GeneHunt for rapid domain-specific annotation of glycoside hydrolases.

The identification of glycoside hydrolases (GHs) for efficient polysaccharide deconstruction is essential for the development of biofuels. Here, we investigate the potential of sequential HMM-profile identification for the rapid and precise identification of the multi-domain architecture of GHs from various datasets. First, as a validation, we successfully reannotated >98% of the biochemically characterized enzymes listed on the CAZy database. Next, we analyzed the 43 million non-redundant sequences from the M5nr data and identified 322,068 unique GHs. Finally, we searched 129 assembled metagenomes retrieved from MG-RAST for environmental GHs and identified 160,790 additional enzymes. Although most identified sequences corresponded to single domain enzymes, many contained several domains, including known accessory domains and some domains never identified in association with GH. Several sequences displayed multiple catalytic domains and few of these potential multi-activity proteins combined potentially synergistic domains. Finally, we produced and confirmed the biochemical activities of a GH5-GH10 cellulase-xylanase and a GH11-CE4 xylanase-esterase. Globally, this "gene to enzyme pipeline" provides a rationale for mining large datasets in order to identify new catalysts combining unique properties for the efficient deconstruction of polysaccharides.

A finite element model of an idealized diarthrodial joint to investigate the effects of variation in the mechanical properties of the tissues.

The stiffness of articular cartilage increases dramatically with increasing rate of loading, and it has been hypothesized that increasing the stiffness of the subchondral bone may result in damaging stresses being generated in the articular cartilage. Despite the interdependence of these tissues in a joint, little is understood of the effect of such changes in one tissue on stresses generated in another. To investigate this, a parametric finite element model of an idealized joint was developed. The model incorporated layers representing articular cartilage, calcified cartilage, the subchondral bone plate and cancellous bone. Taguchi factorial design techniques, employing a two-level full-factorial and a four-level fractional factorial design, were used to vary the material properties and thicknesses of the layers over the wide range of values found in the literature. The effects on the maximum values of von Mises stress in each of the tissues are reported here. The stiffness of the cartilage was the main factor that determined the stress in the articular cartilage. This, and the thickness of the cartilage, also had the largest effect on the stresses in all the other tissues with the exception of the subchondral bone plate, in which stresses were dominated by its own stiffness. The stiffness of the underlying subchondral bone had no effect on the stresses generated in the cartilage. This study shows how stresses in the various tissues are affected by changes in their mechanical properties and thicknesses. It also demonstrates the benefits of a structured, systematic approach to investigating parameter variation in finite element models.

Serodiagnosis of cystic echinococcosis in naturally infected camels.

An ELISA was developed for serological detection of Echinococcus granulosus infection in dromedary camels. Antigen B (AgB) partially purified from hydatid cyst fluid of camels or sheep naturally infected with cystic echinococcosis (CE) due to E. granulosus, as well as a recombinant antigen B product (r-AgB) were used in an ELISA to screen panels of serum samples from slaughtered camels naturally infected with CE. Native hydatid cyst fluid antigen preparations were able to detect antibodies in sera from a significant proportion of camels with CE, as confirmed at post-mortem. Seroreactivity however, was variable. ELISA specificity for sera from naturally infected camels versus inspection-negative animals ranged from 90 to 99%. Native antigen B gave the highest sensitivity (97%) in ELISA for camel CE confirmed at slaughter. In contrast, r-AgB gave lower sensitivity for camel (84%) and sheep (28%) CE. The r-AgB-ELISA was, however, highly specific (90 and 95%) respectively for both camel and sheep natural CE infection. These results indicate that an ELISA based on serum antibody detection to AgB could be developed for immunodiagnosis of cystic echinococcosis in camels.

Impaired fasting glucose and impaired glucose tolerance - lack of agreement between the two categories in a North Indian population.

This study was carried out to determine the relationship between impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) in a North Indian population. The data in 5083 subjects studied earlier was reanalyzed by applying new WHO diagnostic criteria. Reanalysis revealed that 305 (6.0%) subjects had diabetes mellitus (198 on the basis of fasting plasma glucose of > or =7.0 mmol/l (> or =126 mg/dl) and an additional 107 based on a 2-h glucose tolerance test), 381 (7.5%) had IFG and 361 (7.1%) had IGT. Of these 361 subjects with IGT, only 99 (27.4%) had impaired fasting glucose whereas 262 (72.6%) had normal fasting glucose of <6.1 mmol/l (<110 mg/dl). Of 381 subjects with IFG, 99 (26%) had IGT where as 282 (74%) had normal 2-hr glucose. We conclude that there is a poor correlation between IGT and IFG.

