Risk assessment for hepatocellular carcinoma in chronic hepatitis B: scores and surveillance.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death (1). Chronic infection with hepatitis B virus (HBV) is a major risk factor for HCC, accounting for more than one half of cases worldwide (2). Early detection of HCC in populations with chronic HBV infection through surveillance methods is critically important in providing definitive treatment for HCC and has a major impact on patient outcomes (3), including a survival benefit as demonstrated in one prospective randomised controlled trial (4). Efforts to identify populations and individuals with HBV infection who are at high risk may contribute to the development of surveillance strategies with the greatest impact on patient outcomes; however, the implementation of an individualised approach to surveillance can be challenging. This Perspective aims to explore risk factors associated with HCC in patients with chronic HBV infection, recent data on developing scoring systems to assess risk, and how these may impact surveillance strategies.

[Association between autonomic disorders and subclinical atherosclerosis]. / Asociación de desórdenes autonómicos con aterosclerosis subclínica.

INTRODUCTION AND OBJECTIVES: Alterations of the sympathetic and parasympathetic nervous system have been proposed as precursors of the genesis and perpetuation of atherosclerosis for a long time. The objective of this study is to determine if there is an association between the presence of carotid atherosclerosis and the reduction in heart rate variability. METHODS: Using a prospective case-control design, the heart rate variability and the presence of carotid atherosclerosis was investigated in 54 patients, divided into 2groups according to the presence or absence of carotid atherosclerosis. An analysis was made of the heart rate variability variables of the frequency (spectral) domain in high frequency band, low frequency band, parasympathetic autonomic balance, and the total spectral band. RESULTS: Of the 54 individuals evaluated without previous cardiovascular disease consecutively, 26 of them (48%) presented with subclinical carotid atherosclerosis (ATE+). A reduction in heart rate variability was observed in the ATE+group represented by the low frequency (LF) spectrum (P<.0001). The parasympathetic activity specifically represented in the high frequency (HF) band was also lower in the ATE+group in the univariate analysis (P<.0001), same as the total spectral power (P<.0001), an index of integral autonomic regulation. No significant differences were found in the LF/HF analysis (P=.1598). After analysing variables with significant differences in the univariate analysis with a logistic regression model, only systolic blood preassure and the total spectral power were shown to be independent predictors of ATE+. CONCLUSION: A reduction in heart rate variability was found in subjects with carotid atherosclerosis. Some spectral components of heart rate variability, like low frequency or total spectral power, were better predictors of carotid atherosclerosis than the parasympathetic autonomic balance. In this study it seems that total spectral power is an adequate measurement for analysing autonomic function.

Inverse association between hepatic stellate cell apoptosis and fibrosis in chronic hepatitis C virus infection.

Perisinusoidal hepatic stellate cells (HSC) are the principal fibrogenic cells in the liver. In animal models, HSC apoptosis is the predominant clearance mechanism of activated HSC, although data evaluating whether the same processes occur in humans are limited. We conducted a cross-sectional study to evaluate the association between HSC apoptosis and fibrosis stage in subjects with chronic hepatitis C virus (HCV) infection (n = 44) and HCV-negative controls with normal liver histology (n = 9). We used immunohistochemical techniques to identify activated (alpha-smooth muscle actin+), proliferative (Ki-67+) and apoptotic (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end-labelling+) HSC in liver biopsy specimens from all subjects. The same pathologist enumerated positive cells per high-power field (HPF, x 200) in 20 periportal/lobular areas. HSC apoptosis was decreased in HCV-positive subjects compared with controls (median 0.4, range 0.0-3.1 vs 1.1, 0.2-3.5 cells/HPF, P = 0.02). Among HCV-positive subjects, HSC apoptosis was decreased in those with moderate to advanced fibrosis (P = 0.04) compared with those with mild fibrosis. By multivariate analysis, HSC apoptosis decreased by an average of 0.14 cells/HPF (95% confidence interval 0.01-0.28 cells/HPF) per increase in fibrosis stage (P = 0.04). While the number of activated and proliferative HSC was significantly increased in HCV-infected subjects compared with that in uninfected controls, the numbers of these cells did not differ between HCV-infected subjects with mild vs moderate/advanced fibrosis. In conclusion, the number of apoptotic HSC was significantly decreased in HCV-infected subjects with advanced fibrosis. In chronic HCV infection, inhibition of HSC apoptosis may be one mechanism by which fibrosis progresses.

