Western diet increases cardiac ceramide content in healthy and hypertrophied hearts.

BACKGROUND AND AIMS: Obesity and cardiac left ventricular hypertrophy (LVH) are recognised independent risk factors in the development of heart failure (HF). However, the combination of these factors may exacerbate the onset of cardiovascular disease by mechanisms as yet unclear. LVH leads to significant cellular remodelling, including alterations in metabolism which may result in an inappropriate accumulation of lipids and eventual lipotoxicity and apoptosis. The aim of the study was to determine the impact of dietary manipulation on cardiac metabolism in the obese and hypertrophied heart. METHODS AND RESULTS: LVH was induced via aortic constriction (AC) in an experimental model of cardiac hypertrophy and animals subjected to 9 weeks of dietary manipulation with either a standard, high fat, or a sucrose containing Western-style diet (SD, HFD and WD, respectively). This latter diet resulted in accelerated weight gain in both LVH/AC and control animals. LVH was greater in AC animals fed a WD, and both control and AC animals from this diet showed a significant reduction in cardiac fatty acid oxidation and increased triacylglycerol content. Ceramide content was significantly increased in the WD groups, with no additional effect of LVH. Comparison with a model of HF induced by exposure to Doxorubicin and WD showed exacerbated remodelling of cardiac ceramide species leading to increased C16 and C18 content. CONCLUSIONS: These findings highlight the inappropriate accumulation and re-distribution of cardiac ceramide species in a diet-induced model of obesity and LVH, potentially increasing susceptibility to cell death. The combination of increased fat and sugar leads to greater pathological remodelling and may explain why this diet pattern is consistently linked with poor cardiovascular outcomes.

A carrot leucine-rich-repeat protein that inhibits ice recrystallization.

Many organisms adapted to live at subzero temperatures express antifreeze proteins that improve their tolerance to freezing. Although structurally diverse, all antifreeze proteins interact with ice surfaces, depress the freezing temperature of aqueous solutions, and inhibit ice crystal growth. A protein purified from carrot shares these functional features with antifreeze proteins of fish. Expression of the carrot complementary DNA in tobacco resulted in the accumulation of antifreeze activity in the apoplast of plants grown at greenhouse temperatures. The sequence of carrot antifreeze protein is similar to that of polygalacturonase inhibitor proteins and contains leucine-rich repeats.

Molecular characterisation of a candidate gut sucrase in the pea aphid, Acyrthosiphon pisum.

The hydrolysis of sucrose, the principal dietary source of carbon for aphids, is catalysed by a gut alpha-glucosidase/transglucosidase activity. An alpha-glucosidase, referred to as APS1, was identified in both a gut-specific cDNA library and a sucrase-enriched membrane preparation from guts of the pea aphid Acyrthosiphon pisum by a combination of genomic and proteomic techniques. APS1 contains a predicted signal peptide, and has a predicted molecular mass of 68 kDa (unprocessed) or 66.4 kDa (mature protein). It has amino acid sequence similarity to alpha-glucosidases (EC 3.2.1.20) of glycoside hydrolase family 13 in other insects. The predicted APS1 protein contains two domains: an N-terminal catalytic domain, and a C-terminal hydrophobic domain. In situ localisation and RT-PCR studies revealed that APS1 mRNA was expressed in the gut distal to the stomach, the same localisation as sucrase activity. When expressed heterologously in Xenopus embryos, APS1 was membrane-bound and had sucrase activity. It is concluded that APS1 is a dominant, and possibly sole, protein mediating sucrase activity in the aphid gut.

Microbial forensics for natural and intentional incidents of infectious disease involving animals.

Microbial forensics is a relatively new scientific discipline dedicated to analysing microbiological evidence from a crime for attribution purposes. It builds on traditional microbiology and epidemiology but within a legal framework. Important motives for forensic investigations include interdiction of criminals, prosecution of justice, and ideally, deterrence of others from committing similar acts. Forensic capabilities in animal health should focus on building capacity for detection and reporting of increases in infectious disease morbidity and mortality among animals that might reflect a covert release of a pathogen. Suspicion should be raised when epidemiological patterns are different from those expected for the animal population and the pathogen in question. Existing capacities for the detection and reporting of epidemic and even endemic diseases should be an international priority for the prevention of catastrophic losses in animal and potentially in human life. The veterinary community needs to be more aware of the legal requirements related to forensic investigations so that veterinarians will be prepared to handle evidence properly within their own fields.