Copper, zinc and magnesium levels in fibrocalculous pancreatic diabetes.

Alterations in trace elements and mineral homeostasis have been documented both in insulin-dependent diabetes mellitus and non-insulin dependent diabetes mellitus. No data are available about trace elements in fibrocalculous pancreatic diabetes, a unique form of secondary diabetes mellitus. This study evaluated the plasma concentrations of copper, zinc and magnesium in this form of diabetes. Twenty-five patients (9 men and 16 women) with fibrocalculous pancreatic diabetes and 25 healthy non-diabetic subjects (16 men and 9 women) were studied. Patients with overt nephropathy were excluded. Plasma copper, zinc, and magnesium levels were analyzed using a GBC 902 double beam absorption spectrophotometer. The effect of glycemic control, microalbuminuria, sex and modality of treatment received on the plasma levels of copper, zinc and magnesium was assessed. Results of the study revealed that plasma copper, zinc, and magnesium levels were comparable between patients with fibrocalculous pancreatic diabetes and control subjects. Plasma copper levels were significantly higher in patients with controlled diabetes (16.15 +/- 0.67 micromol L(-1)) as compared to those with uncontrolled diabetes (13.75 +/- 0.61 micromol L(-1)) and healthy controls (13.91 +/- 0.55 micromol L(-1)). This merits further investigation. Microalbuminuria, modality of treatment received and sex did not influence the levels of these elements in fibrocalculous pancreatic diabetes.

Prevalence of type 2 diabetes mellitus and impaired glucose tolerance in the Kashmir Valley of the Indian subcontinent.

This cross-sectional population survey was undertaken to determine the prevalence of type 2 diabetes and impaired glucose tolerance in subjects aged 40 years or more in Kashmir Valley, India. The study was carried out in two phases. In phase one, 6091 randomly selected subjects, 40 years or older, from all six districts of the valley were surveyed for prevalence of known diabetes mellitus. In phase two, 5083 subjects, 40 years or older, were screened with oral glucose tolerance test for prevalence of undiagnosed (asymptomatic) diabetes mellitus and impaired glucose tolerance. Abnormalities of carbohydrate intolerance were determined as recommended by WHO. Of 6091 subjects interviewed, 115 were known cases of diabetes mellitus with an overall prevalence of 1.89% (1.98% in males and 1.77% in females). Results of glucose tolerance test revealed that mean fasting as well as mean 2 h blood glucose was significantly more in females as compared to males (4.68+/-0.91 and 6.40+/-2.12 vs. 4.49+/-0.96 and 5.94+/-2.03 mmol/l, respectively, P < 0.0001). Of 5083 subjects who were subjected to glucose tolerance test (GTT), 627 (12.34%) had an abnormal test; with 411 (8.09%) having impaired glucose tolerance (IGT) and 216 (4.25%) having diabetes mellitus. The prevalence of IGT as well as of diabetes was significantly more in females as compared to males (P < 0.001). Subjects who had family history of diabetes had a significantly higher prevalence of abnormal GTT. Prevalence of known diabetes as well as that of abnormal GTT steadily increased with age, with a highest prevalence in the age group of > or = 70 years (P < 0.001). Obese subjects had a significantly higher basal as well as 2 h blood glucose in males as well as in females. Subjects with diabetes on GTT had a higher waist/hip ratio. Overall the prevalence of diabetes as well as IGT was significantly higher in the urban population. We conclude that 1.89% of the general population have known diabetes, 4.25% have undiagnosed diabetes and 8.09% have impaired glucose tolerance test; making the total load of abnormal glucose tolerance 14.23% in Kashmir Valley. In subjects greater than 40 years of age having a family history of diabetes, obesity, higher age (50 years or above), female sex, and urban origin have more chance (odds ratio: 4.65, 2.30, 1.87, 1.49 and 1.16, respectively) of developing abnormal glucose tolerance.

Inhibition of rotavirus infection in vitro and in vivo by a synthetic peptide from VP4.