Hepatic inflammatory cytokine mRNA expression in hepatitis C virus-human immunodeficiency virus co-infection.

Although epidemiologic studies have documented that hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infected patients have accelerated fibrogenesis, especially those with CD4+ cell counts <200 cells/mm(3), the pathogenic mechanisms are poorly understood. We investigated whether severe immunodeficiency in co-infection is associated with changes in intrahepatic inflammatory cytokine mRNA levels. We measured interferon (IFN)-gamma, tumour necrosis factor-alpha, transforming growth factor (TGF)-beta(1), interleukin (IL)-4, IL-10, IL-12p35 and IL-12p40 mRNA levels by real-time PCR performed on liver samples from HCV mono-infected (n = 19) and HCV/HIV co-infected (n = 24) patients. Co-infected patients had decreased intrahepatic mRNA levels of IFN-gamma (P = 0.09), IL-4 (P = 0.05) and IL-12p35 (P = 0.04) compared with mono-infected patients, while IL-10 was increased (P = 0.07). In co-infected patients, IFN-gamma mRNA levels increased linearly with increasing peripheral CD4+ cell counts by 1.23 times relative to the calibrator for every 100 CD4+ cells/mm(3) increase (P = 0.02). No other cytokines were significantly associated with CD4+ cell counts. In conclusion, HIV-induced lymphopenia may result in hepatic inflammatory cytokine suppression in HCV/HIV co-infection. Intrahepatic IFN-gamma levels are significantly reduced in patients with advanced immunodeficiency. Further studies are needed to assess whether decreased IFN-gamma secretion by HCV-specific CD4+ cells may account for accelerated fibrogenesis in these patients.

Safety and efficacy of ledipasvir/sofosbuvir with or without ribavirin in hepatitis C genotype 1 patients including those with decompensated cirrhosis who failed prior treatment with simeprevir/sofosbuvir.

BACKGROUND: Combination therapy of simeprevir (SIM)/sofosbuvir (SOF) is an approved treatment for hepatitis C genotype (gen) 1 with overall SVR12 rate of 85%-95%. The single tablet fixed-dose combination of ledipasvir (LDV)/SOF is also approved for gen 1 with sustained virologic response at 12 weeks (SVR12) rates ≥95%. No data are available on the efficacy of retreatment with LDV/SOF in patients who failed initial treatment with SIM/SOF. AIM: To evaluate the efficacy of retreatment with LDV/SOF ± ribavirin (RBV) in gen 1 patients who had previously failed treatment with SIM/SOF. METHODS: Data from a combined treatment cohort of 2 hepatology centres, which included patients previously treated with SIM/SOF ± RBV for 12 weeks but failed to achieve SVR and then underwent retreatment with LDV/SOF ± RBV, were analysed (n = 30). LDV/SOF ± RBV was administered for 12-24 weeks based on the discretion of the treating hepatologist. RESULTS: Of the 30 patients, 23 (77%) were male, 77% were Caucasian and 26 (87%) were gen 1a. 26 (86%) had cirrhosis, of which 16 (62%) had decompensated, Child's class B or C cirrhosis. Three patients were liver transplant recipients with recurrent hepatitis C. Overall, 27/30 (90%) achieved SVR. Treatment was well tolerated with 37% reporting no adverse events. The most common adverse events were fatigue, headache, insomnia and nausea. Two patients with Child's B cirrhosis required hospitalization during treatment for variceal haemorrhage and abdominal pain respectively. However, no treatment discontinuations or deaths occurred. CONCLUSION: Single tablet fixed-dose combination LDV/SOF ± RBV is efficacious and well tolerated in patients who previously failed treatment with SIM/SOF, including those with decompensated cirrhosis and recurrent hepatitis C following liver transplantation.

[Determination of colonic transit time in healthy subjects in Mexico City. Prospective study]. / Determinación del tiempo de tránsito colónico en personas sanas de la Ciudad de México. Estudio prospectivo.