A mass spectrometry method for the determination of the species of origin of gelatine in foods and pharmaceutical products.

Gelatine is a component of a wide range of foods. It is manufactured as a by-product of the meat industry from bone and hide, mainly from bovine and porcine sources. Accurate food labelling enables consumers to make informed decisions about the food they buy. Since labelling currently relies heavily on due diligence involving a paper trail, there could be benefits in developing a reliable test method for the consumer industries in terms of the species origin of gelatine. We present a method to determine the species origin of gelatines by peptide mass spectrometry methods. An evaluative comparison is also made with ELISA and PCR technologies. Commercial gelatines were found to contain undeclared species. Furthermore, undeclared bovine peptides were observed in commercial injection matrices. This analytical method could therefore support the food industry in terms of determining the species authenticity of gelatine in foods.

Structural and composition of the N2 neuraminidase of influenza virus. Effect of carbohydrate content on the validity of molecular weight estimations.

Neuraminidase was isolated by proteolysis of the X7-(F1) (HON2) strain of influenza virus, and purified by gel filtration. The molecule contained a total of 46% (w/w) carbohydrate. The Mr was estimated as 152 500 (sedimentation diffusion) and 147 000 (sedimentation equilibrium). In 6 M guanidine-HCl the molecular weight was halved to 66 000 (sedimentation equilibrium). After irreversible reduction and blocking of sulphydryl groups the molecular weight was halved again to 33 500 (sedimentation equilibrium). These results confirm the tetrameric model of neuraminidase structure. They also provide strong evidence that the tetramer is composed of two disulphide linked dimers, themselves associated by non-covalent linkages. Theoretical considerations based on this model predict that assembly of the molecule must be accompanied by allosteric conformational changes in the subunits. The high carbohydrate content was thought to explain the discrepancy between the molecular weight values for the neuraminidase polypeptide obtained by different methods, and also the exceptional resistance of the molecule to digestion by proteolytic enzymes.

Two-domain structure of the native and reactive centre cleaved forms of C1 inhibitor of human complement by neutron scattering.

The C1 inhibitor component of human complement is a member of the serpin superfamily, and controls C1 activation. Carbohydrate analyses showed that there are seven O-linked oligosaccharides in C1 inhibitor. Together with six N-linked complex-type oligosaccharides, the carbohydrate content is therefore 26% by weight and the molecular weight (Mr) is calculated as 71,100. Neutron scattering gives an Mr of 76,000 (+/- 4000) and a matchpoint of 41.8 to 42.3% 2H2O, in agreement with this carbohydrate and amino acid composition. Guinier plots to determine the radius of gyration RG were biphasic. Neutron contrast variation of C1 inhibitor in H2O-2H2O mixtures gave an overall radius of gyration RG at infinite contrast of 4.85 nm, from analyses at low Q, and a cross-sectional RG of 1.43 nm. The reactive centre cleaved form of C1 inhibitor has the same Mr and structure as the native molecule. The length of C1 inhibitor, 16 to 19 nm, is far greater than that of the putative serpin domain. This is attributed to an elongated structure for the carbohydrate-rich 113-residue N-terminal domain. The radial inhomogeneity of scattering density, alpha, is large at 59 x 10(-5) from the RG data and 28 x 10(-5) from the cross-sectional analysis, and this is accounted for by the high oligosaccharide content of C1 inhibitor. The scattering data were modelled using small spheres. A two-domain structure of length 18 nm based on two distinct scattering densities accounted for all the contrast variation data. One domain is based on the crystal structure of alpha 1 antitrypsin (7 nm x 3 nm x 3 nm). The other corresponds to an extended heavily glycosylated N-terminal domain of length 15 nm, whose long axis is close to the longest axis of the serpin domain. Calculation of the sedimentation coefficient s0(20),w for C1 inhibitor using the hydrodynamic sphere approach showed that a two-domain head-and-tail structure with an Mr of 71,000 and longest axis of 16 to 19 nm successfully reproduced the s0(20),w of 3.7 S. Possible roles of the N-terminal domain in the function of C1 inhibitor are discussed.