A synthetic peptide corresponding to bovine rotavirus C486 (BRV) VP4 amino acid sequence 232-255 (VP4-peptide) was studied with the objective of defining the origin of the protective immune response reported previously by Ijaz et al. (J. Virol. 1991, 65, 3106-3113). Pretreatment of MA-104 cells with the VP4-peptide before infection with rotavirus prevented both the attachment of 35S-labelled virus and plaque formation in vitro. In vivo studies using a murine rotavirus model demonstrated that intragastric administration of VP4-peptide protected subjects from challenge with virulent rotavirus. These results clearly indicate the importance of this epitope in virus-cell interactions and their potential as a rotavirus vaccine candidate.

Comparative studies on the sensitivity of polymerase chain reaction and microscopic examination for the detection of Trypanosoma evansi in experimentally infected mice.

Trypanosoma evansi, a protozoan parasite in the blood of camels is routinely diagnosed by finding the flagellates in the wet films or stained smear of peripheral blood, examined under a microscope. Although specific, this method is not sensitive at early stages of infection. We have tested the use of polymerase chain reaction (PCR) in the identification of T. evansi in different stages of infection in mice and compared its sensitivity with that of the standard microscopic examination method. Using a specific pair of primers, it was possible to identify T. evansi in the blood of infected mice. Experimentally, groups of mice were infected with T. evansi, isolated from a naturally infected local camel and blood samples were collected every day for 30 days post-infection. Direct microscopy or PCR was applied to detect parasitaemia. Results showed that during the acute phase of infection, parasites were detected by PCR three days earlier than by microscopy. Furthermore, the infected mice were consistently positive by PCR during the chronic phase while the parasites could not be demonstrated during this period using microscopic examination. These findings suggest that PCR may be applied to camel trypanosomosis during both acute and chronic phase of infection. Furthermore, it would provide an excellent tool in the evaluation of treatment of anti-trypanocidal drugs.

The prevalence of Toxoplasma gondii specific IgG and IgM antibodies in blood donors in Al Ain, United Arab Emirates indicates a potential risk to recipients.

The results of a prospective cross-sectional study on the anti-toxoplasma IgG and IgM specific antibody profile among blood donors in Al Ain United Arab Emirates are presented. The overall infection rate was 34%. Based on IgM specific antibody positive rate, acute toxoplasmosis was evident among 3% of the blood donors studied. The theoretical implications of these findings are discussed.

Copper, zinc, and magnesium levels in non-insulin dependent diabetes mellitus.

A relationship has been reported between trace elements and diabetes mellitus. This study evaluated the role of such a relationship in 83 patients with non-insulin dependent diabetes mellitus (40 men and 43 women), with a mean duration of diabetes of 3.9 +/- 3.6 years. Patients with nephropathy were excluded. Thirty healthy non-diabetic subjects were studied for comparative analysis. Subjects were subdivided into obese and non-obese. Diabetic subjects were also subdivided into controlled and uncontrolled groups; control was based on fasting blood glucose and serum fructosamine levels. Plasma copper, zinc and magnesium levels were analysed using a GBC 902 double beam atomic absorption spectrophotometer. Plasma zinc and magnesium levels were comparable between diabetic and non-diabetic subjects, while copper levels were significantly elevated (p < 0.01) in diabetic patients. Age, sex, duration and control of diabetes did not influence copper, zinc, or magnesium concentrations. We conclude that zinc and magnesium levels are not altered in diabetes mellitus, but the increased copper levels found in diabetics in our study may merit further investigation of the relationship between copper and non-insulin dependent diabetes mellitus.

Toxoplasma antibody prevalence among healthy adults in United Arab Emirates.

Full text is available as a scanned copy of the original print version.

Public health aspects of cystic echinococcosis in the Arab countries.

Human cystic echinococcosis (CE) caused by larval Echinococcus granulosus is a zoonosis of major public health importance throughout the region comprising Arab North Africa and the Middle East. Prevalence rates are determined by epizootiological factors related to the size of stray dog population and its worm burden and to the infection rates in the intermediate host reservoir livestock population. Socio-economic development and socio-cultural practices are considered important determinants in the continued transmission of the disease. The reasons why CE remains a significant public health problem in the region are summarized.

Gestational and neonatal toxoplasmosis: regional seroprevalence in the United Arab Emirates.

