and HB8 (33 and 25 U l(-1), respectively), and finally by Thermus sp. (16 U l(-1)). Extra-cellular activity was detected in T. aquaticus YT-1 and T. thermophilus HB27 (33 and 17 U l(-1)). All enzymes were stable at 80 degrees C over 30 min, and their activity towards fatty acid esters increased as substrate chain-length diminished (i.e. hydrolysis rate was up to 6-fold higher on p-nitrophenyl caproate than on laurate).

Sporadic hepatitis C virus infection: a case-control study of transmission routes in a selected hospital sample of the general population in Italy.

A case-control study was performed on 9,175 Italian adult outpatients in 5 hospitals in Rome. The study was carried out to clarify the role of some less investigated risk factors (RF) in the spread of hepatitis C virus (HCV) infection. All subjects were contacted by interviewers, who completed a questionnaire. Their sera were stored and subsequently tested for both HCV and hepatitis B virus core (HBc) antibodies. 365 subjects, positive for anti-HCV and anti-HBc-negative, and who had denied intravenous drug use (IDU) (cases) were compared with an equal number of suitable random controls negative for anti-HCV and anti-HBc. Gender, age and region of birth and residence were matched. The prevalence of 13 RFs were statistically compared by univariate and multivariate analysis. A positive anti-HCV test was significantly associated, by multivariate analysis with intravenous treatments and minor surgical procedures (both before 1975) (p < 0.001), blood transfusions (before 1991) (p < 0.01), diabetes (p < 0.01), and deliveries in hospital (p < 0.05) (both before 1975). After 1975 (1991 for transfusions), all associations lost their significance. Intra-familial (sexual and non sexual), occupational RFs and dental care were not significantly associated with the presence of anti-HCV. We suggest that non-disposable syringes, commonly used until 1975 in Italy for i.v. treatments, have been the major route for HCV transmission in Italy among non-IDU subjects.

Nonfatal demyelinating encephalomyelitis induced by coronavirus (JHM strain) infection in mice

C3H mice infected intravenously with the JHM strain of coronavirus showed high incidence of demyelination (44.8%) and low incidence of encephalitis-induced mortality (6.9%). High titers of virus were detectable in the brain and liver of mice only during the first 3 to 12 days of infection (10 and 10 PFU/g, respectively). Most of the animals recovered from the first phase of disease and some (1.1%) came down with paralysis 6 to 7 weeks after the infection, with no histological changes or virus detectable in their tissues