Leptospirosis among patients presenting with dengue-like illness in Puerto Rico.

Leptospirosis is difficult to distinguish from dengue fever without laboratory confirmation. Sporadic cases/clusters of leptospirosis occur in Puerto Rico, but surveillance is passive and laboratory confirmation is rare. We tested for leptospirosis using an IgM ELISA on sera testing negative for dengue virus IgM antibody and conducted a case-control study assessing risk factors for leptospirosis, comparing clinical/laboratory findings between leptospirosis (case-patients) and dengue patients (controls). Among 730 dengue-negative sera, 36 (5%) were positive for leptospirosis. We performed post mortem testing for leptospirosis on 12 available specimens from suspected dengue-related fatalities; 10 (83%) tested positive. Among these 10 fatal cases, pulmonary hemorrhage and renal failure were the most common causes of death. We enrolled 42 case-patients and 84 controls. Jaundice, elevated BUN, hyperbilirubinemia, anemia, and leukocytosis were associated with leptospirosis (p < .01 for all). Male sex, walking in puddles, rural habitation, and owning horses were independently associated with leptospirosis. Epidemiological, clinical, and laboratory criteria may help distinguish leptospirosis from dengue and identify patients who would benefit from early antibiotic treatment.

Carbohydrate analysis.

The analysis of the carbohydrate chains attached to proteins is becoming increasingly important as appreciation of the role of glycosylation in the structural and functional properties of biologically significant glycoproteins grows. Over the past year, a number of developments have been made that may improve and promote the analysis of the glycosylation of proteins.

The significance of gut sucrase activity for osmoregulation in the pea aphid, Acyrthosiphon pisum.

The osmotic pressure of the body fluids of aphids is lower than in their diet of plant phloem sap. It is hypothesised that aphids reduce the osmotic pressure of ingested food by sucrase-mediated hydrolysis of dietary sucrose to glucose and fructose, and the polymerisation of glucose into oligosaccharides of low osmotic pressure per hexose unit. To test this hypothesis, the impact of the alpha-glucosidase inhibitor acarbose on the sugar relations and osmoregulation of aphids was explored. Acarbose inhibited sucrase activity in gut homogenates and the production of monosaccharides and oligosaccharides in the honeydew of live aphids. Acarbose caused an increase in the haemolymph osmotic pressure for aphids reared on a diet (containing 0.75 M sucrose) hyperosmotic to the haemolymph and not on the isoosmotic diet containing 0.2 M sucrose. It did not affect aphid feeding rate over 2 days, except at high concentrations on 0.75 M sucrose diet, and this may have been a secondary consequence of osmotic dysfunction. Acarbose-treated aphids died prematurely. With 5 microM dietary acarbose, mean survivorship on 0.2 M sucrose diet was 4.2 days, not significantly different from starved aphids, indicating that, although these aphids fed, they were deprived of utilisable carbon; and on 0.75 M sucrose diet, mean survivorship was just 2.8 days, probably as a consequence of osmotic failure. It is concluded that the aphid gut sucrase activity is essential for osmoregulation of aphids ingesting food hyperosmotic to their body fluids.

Leptospirosis: an emerging health problem in Thailand.

Leptospirosis is an emerging health problem in Thailand, with dramatic increases in reported incidence since 1996. The annual number of reported leptospirosis cases increased from 398 cases in 1996 to 14,285 cases in 2000. In 2001, 2002, and 2003, the number of reported cases decreased, but still remained high at 10,217, 6,864, and 4,958 cases, respectively. The epidemiological characteristics of leptospirosis in Thailand include a peak incidence in September and October in association with the rainy season. A vast majority of the cases (90%) were reported in the Northeast region. The case fatality rate was as high as 4.4%, having a predominant association with male farmers aged 15 to 45 years. Outpatient cases were approximately 9 times more common than admitted cases, with an apparent recent shift in the pattern of infecting serovars among reservoir animals and humans.