Paired maternal/cord blood samples were tested for anti-Toxoplasma IgG or IgM antibodies using Biomerieux Micro-EIA2 IgG and IgM test kits. Of the 1503 women tested at the time of delivery, 344 (22.9%) were IgG seropositive. Three hundred and one maternal sera, including 265 that were IgG positive, were tested for IgM antibodies: 47 were found positive, indicating a gestational toxoplasmosis incidence of 31 per 1000 pregnancies over one year. All but one of the IgM positive maternal sera had tested IgG positive. Cord blood IgG seropositivity was similar to the maternal rate but 18 of the 301 babies had significant levels of anti-Toxoplasma IgM antibodies. As these 18 babies were all born to mothers also positive for IgM antibodies, the calculated rate of transplacental transmission was 38.3% with the estimated prevalence of congenital toxoplasmosis of 12 per 1000 live births. There was no statistically significant positive correlation between maternal seroprevalence and such well-known risk factors as consumption of raw meat and milk, or proximity of cats and other animals. One baby was born with the classical stigmata of congenital toxoplasmosis.

Effect of previous chloroquine intake on in vivo P. falciparum drug sensitivity.

Investigation of the in vivo response of P. falciparum malaria parasites to chloroquine was conducted during 1993/94 in Al-Ain District of Abu Dhabi Emirate, UAE. Sixty seven expatriates who developed falciparum malaria on their return from Pakistan, Oman and Sudan were recruited for the WHO in vivo tests. Of the 67 patients, eight were classified as having RII and RIII responses, while 59 remained aparasitaemic at the end of the seven-day WHO standard test. On continuation into the 28-day WHO extended test, a further 34 patients exhibited RI resistance. Resistance of parasites to chloroquine was confirmed by measurement of plasma chloroquine using High-Performance Liquid Chromatography. In all 67 patients, the level of chloroquine was well above the minimum therapeutic level. The outcome of the in vivo test in patients treated for the first time was significantly different from that in patients who were previously on chloroquine. Among patients treated for the first time, 36 out of 41 (88%) had a resistant response, whereas, among those previously on chloroquine only six out of 26 (23%) had a resistant response. The difference is probably due to the higher initial plasma level of chloroquine among patients who were previously on the drug. Curing more patients with higher plasma chloroquine implies that chloroquine shall continue to be useful, particularly if resistance is at the RI level. Appropriate higher therapeutic levels of chloroquine should be defined for such patients.

Profile of neurological problems in diabetes mellitus retrospective analysis of data from 1294 patients.

Data from 1294 patients with diabetes mellitus admitted to the Endocrinology Department of the Institute of Medical Sciences, Srinagar, Kashmir, from 1986 to 1994, were analyzed for frequency of various neurological problems. Of 1294 patients, 46.29% had clinical evidence of one or more neurological problems. The frequency of neurological problems was significantly more in patients with type II diabetes mellitus (P<0.001). Predominant neurological problems included peripheral neuropathy (96.66%), stroke (5.51%), Parkinsonism (1.50%), seizure disorder (1.17%) and dementia (1%). Mean (+/- SD) age of patients with neurological problems was significantly more (P<0.001) than those without neurological problems (52.07+/- 9.52 versus 47.45+/- 12.87 years for type II diabetes mellitus; 26.73+/- 8.40 versus 18.0+/- 3.62 for type I diabetes mellitus). Mean duration of diabetes in patients with neurological problems was significantly more than those without neurological problems (6.70+/- 6.04 versus 3.95+/- 4.22 years for type II diabetes mellitus; 5.63+/- 3.67 versus 1.89+/- 2.57 for type I diabetes mellitus). At the time of admission, fasting blood glucose was lower in patients without neurological problems as compared to patients with problems (9.08+/- 2.22 versus 11.05+/- 4.91 mmol/L for type II diabetes mellitus; 9.44+/- 2.80 versus 13.01+/- 5.01 mmol/L for type I diabetes mellitus; P7lt;0.001).

Characterization of a synthetic peptide mimicking trypsin-cleavage site of rotavirus VP4.