UNLABELLED: Colonic transit time (CTT) is determined by multiple factors; currently, normal values for the Mexican population are not available. In order to get an estimate one must look at the values reported in the international literature, but cultural, ethnical, nutritional and economic differences may lead to different values. OBJECTIVE: To determine the normal values of colonic transit time in healthy people in Mexico City by the use of radiopaque markers. PATIENTS AND METHODS: Prospective, longitudinal and observational study, which included healthy patients ranging from 18 to 60 years old; excluding pregnant women. The whole group of patients was given before breakfast a gelatin capsule which had 20 radiopaque markers inside -the markers were each 2mm long, and were made by the researcher-. After that, they were taken a simple abdominal X-ray film every 24 hours until they totally eliminated the markers. Their eating and defecation habits were evaluated and also the total amount of liquid they consumed. Inferential statistics were used; data was validated with both parametric and non-parametric tests, considering a significance of p < 0.05. RESULTS: A hundred patients were included in the sample in which 48% were female and 52% male, they were divided in three groups: group A (31%)from 18 to 25 years, group B (37%)from 26 to 40 and group C (32%)from 41 to 60 years; there were no important differences in their water consumption, which was in average of 1.87 lts. in 24 hours; also, there were no considerable differences regarding to their meat, vegetables and fruits' consumption, which was in average of 4.4 times a week; the whole group eliminated the markers according to X-rays which was in 54% after 72 hrs, 45% after 48 hrs and 1% after 24 hrs. We can observe an increase of the CTT related to age: in group C 94% eliminated the markers after 72 hrs and there was no significant difference (statistically) with regards to the other groups. A tendency of an increase of CTT with regards to age was observed: in group A, 80% eliminated the markers after 48 hrs, in group B 49% eliminated them after 48 hrs and 51% after 72 hrs and, in group C, 94% eliminated them after 72 hrs without any statistically significant differences among the study groups. CONCLUSION: The CTT in healthy patients is in a 100% of the cases studied lower or equal to 72 hrs with a tendency to increase in relation to age.

The reliability and validity of a short food frequency questionnaire among 9-11-year olds: a multinational study on three middle-income and high-income countries.

OBJECTIVE: The main aim of this study was to assess the reliability and validity of a food frequency questionnaire with 23 food groups (I-FFQ) among a sample of 9-11-year-old children from three different countries that differ on economical development and income distribution, and to assess differences between country sites. Furthermore, we assessed factors associated with I-FFQ's performance. METHODS: This was an ancillary study of the International Study of Childhood Obesity, Lifestyle and the Environment. Reliability (n=321) and validity (n=282) components of this study had the same participants. Participation rates were 95% and 70%, respectively. Participants completed two I-FFQs with a mean interval of 4.9 weeks to assess reliability. A 3-day pre-coded food diary (PFD) was used as the reference method in the validity analyses. Wilcoxon signed-rank tests, intraclass correlation coefficients and cross-classifications were used to assess the reliability of I-FFQ. Spearman correlation coefficients, percentage difference and cross-classifications were used to assess the validity of I-FFQ. A logistic regression model was used to assess the relation of selected variables with the estimate of validity. Analyses based on information in the PFDs were performed to assess how participants interpreted food groups. RESULTS: Reliability correlation coefficients ranged from 0.37 to 0.78 and gross misclassification for all food groups was <5%. Validity correlation coefficients were below 0.5 for 22/23 food groups, and they differed among country sites. For validity, gross misclassification was <5% for 22/23 food groups. Over- or underestimation did not appear for 19/23 food groups. Logistic regression showed that country of participation and parental education were associated (P⩽0.05) with the validity of I-FFQ. Analyses of children's interpretation of food groups suggested that the meaning of most food groups was understood by the children. CONCLUSION: I-FFQ is a moderately reliable method and its validity ranged from low to moderate, depending on food group and country site.

Relationships between active school transport and adiposity indicators in school-age children from low-, middle- and high-income countries.

