Application of cardiovascular disease risk prediction models and the relevance of novel biomarkers to risk stratification in Asian Indians.

The increasing pressure on health resources has led to the emergence of risk assessment as an essential tool in the management of cardiovascular disease (CVD). Concern exists regarding the validity of their generalization to all populations. Existing risk scoring models do not incorporate emerging 'novel' risk factors. In this context, the aim of the study was to examine the relevance of British, European, and Framingham predictive CVD risk scores to the asymptomatic high risk Indian population. Blood samples drawn from the participants were analyzed for various 'traditional' and 'novel' biomarkers, and their CVD risk factor profiling was also done. The Framingham model defined only 5% of the study cohort to be at high risk, which appears to be an underestimation of CVD risk in this genetically predisposed population. These subjects at high risk had significantly elevated levels of lipid, pro-inflammatory, pro-thrombotic, and serological markers. It is more relevant to develop risk predictive scores for application to the Indian population. This study substantiates the argument that alternative approaches to risk stratification are required in order to make them more adaptable and applicable to different populations with varying risk factor and disease patterns.

Ionic liquids as a reaction medium for lipase-catalyzed methanolysis of sunflower oil.

Ionic liquids, 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIm][PF(6)]) and 1-ethyl-3-methyl imidazolium hexafluorophosphate ([EMIm][PF(6)]), were used for the methanolysis of sunflower oil using Candida antarctica lipase (Novozyme 435) and gave yields of fatty acid methyl esters at 98-99% within 10 h. The optimum conditions of methanolysis in hydrophobic ionic liquids are 2% (w/w) lipase, 1:1 (w/w) oil/ionic liquid and 1:8 (mol/mol) oil/methanol at 58-60 degrees C. Methanolysis using hydrophilic ionic liquids, 3-methyl imidazolium tetrafluoroborate ([HMIm][BF(4)]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIm][BF(4)]), gave very poor yields. A hydrophobic ionic liquid thus protects the lipase from methanol. Recovered ionic liquids and lipase were used for four successive reaction cycles without any significant loss of activity.

Isolation and antimicrobial evaluation of isomeric hydroxy ketones in leaf cuticular waxes of Annona squamosa.

A novel natural compound, 11-hydroxy-16-hentriacontanone, has been isolated from the leaf cuticular wax of Annona squamosa along with its known isomer 10-hydroxy-16-hentriacontanone in a ratio of 67:33. This isomeric mixture of hydroxy ketones constituted together 16.5% of the total cuticular waxes. The new compound was characterised using spectral and chromatographic techniques. The major component was found to be 16-hentriacontanone (palmitone), which constituted up to 48% of the total cuticular wax, together with a homologous series of hydrocarbons, fatty aldehydes, fatty alcohols, fatty acids and sterols as minor components. The antimicrobial activity of the isomeric hydroxy ketones was tested against selected Gram-positive and Gram-negative bacterial strains, and also some selected fungal strains, and compared with palmitone. The antibacterial activity of palmitone was significantly higher than that of the isomeric hydroxy ketones, but their antifungal activities were comparable.

Uncomplicated falciparum malaria complicated by salmonella septicaemia: cause not coincidence.

A male patient of 10-year-old presented with fever, headache and vomiting for last few days. He was being treated with antimalarial drugs. On 14th day of illness he again showed rise of temperature. His blood culture showed growth of Salmonella typhi. He was treated with ceftriaxone and responded favourably. Here uncomplicated falciparum malaria developed a secondary infection with salmonella during hospital stay. This uncommon association was noted rather than a mere coincidence, which rarely reported in literature.

Ultrasonic studies of alcohol-induced transconformation in beta-lactoglobulin: the intermediate state.

In mixed alcohol-water solvents, bovine beta-lactoglobulin undergoes a cooperative transition from beta-sheet to a high alpha-helix content conformer. We report here the characterization of beta-lactoglobulin by compressibility and spectroscopy measurements during this transconformation. Both the volume and compressibility increase as a function of alcohol concentration, up to maximal values which depend on the chemical nature of the three alcohols used: hexafluoroisopropanol, trifluoroethanol, and isopropanol. The order of effectiveness of alcohols in inducing the compressibility transition is identical to that previously reported for circular dichroism and thus independent of the observation technique. The highly cooperative sigmoidal curves found by compressibility determination match closely those obtained by circular dichroism at 222 nm, indicating a correlation between the two phenomena measured by the two different techniques. The presence of an equilibrium intermediate form was shown by the interaction of beta-lactoglobulin with 8-anilino-1-naphthalene sulfonic acid, a probe widely used to detect molten-globule states of proteins. It was correlated with the plateau region of the volume curves and with the inflexion points of the sigmoidal compressibility curves. Ultrasound characterization of proteins can be carried out in optically transparent or nontransparent media.

Establishment of two vaccine-associated feline sarcoma cell lines and determination of in vitro chemosensitivity to doxorubicin and mitoxantrone.