Outbreak of histoplasmosis among cavers attending the National Speleological Society Annual Convention, Texas, 1994.

In June 1994, 18 people developed serologically confirmed histoplasmosis following cave exploration associated with the annual National Speleological Society Convention in Bracketville, Texas. Six others had an undiagnosed illness suspected to be histoplasmosis. Two persons were hospitalized. We conducted a survey of convention attendees and a nested case-control study of those entering caves. We also conducted a histoplasmin skin test survey of a subgroup of the society, the Texas Cavers Association, who were attending a reunion in October 1994. Among the national convention attendees, exposure to two caves was identified as responsible for 22 (92%) of the 24 cases; 12 (75%) of 16 people exploring one cave (Cave A) and 10 (77%) of 13 exploring a separate cave (Cave B) developed acute histoplasmosis. Additional risk-factors included fewer years of caving experience, longer time spent in the caves, and entering a confined crawl space in Cave A. Of 113 participants in the separate skin test survey, 68 (60%) were found to be skin test positive, indicating previous exposure to Histoplasma capsulatum. A positive skin test was significantly associated with male sex and more years of caving experience. Those less experienced in caving associations should be taught about histoplasmosis, and health care providers should pursue histories of cave exposure for patients with bronchitis or pneumonia that does not respond to initial antibiotic therapy.

Risk factors associated with leptospirosis in northeastern Thailand, 1998.

Leptospirosis is a zoonotic disease of worldwide distribution caused by spirochetes of the genus Leptospira. Humans are infected through direct contact with infected animals or through exposure to fresh water or soil contaminated by infected animal urine. Leptospirosis is characterized by acute fever that can be followed by a more severe, sometimes fatal illness that may include jaundice and renal failure (Weil's disease), meningitis, myocarditis, hemorrhagic pneumonitis, or hemodynamic collapse. To identify potential risk factors for leptospirosis in Thailand, we conducted a matched case-control study in Nakornratchasrima Province of the northeastern region. Fifty-nine cases and 118 controls were included in the study. Four activities in the two weeks prior to illness were independently associated with leptospirosis infection: walking through water (odds ratio [OR] = 4.9, 95% confidence interval [CI] = 1.7-14.1), applying fertilizer in wet fields for more than 6 hr a day (OR = 3.4, 95% CI = 1.5-7.8), plowing in wet fields for more than 6 hr a day (OR = 3.5, 95% CI = 1.1-11.6), and pulling out rice plant sprouts in wet fields for more than 6 hr a day (OR = 3.1, 95% CI = 1.02-9.3). Identification of these risk factors on admission might prove useful for early diagnosis and treatment of leptospirosis in Thailand.

Studies on the control of visceral leishmaniasis: validation of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) for field diagnosis of canine visceral leishmaniasis.

The Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA), the latest version of the enzyme-linked immunosorbent assay, uses antigen-coated beads. A 96-well plate can be run in 20 min without electricity or expensive equipment. We compared the FAST-ELISA, a standard ELISA, and an indirect immunofluorescent assay (IFA) for evaluation of canine leishmaniasis under field conditions using samples from 161 dogs from our longitudinal study in the endemic area of Jacobina, Bahia, Brazil. Organisms were isolated by culture (NN medium) or by inoculation of hamsters with samples from 59 of the dogs. When plasma were tested, we found a sensitivity of 88% and a specificity of 90% using the FAST-ELISA with a spectrophotometer. Using the same plasma samples, the IFA had a sensitivity of 75% and a specificity of 93%. The standard ELISA had a sensitivity of 90% and a specificity of 85%. When whole blood was tested with the FAST-ELISA, we found a sensitivity of 85%. There was no significant difference between visual and spectrophotometric results with plasma or whole blood. The FAST-ELISA system provides a sensitive, specific, and field-adaptable test for canine visceral leishmaniasis, which can be evaluated quickly without the use of a microscope or spectrophotometer.