OBJECTIVES: Within the global context of the nutrition and physical activity transition it is important to determine the relationship between adiposity and active school transport (AST) across different environmental and socio-cultural settings. The present study assessed the association between adiposity (that is, body mass index z-score (BMIz), obesity, percentage body fat (PBF), waist circumference) and AST in 12 country sites, in the International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE). METHODS: The analytical sample included 6797 children aged 9-11 years. Adiposity indicators included, BMIz calculated using reference data from the World Health Organization, obesity (BMIz ⩾+2 s.d.), PBF measured using bioelectrical impedance and waist circumference. School travel mode was assessed by questionnaire and categorized as active travel versus motorized travel. Multilevel linear and non-linear models were used to estimate the magnitude of the associations between adiposity indicators and AST by country site and sex. RESULTS: After adjusting for age, sex, parental education and motorized vehicle availability, children who reported AST were less likely to be obese (odds ratio=0.72, 95% confidence interval (0.60-0.87), P<0.001) and had a lower BMIz (-0.09, s.e.m.=0.04, P=0.013), PBF (least square means (LSM) 20.57 versus 21.23% difference -0.66, s.e.m.=0.22, P=0.002) and waist circumference (LSM 63.73 cm versus 64.63 cm difference -0.90, s.e.m.=0.26, P=0.001) compared with those who reported motorized travel. Overall, associations between obesity and AST did not differ by country (P=0.279) or by sex (P=0.571). CONCLUSIONS: AST was associated with lower measures of adiposity in this multinational sample of children. Such findings could inform global efforts to prevent obesity among school-age children.

An international comparison of dietary patterns in 9-11-year-old children.

OBJECTIVES: Dietary pattern is defined as a combination of foods and drinks and the frequency of consumption within a population. Dietary patterns are changing on a global level, which may be linked to an increased incidence of chronic diseases. The aim of this study was to identify and compare the dietary patterns among 9-11-year-old children living in urban regions in different parts of the world. METHODS: Participants were 7199 children (54% girls), aged 9-11 years, from 12 countries situated in all major world regions. Food consumption was assessed using a 23-item Food Frequency Questionnaire (FFQ). To identify dietary patterns, principal components analyses (PCA) were carried out using weekly portions as input variables. RESULTS: Both site-specific and pooled PCA resulted in two strong components. Component 1 ('unhealthy diet pattern') included fast foods, ice cream, fried food, French fries, potato chips, cakes and sugar-sweetened sodas with >0.6 loadings. The loadings for component 2 ('healthy diet pattern') were slightly weaker with only dark-green vegetables, orange vegetables, vegetables in general, and fruits and berries reaching a >0.6 loading. The site-specific diet pattern scores had very strong correlations with the pattern scores from the pooled data: r=0.82 and 0.94 for components 1 and 2, respectively. CONCULSIONS: The results suggest that the same 'healthier' and 'unhealthier' foods tend to be consumed in similar combinations among 9-11-year-old children in different countries, despite variation in food culture, geographical location, ethnic background and economic development.

Isolation of a mitotic-like cyclin homologue from the protozoan Trypanosoma brucei.

Activation of the p34cdc2 protein kinase (PK) at different stages of the eukaryotic cell cycle is controlled by interaction with regulatory proteins known as cyclins (CYCs). Using a probe obtained by PCR amplification, we have isolated from the protozoan, Trypanosoma brucei, a cDNA clone encoding a CYC homologue. The amino acid sequence deduced for this gene (CYC1) shares structural homology with A- and B-type CYCs of other organisms, including a motif, the destruction box, which has been related to the rapid turnover of these CYC proteins in mitosis. When expressed in fission yeast, CYC1 is able to rescue the defect of a temperature-sensitive cdc13 mutant, demonstrating that it is functional as a cell-cycle regulator. In trypanosome cells, CYC1 associates with a 34-kDa protein that cross-reacts with a monoclonal antibody against the conserved 'PSTAIR' epitope of p34cdc2, and the complex displays histone H1 PK activity. Furthermore, when trypanosome cells are synchronized by hydroxyurea treatment, CYC1 accumulates as cells progress towards mitosis. These observations, taken together, suggest that CYC1 is a component of the active PK complex required for the control of trypanosome mitosis.

Mutational analysis of the feline immunodeficiency virus matrix protein.