OBJECTIVE: To establish 2 vaccine-associated feline sarcoma (VAFS) cell lines and to determine their in vitro sensitivity to the chemotherapeutic agents doxorubicin and mitoxantrone. SAMPLE POPULATION: Tumor specimens collected from 2 cats undergoing surgery for removal of vaccine-associated sarcomas. PROCEDURES: Tumor specimens were minced and treated with trypsin under aseptic conditions to obtain single-cell suspensions, which were then cultured in vitro in medium supplemented with 5% heat-inactivated fetal bovine serum. Growth rates and sensitivity after 24 hours of exposure to various concentrations (0.1 to 100 microg/ml) of doxorubicin and mitoxantrone were assessed for each cell line. Survival of cells was estimated 3 days after exposure to the 2 agents, and the concentration of each drug that resulted in a 50% reduction in the number of viable cells (IC50) was calculated. RESULTS: Two tumor-derived cell lines (FSA and FSB) were successfully established and determined to be sensitive to doxorubicin and mitoxantrone. Under the conditions tested, the IC50 of doxorubicin were 0.6 and 1.5 microg/ml for cell lines FSB and FSA, respectively. The IC50 of mitoxantrone was 0.4 microg/ml for both cell lines. CONCLUSIONS AND CLINICAL RELEVANCE: The establishment of VAFS cell lines provides a tool for the in vitro screening of antitumor drugs. Doxorubicin and mitoxantrone were effective in decreasing the number of viable cells in the 2 cell lines tested. Both of these anthracycline antibiotics have been used to treat various neoplasias in cats, and their efficacy for adjuvant treatment of vaccine-associated sarcomas should be further evaluated.

Synthesis and estimation of calorific value of a structured lipid-potential reduced calorie fat.

The majority of reduced calorie fats and fat substitutes available today, though similar in texture and flavor to natural fats, contain fatty acids that are not usually present in edible oils and fats and thus do not fully match the chemistry and functions of natural fats. For example, such products do not provide nutritionally important essential fatty acids (EFA). In this investigation, we prepared and evaluated a reduced calorie fat, prepared entirely from natural fats, taking advantage of the fact that long-chain saturated fatty acids (LCSFA), such as behenic acid (22:0), are poorly absorbed. Mustard oil (MO) and sunflower oil (SO) were used as substrates to yield a structured lipid (SL). The product, being derived from a natural vegetable oil, would thus provide EFA, as would a native fat, a feature not provided by the low-calorie fats available in the market. Erucic acid (22:1) was isolated from MO by a lipase (EC 3.1.1.3)-catalyzed reaction. It was then hydrogenated to behenic acid, the ethyl ester of which was subsequently enzymatically transesterified with SO to yield a plastic fat containing about 30-35% behenic acid. Absorption of this fat was studied in Wistar rats. In a preliminary single oral dose experiment, rats were fed equal amounts (2 mL) of SO and the SL. Plasma triacylglycerol (TAG) levels were estimated after 1, 2, and 3 h of feeding. The significantly lower concentration of plasma TAG in the 2-h sample, observed in the SL-fed group compared to the SO-fed group (P<0.001), indicated poor absorption of the SL. In order to estimate the calorific value of the SL, we conducted a restricted diet growth experiment over 21 d on weanling Wistar male rats with SO as caloric control. Diets for the test groups were modified by adding 5, 10, and 15% SO for the control groups, and 5 and 10% SL for the experimental groups. Food consumption of the test groups was restricted to 50% of the feed containing 5% SO that had been consumed by the ad libitum group the previous day. Body weights were recorded during the experiment. Calorific value of the SL was estimated by comparing the 21 st-d mean body weight gain of the control group with that of the experimental group. Estimated calorific value of the SL was 5.36 kcal/g. Most of the behenic acid fed was excreted, as indicated by the analysis of the fatty acids of plasma and fecal total lipid. A second growth experiment on ad libitum diet was conducted over 21 d on weanling Wistar male rats to compare the absorption behavior of the SL with that of natural oil. SO (10%) was added to the diet of the control group, and SL (10%) was added to the diet of the experimental group. Feed consumption, as well as body weights, was recorded during the experiment. The growth pattern of the experimental group was identical to that of the control group during the period of study. The mean feed intake (9.8 g/d/rat for the control group vs. 9.9 g/d/rat for the experimental group) indicated good palatability of the product. In conclusion, the enzymatically synthesized SL containing EFA and natural antioxidants has nutritional properties almost identical to those of natural fats, and can be used as a reduced calorie fat.

p53 mutations in nonmelanoma skin cancer of the head and neck: molecular evidence for field cancerization.

Multiple and distinct p53 mutations were detected by DNA sequence analysis in tumor and adjacent nonmalignant skin samples from eight patients with nonmelanoma skin cancer of the head and neck, providing unambiguous evidence for field cancerization. The mutations consisted of C-->T transitions at dipyrimidine sequences (30% of all single base substitutions), T-->C transitions (47%), and G-->T transversions (12%), suggesting that other carcinogens may act along with UV radiation in the development of nonmelanoma skin cancer. Patient interviews revealed that, in addition to substantial exposure to solar UV radiation, most had a history of smoking and were exposed to carcinogens from industrial or agricultural sources. These data show that extensive molecular epidemiological investigations are necessary to elucidate risk factors associated with the disease in localities where patients often report substantial exposure to environmental carcinogens.