Comparison of the polymerase chain reaction and serology for the detection of canine visceral leishmaniasis.

The polymerase chain reaction (PCR) and serology was evaluated for the diagnosis of canine visceral leishmaniasis in Bahia, Brazil in a study of 125 dogs. The PCR was 100% sensitive in 25 dogs that had Leishmania demonstrated by either culture or hamster inoculation. It was 100% specific for 35 dogs from the northeastern United States, all were PCR negative. However, 22 of 54 Brazilian dogs that were culture-hamster inoculation-negative were positive by PCR. The nature of the PCR product was identified by hybridization with specific Leishmania probes. Whereas the sensitivity of serology in relationship to infection, as determined by hamster or culture assay was more than 80%, sensitivity of serology was only 63% when compared with PCR. These results raise questions about the use of serology to detect Leishmania infection in dogs, and suggest that the PCR might serve as a better gold standard to define Leishmania infection than culture or hamster inoculation.

Studies on control of visceral leishmaniasis: impact of dog control on canine and human visceral leishmaniasis in Jacobina, Bahia, Brazil.

To assess the effect of removing leishmania-infected dogs on the incidence of visceral leishmaniasis, a controlled intervention study was performed in northeast Brazil. The attempted elimination of seropositive dogs resulted in an initial significant decrease in the annual incidence of seroconversion among dogs from 36% to 6% over the first two years. In the following two years, the incidence increased to 11% and 14%, respectively. In a control area in which dogs were surveyed but seropositive dogs were not removed, the cumulative incidence did not vary significantly from year to year, ranging from 16% to 27%. In the intervention area, the prevalence of dog seropositivity decreased from 36% before the intervention to 10% and remained stable. These findings suggest that attempting to remove seropositive dogs is insufficient as a measure for eradicating visceral leishmaniasis in dogs. However, the force of transmission of infection among dogs can be reduced by such programs. Also, when the number of human cases before and after the start of the intervention was calculated, a significant decrease in incidence of disease in the intervention area was observed among children less than 15 years of age (P < 0.01). The results of this intervention study suggest that the elimination of the majority of seropositive dogs may affect the cumulative incidence of seroconversion in dogs temporarily and may also diminish the incidence of human cases of visceral leishmaniasis.

Asymptomatic infection and risk factors for leptospirosis in Nicaragua.

As part of an investigation of a 1995 outbreak of leptospirosis in Nicaragua, a cross-sectional serologic survey was conducted in the town of El Sauce. Of 566 persons, 85 (15%) were positive for IgM anti-Leptospira antibodies, indicating recent leptospirosis infection. Asymptomatic leptospirosis infection was common, with only 25 (29.4%) of the 85 seropositive inhabitants reporting a febrile illness in the 2 months before the survey. Multivariable analysis revealed that having an indoor water source remained independently protective against leptospirosis. Gathering wood was independently associated with infection. These findings suggest that asymptomatic infection with Leptospira is common in endemic areas of Leptospira transmission. Improvement in water sanitation and behavioral modifications to reduce environmental exposure may reduce the risk of leptospirosis in endemic regions.