To study the process of feline immunodeficiency virus (FIV) assembly, we examined the suitability of the vaccinia vector system to reproduce FIV particle formation. To this end, we constructed a recombinant vaccinia virus carrying the FIV gag gene. Biochemical and electron microscopy analyses of cells infected with this recombinant virus showed that the FIV Gag polyprotein self-assembled into lentivirus-like particles that were released into the culture medium. As a first step in the identification of molecular determinants in FIV Gag that are involved in virus assembly, we performed a site-directed mutagenesis analysis of the N-terminal matrix (MA) domain of the FIV Gag precursor. To this end, a series of amino acid substitutions and small in-frame deletions were introduced into the FIV MA and the mutated FIV gag gene constructs were expressed by means of the vaccinia system. Characterization of the assembly phenotype of these FIV Gag mutants led to the identification of amino acidic regions within the MA domain that are necessary for efficient transport of the Gag precursor to the plasma membrane and particle assembly. Our results reveal the role that the FIV MA plays in virus morphogenesis and contribute to the understanding of the assembly process in non-primate lentiviruses.

Small variations in the length of the cytoplasmic domain of the simian immunodeficiency virus transmembrane protein drastically affect envelope incorporation and virus entry.

Simian immunodeficiency viruses (SIVs) have an envelope (Env) glycoprotein with an unusually long cytoplasmic domain of 164 amino acids. In this article, we have characterized a series of SIV Env truncation mutants in which the cytoplasmic domain was progressively shortened from its carboxyl terminus by 20 amino acids. Expression by means of the vaccinia virus system showed that all of the SIV Env mutants were expressed and processed into the surface and transmembrane (TM) subunits. When the ability of the Env mutants to associate with SIV Gag particles was examined, we found that deletion of 20 to 80 residues from the carboxyl terminus of the SIV TM cytoplasmic tail abrogated the incorporation of the Env glycoprotein into particles. By contrast, further truncation of the SIV TM protein by 100 to 140 amino acids restored the ability of the Env protein to associate with Gag particles. Interestingly, mutants bearing a 44- or 24-amino acid cytoplasmic domain were incorporated at levels significantly higher than those of the wild-type Env. Single-cycle infectivity assays showed that Env mutants bearing cytoplasmic tails of 144 to 64 amino acids were highly inefficient at mediating virus entry. By contrast, truncation of the cytoplasmic domain to 44 or 24 amino acids drastically enhanced virus infectivity with respect to that conferred by the full-length Env protein. Our results demonstrate that small variations in the length of the SIV Env cytoplasmic domain dramatically influence Env-mediated viral functions.

Automatic temperature controller for maintaining body temperature in experimental animals.

A system for autoregulation of body temperature of large and small animals is described. The device uses the IC AD590 as a temperature transducer. It operates on the basis of continuous regulation of the heating current, and does not emit transients which interfere with electrical recordings. A panel meter shows either the rectal temperature of the animal or the current flow to the blanket.

[Diabetes and pregnancy]. / Diabetes y embarazo.

Diabetes mellitus during pregnancy could result in severe or fatal complications to mother or the unborn product, like polyhydramnios, preeclampsia, abortion, neonatal asphyxia, macrosomia, stillbirth, and others, therefore is very important the early detection and treatment of diabetes. Gestacional Diabetes Mellitus (GDM) is the carbohydrate intolerance of variable severity first recognized during pregnancy. The screening test consist of 50 g of oral glucose and a plasma glucose measurement at one hour, regardless of the time of the last meal, and this may do in all pregnancies between 24 and 28 weeks of gestation. If plasma glucose level above 140 mg/dl results, a oral glucose tolerance test with 100 g must be done. This is the GDM diagnostic test. The risk factors for gestacional diabetes (older than 30 years of age, obesity, arterial hypertension, glucosury, previous GDM, family history of diabetes, family history of macrosomia) identify only 50% of pregnancies with gestacional diabetes, therefore, is necessary to screen all pregnancies who become pregnant, a strict control before pregnant is indispensable, with aim to slow congenital malformations probability and another complications. Gestacional diabetes prevalence in hispanic women in the U.S.A. is 12.3 percent. Diabetes mellitus prevalence in Mexico is about 2-6 percent. The goal of management of diabetes during pregnancy is the maintainance of fasting plasma glucose 105 mg/dl and 120 mg/dl two hours after meals. Treatment consist in diabetes education, diet with caloric needs calculation, exercise, and occasionally insulin. Is necessary the prenatal monitoring, the supervision of delivery or cesarean metabolic changes, and the postnatal monitoring of the mother and product.