Molecular population genetic analysis of the streptokinase gene of Streptococcus pyogenes: mosaic alleles generated by recombination.

To understand the mechanisms governing molecular evolution of the streptokinase gene (skn), a 384 bp DNA fragment encoding two variable regions of the molecule was characterized in 47 isolates of Streptococcus pyogenes. The results reveal that alleles of the streptokinase gene have a mosaic structure, and provide strong evidence for intragenic recombination. Moreover, organisms that are well differentiated in overall chromosomal character have identical skn alleles, which suggests that horizontal gene transfer and recombination have participated in the evolution of this locus. No simple relationship between skn allele and serum opacity factor production or specific disease was identified. The predicted amino acid sequences of highly divergent skn alleles are strikingly similar in hydrophilicity and hydrophobicity profiles, distribution of amphipathic and flexible regions, surface probability plots, and antigenic indices, indicating that despite extensive nucleotide polymorphism in the two skn variable regions, selective pressure has constrained overall structural divergence. These results add to an important emerging theme that intragenic recombination plays a critical role in diversifying genes coding for streptococcal virulence factors.

High frequency of p53 mutations in ultraviolet radiation-induced murine skin tumors: evidence for strand bias and tumor heterogeneity.

Exposure to UV radiation has long been associated with the development of skin cancers. To identify the molecular targets in UV carcinogenesis, we analyzed 11 UV-induced murine skin cancers for mutations in the p53 tumor suppressor gene and found a 100% incidence rate. Such a high frequency of p53 mutations is unprecedented and suggests that this gene plays an important role in the development of UV-induced skin cancers. The mutations were predominantly "UV-signature" transitions (C-->T and CC-->TT) at pyrimidine-rich sequences located on the nontranscribed strand of the gene. In addition, seven tumors harbored multiple mutant alleles of p53, providing strong evidence for tumor heterogeneity at the molecular level.

Cord blood diazepam: clinical effects in neonates of eclamptic mothers.

Diazepam used in the treatment of eclampsia crosses the placental barrier readily, and may cause various clinical effects in the neonates. Twenty-five (25) live born babies of eclamptic mothers receiving diazepam were studied and cord blood diazepam concentration was estimated. Effect of low dose of diazepam is minimal apart from lowering of rectal temperature and the effects lasted for a period of 12 hours. But high dose (> 30 mg) of diazepam and prolonged duration of diazepam therapy in mothers causes significant depression of the newborn and the effects lasted for a period of 36-48 hours. As the clinical condition of the newborn is not related to the diazepam concentration in cord blood, the cord blood estimation is not helpful in the assessment of clinical effects of the drug in newborn. The tissue storage of the drug in newborn appears to be responsible for the clinical effects.

Temporal variation in bacterial disease frequency: molecular population genetic analysis of scarlet fever epidemics in Ottawa and in eastern Germany.

In an effort to understand the molecular genetic basis of temporal variation in frequency and severity of bacterial disease, genetic relationships among strains of Streptococcus pyogenes that caused scarlet fever epidemics in Canada in the early 1940s and in eastern Germany in the 1960s to 1980s were studied. Application of multilocus enzyme electrophoresis and comparative sequencing of the gene (speA) encoding streptococcal pyrogenic exotoxin A (scarlet fever toxin) revealed that new waves of scarlet fever are associated with an increase in frequency of S. pyogenes clones carrying variant speA alleles. This finding suggests that the occurrence of new scarlet fever epidemics can be predicted by comprehensive monitoring of the frequency of S. pyogenes clones with variant toxin alleles.

Geographic and temporal distribution and molecular characterization of two highly pathogenic clones of Streptococcus pyogenes expressing allelic variants of pyrogenic exotoxin A (Scarlet fever toxin).

The molecular population genetics and pathogenic potential of North American and European invasive strains of Streptococcus pyogenes were assessed. Isolates from recent invasive infections and from infections in the 1920s and 1930s were characterized for multilocus enzyme genotype and allelic variation in the gene (speA) that encodes streptococcal pyrogenic exotoxin (SPE) A (scarlet fever toxin). A subset of strains was studied for allelic variation in genes that encode SPE B and streptokinase. All contemporary strains assigned to electrophoretic types (ETs) 1 and 2 that synthesize SPE A have the speA2 and speA3 allelic variants, respectively, and their relative virulence in two mouse models is similar to that of strains of the same ET and M protein types recovered earlier. In contrast, ET 1 and 2 isolates from disease episodes in the 1920s and 1930s contain the speA1 allele. The data suggest there may be temporal and geographic variation in the occurrence of clone--virulence factor allele combinations, an observation that may in part explain fluctuations in disease frequency, severity, and character.