Honeydew sugars and osmoregulation in the pea aphid Acyrthosiphon pisum

Pea aphids, Acyrthosiphon pisum, containing their symbiotic bacteria (untreated aphids) and experimentally deprived of their bacteria by treatment with the antibiotic rifampicin (antibiotic-treated aphids) were reared on the plant Vicia faba. The sugars in the honeydew produced by untreated aphids comprised predominantly the monosaccharides glucose and fructose, while the honeydew of antibiotic-treated aphids contained considerable amounts of oligosaccharides of up to 16 hexose units. The honeydew and haemolymph of the aphids were iso-osmotic, and their osmotic pressure was significantly lower in untreated aphids (0.91­0.95 MPa) than in antibiotic-treated aphids (1.01­1.05 MPa) (P<0.05). For insects reared on chemically defined diets containing 0.15­1.0 mol l-1 sucrose (osmotic pressure 1.1­4.0 MPa), the osmotic pressure of the aphid haemolymph did not vary with dietary osmotic pressure, but was regulated to approximately 1.0 MPa in untreated and 1.3 MPa in antibiotic-treated aphids. The sugars in the aphid honeydew varied with dietary sucrose concentration; with monosaccharides dominant at low concentrations and oligosaccharides dominant at high concentrations of dietary sucrose. The lowest dietary sucrose concentration at which honeydew oligosaccharides were detected was 0.2 mol l-1 for the antibiotic-treated aphids and 0.3 mol l-1 for untreated aphids. These data indicate that the aphid, and not its associated microbiota, mediates the synthesis of oligosaccharides when the osmotic pressure of the ingesta is high.

The glycosylation of glycoprotein lectins. Intra- and inter-genus variation in N-linked oligosaccharide expression.

Glycosylated lectins represent a series of glycoproteins with related activities and, in the case of the Leguminosae, related amino acid sequences. Therefore, they offer a model system in which to study the diversity of N-linked oligosaccharide structures of plant glycoproteins. The influence of the polypeptide on the type of oligosaccharide substitution and the problem of inter- and intra-genus variation in glycosylation can also be addressed. Analysis of the glycosylation of 18 lectins has shown that they can be classified into four qualitatively similar groups on the basis of the Bio-Gel P-4 elution profiles of the oligosaccharides released by hydrazinolysis: (a) The Erythrina cristagalli profile, with a major component at 8.8 glucose units (gu) and minor components at 8.0, 7.2, and 5.8 gu. The major component is the heptasaccharide, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-[beta-D-Xyl p-(1----2)]- beta-D-Manp-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-(1----3)]- D-GlcNAc. (b) The Phaseolus vulgaris profile, which was characterized by peaks at 12.5, 11.7, 10.8, and 9.9 gu, in addition to the peaks at 8.8, 8.0, 7.2, and 5.8 gu mentioned above. These higher-mol.-wt. components were oligo-D-mannose oligosaccharides containing 9, 8, 7, and 6 D-mannose residues, respectively. (c) The Lonchocarpas capassa profile, which had a major peak at approximately 8 gu. (d) The soybean agglutinin profile, which has a single peak at 12.5 gu. This peak consisted solely of an oligomannose undecasaccharide containing 9 D-mannose residues. This lectin is unique in that it shows no microheterogeneity.

Infectivity of low numbers of Toxoplasma gondii oocysts to pigs.

To define the infectiousness of the VEG strain of Toxoplasma gondii, 42 pigs were fed doses estimated at 10, 1, or < 1 mouse infective oocysts. They were killed 38-99 days after inoculation and 50 g of tissues from their tongue, heart, and brain were individually homogenized in acidic pepsin solution and bioassayed in mice. Pools of brain, heart, tongue, and skeletal muscle (total 500 g) were bioassayed in cats. Toxoplasma gondii was isolated by bioassays in mice and in cats from 13 of 14 pigs fed 10 oocysts, 13 of 14 pigs fed 1 oocyst, and 4 of 14 pigs fed "less than" 1 oocyst, indicating high infectivity of VEG strain of T. gondii to pigs. All infected pigs developed modified agglutination test antibodies (> 1:50). Control pigs (n = 6) remained seronegative (< 1:20) and T. gondii was not isolated from their tissues. Toxoplasma gondii was isolated from tongues of 27 (93%), brains of 21 (72%), and hearts of 13 (45%) of 29 experimentally infected pigs by bioassay in mice. The number of T. gondii-positive mice after inoculation of tongue, brain, and heart from infected pigs was 240 (80%), 84 (28%), and 36 (12%) of 300 mice inoculated with each organ, respectively. Thus, the VEG strain of T. gondii was localized more often and in higher numbers in the tongue than in the brain and the heart of pigs. The apparent muscle localization after infection with the low dose of the VEG strain of T. gondii agrees with other studies in livestock that suggest T. gondii is more neurotropic in mice than in livestock.