Assessment of Fenton's reagent and ozonation as pre-treatments for increasing the biodegradability of aqueous diethanolamine solutions from an oil refinery gas sweetening process.

The aim of this work was to evaluate the efficiency of three chemical oxidation processes for increasing the biodegradability of aqueous diethanolamine solutions (aqueous DEA solutions), to be used as pre-treatments before a biological process. The raw aqueous DEA solution, sourced from a sour gas sweetening plant at a Mexican oil refinery, was first characterized by standardized physico-chemical methods. Then experiments were conducted on diluted aqueous DEA solutions to test the effects of Fenton's reagent, ozone and ozone-hydrogen peroxide on the removal of some physicochemical parameters of these solutions. Lastly, biodegradability tests based on Dissolved Organic Carbon Die Away OECD301-A, were carried out on a dilution of the raw aqueous DEA solution and on the treated aqueous DEA solutions, produced by applying the best experimental conditions determined during the aforementioned oxidation tests. Experimental results showed that for aqueous DEA solutions treated with Fenton's reagent, the best degradation rate (70%) was obtained at pH 2.8, with Fe(2+) and H(2)O(2) at doses of 1000 and 10,000 mg/L respectively. In the ozone process, the best degradation (60%) was observed in aqueous DEA solution (100 mg COD/L), using 100 mg O(3)/L at pH 5. In the ozone-hydrogen peroxide process, no COD or DOC removals were observed. The diluted spent diethanolamine solution showed its greatest increase in biodegradability after a reaction period of 28 days when treated with Fenton's reagent, but after only 15 days in the case of ozonation.

ANÁLISIS DEL ESTADO ACTUAL DE LA INGENIERÍA CLÍNICA EN LAS INSTITUCIONES HOSPITALARIAS DE CALI / ANALYSIS OF THE CURRENT STATE OF CLINICAL ENGINEERING IN HOSPITALS IN CALI / A ANÁLISE DO ESTADO ATUAL DA ENGENHARIA CLÍNICA EM HOSPITAIS DE CALI

En este trabajo se presentan los resultados obtenidos tras analizar la información recolectada en once instituciones prestadoras de servicios de salud (IPS) en la ciudad de Cali y municipios aledaños, sobre tres elementos clave para la buena práctica de la Ingeniería Clínica: adquisición de tecnología, gestión de mantenimiento y formación del personal. Se realizó una comparación entre las prácticas actuales de IC en las IPS encuestadas y las prácticas propuestas en la literatura existente. Se propone además una serie de aspectos a tomar en cuenta con miras a mejorar el desempeño de los departamentos de IC tanto en la ciudad como en el país.

This paper summarizes the results obtained after analyzing the information gathered in eleven institutions providing health services (IPS, in Colombia) in the city of Cali and surrounding municipalities, three key elements for good clinical engineering practice are presented: Acquisition technology, management, maintenance and staff training. A comparison between current practices in the surveyed IC at IPS and practices proposed in the literature was conducted. It also proposes a number of aspects to consider in order to improve performance of both IC departments in the city and in the country.

Este artigo apresenta os resultados obtidos depois de analisar as informações coletadas em onze instituições prestadoras de serviços de saúde (IPS), na cidade de Cali e municípios vizinhos, sobre três elementos-chave para a boa prática de engenharia clínica: aquisição de tecnologia, gestão de manutenção e treinamento de pessoal. Uma comparação foi feita entre as práticas atuais nas IPS e práticas propostos na literatura atual. Ele também propõe uma série de aspectos a considerar, a fim de melhorar o desempenho dos departamentos IC tanto na cidade quanto no país.

Proteomic analysis of the venom from the scorpion Tityus stigmurus: biochemical and physiological comparison with other Tityus species.

The venom from the Brazilian scorpion Tityus stigmurus was fractionated by high performance liquid chromatography (HPLC) and the corresponding components were used for molecular mass determination using electrospray ion trap mass spectrometry. One hundred distinct components were clearly assigned showing molecular masses from 216.5 to 44,800.0 Da. Fifteen new components were isolated and sequenced, four of them to completion: Tst-3 (similar to Na(+) channel specific scorpion toxins), Tst-17 (a K(+) channel blocking peptide similar to Tc1), Tst beta KTx (a peptide with identical sequence as that of TsTX-K beta toxin earlier described to exist in T. serrulatus venom) and finally a novel proline-rich peptide of unknown function. Among the eleven components partially sequenced were two enzymes: hyaluronidase and lysozyme. The first enzyme has a molecular mass of 44,800.0 Da. This enzyme showed high activity against the substrate hyaluronan in vitro. Amino acid sequence of the second enzyme showed that it is similar to other known lysozymes, with similar molecular mass and sequence to that of bona fide lysozymes reported in public protein data banks. Finally, this communication reports a correlation among HPLC retention times and molecular masses of folded scorpion toxins as well as a comparative structural and physiological analysis of components from the venom of several species of the genus Tityus.

Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine.

We studied the post-translational modification of NS26, the protein product of rotavirus gene 11 segment. Based on the presence of a putative N-glycosylation site and the high content of serine and threonine residues in gene 11 amino acid sequence we investigated whether NS26 is modified by carbohydrate addition. Specific antibodies raised against the gene 11 product expressed in Escherichia coli recognized in infected cells two polypeptides with apparent molecular weight of 26,000 (26-kDa polypeptide) and 28,000 (28-kDa polypeptide). Pulse-chase experiments demonstrated that the 26-kDa product was processed to the 28-kDa polypeptide. Both polypeptides were metabolically labeled with [3H]glucosamine, indicating the presence of a carbohydrate moiety on the protein. NS26 was found to be resistant to endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F/peptide:N-glycosidase F treatment, but sensitive to removal by alkali-induced beta-elimination, suggesting that the saccharide chain was attached to the protein via an O-glycosidic linkage. Chromatographic analysis of total acid hydrolysates of [3H]glucosamine-labeled NS26-bound carbohydrate indicated the presence of N-acetylglucosamine. In addition, mild alkaline treatment of NS26 in the presence of NaB3H4 identified the O-linked carbohydrate moiety as N-acetylglucosamine. Taken together, these data demonstrate that NS26 is processed to a 28-kDa polypeptide by addition of O-linked monosaccharide residues of N-acetylglucosamine.

Mutational analysis of the conserved cysteine residues in the simian immunodeficiency virus matrix protein.

The matrix protein (MA) of human and simian immunodeficiency viruses (HIV and SIV) is encoded by the amino-terminal region of the Gag precursor and has been suggested to be involved in different processes during the early and late stages of the virus life cycle. The MA protein of SIV contains three cysteine residues at positions 57, 83, and 87, which are also highly conserved among HIV-2 isolates. In order to study the functional significance of these residues in virus morphogenesis, a series of mutations affecting the cysteines of SIV MA were introduced into a gag-protease construct and expressed in the vaccinia vector system. The MA mutants were assayed for their ability to synthesize and process the Gag polyprotein precursor as well as to release particles into the culture medium. In addition, the incorporation of the envelope glycoprotein (Env) into the Gag-made particles was investigated. Substitution of alanine for cysteine 87 had little effect on particle release and Env glycoprotein association. By contrast, the individual replacement of cysteines 57 or 83 by alanine, as well as the simultaneous mutation of cysteines 83 and 87, significantly reduced the ability of Gag polypeptides to produce extracellular particles. Assembly into particles appeared to be also affected, albeit to a lesser extent, when both cysteines 57 and 83 were replaced by alanine. Furthermore, substitution of cysteine 83 in the SIV MA domain was found to be detrimental to Gag polyprotein processing. Analysis of the Env glycoprotein association with recombinant particles revealed that this process was moderately affected in the case of the double mutants lacking cysteines 57 and 83, or cysteines 57 and 87, and the cysteine-minus triple mutant. Our results suggest that the conserved cysteines 57 and 83 in the MA domain are important for efficient SIV Gag particle production.