A synthetic peptide corresponding to the trypsin cleavage site on the 84 k protein of bovine rotavirus was synthesized (VP4-peptide). This synthetic peptide could be cleaved by trypsin and therefore possessed the enzyme binding site present on the authentic protein. Further proof that this peptide mimicks the authentic trypsin cleavage site was the specific reaction of anti-peptide serum with the 84 k protein. The reaction of anti-peptide serum with infectious virus neutralized infectivity thereby supporting the biological importance of this site. Another interesting characteristic of this peptide was its ability to bind to the nucleocapsid protein resulting in a laddering effect on the nucleocapsid monomer (45 k), dimer (90 k) and trimer (135 k) [Gorzilia et al., J. Gen. Virol. 66, 1889-1900 (1985); Sabara et al., J. Virol. 53, 58-66 (1985); Sabara et al., J. Gen. Virol. 67, 201-212 (1986)]. Definitive proof of binding was provided by the fact that the increments in the ladder corresponded to the molecular weight of the synthetic peptide and that anti-peptide serum specifically reacted with the ladder formations. The laddering of the nucleocapsid could be eliminated by incubation with trypsin thus further supporting the formation of a synthetic peptide-nucleocapsid complex. Due to the ability of the peptide to bind to trypsin and to the nucleocapsid protein its biological activity was investigated. It appeared that increasing concentrations of the peptide reduced the rate of virus plaque formation, thereby suggesting that virus replication was inhibited. These results illustrate two features of this synthetic peptide which warrant further investigation; (1) its capacity to mimic an enzyme cleavage site and, (2) its ability to complex tightly to another protein. In protection-challenge experiments performed using a murine model, animals immunized with VP4-peptide provided protection passively, to neonates suckling on the immune dams, against a virulent rotavirus. The potential applications of this peptide in rotavirus diagnosis, therapy and synthetic peptides based vaccine is discussed.

Suppression of transplant immunity in experimental trichinellosis.

Skin allograft rejection in Balb/c and C57BL/6J mice following experimental infection with 300 larvae of Trichinella spiralis or Trichinella pseudospiralis was studied. Skin grafts from normal C57BL/6J mice were transplanted to infected Balb/c mice and vice versa at days 3, 10, 20 and 30 post-infection. The clinical criteria for graft rejection, scarring and graft falling, were followed. The results indicated that T. spiralis and T. pseudospiralis infections induced a significant delay in graft rejection when compared to the control groups. A maximum rejection time of 24 days was observed in T. spiralis infected C57BL/6J mice which received skin grafts from Balb/c mice on day 3 post-infection. The rejection in the uninfected control group was on day 7 post transplant. The mean rejection times for transplants on various days post-infection, with both species were very similar. Also, the rejection profiles in Balb/c mice were comparable to that observed in C57BL/6J mice, with a maximum delay of 26 days to rejection again obtained in mice transplanted on day 3 post-infection, for both species. When the skin grafts were performed 5 or 10 days prior to infection, the rejection occurred on day 7, as in the control group. The effect of T. spiralis and T. pseudospiralis soluble larval extracts (TSE or TPE) on graft rejection was also examined. Four intraperitoneal injections of 50 micrograms each of TSE or TPE every 48 h for 7 days did not induce any significant delay in graft rejection. In contrast, secretory antigens prepared from cultured larvae in vitro induced significant delays in graft rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Priming and induction of anti-rotavirus antibody response by synthetic peptides derived from VP7 and VP4.

Synthetic peptides derived from bovine rotavirus C-486 (BRV) outer capsid (VP7 and VP4) and inner capsid (VP6) proteins were tested to evaluate their ability to prime and induce an anti-rotavirus antibody response. Peptides corresponding to the amino acid residues 232-255 of VP4 (VP4-peptide), 275-295 of VP7 (VP7-peptide) and 40-60 of VP6 (VP6-peptide) of BRV were chemically synthesized. These peptides were coupled to carrier proteins (either keyhole limpet haemocyanin (KLH) or recombinant rotavirus inner capsid protein-VP6 assembled into virus-like particles (VP6-carrier) were used as carrier to link the synthetic peptides under study), and the resulting conjugates were used to immunize rotavirus seronegative mice. An enzyme-linked immunosorbent assay (ELISA) was used to determine anti-peptide and anti-rotavirus antibody titres in serum samples collected after immunization. All peptides were immunogenic in mice and induced the production of anti-peptide antibodies, but with the exception of VP6-peptide they were not able to induce anti-rotavirus antibodies as measured by ELISA. Western blot analysis indicated that antibodies against each peptide were able to react with the respective authentic viral proteins of various rotavirus serotypes. To determine if a peptide-primed animal would respond to native viral proteins, animals were subsequently injected with purified BRV. A rapid and high anti-rotavirus antibody titre, in addition to a rise in anti-peptide antibody titre, was observed in peptide-primed mice. Furthermore, the sera obtained from these mice neutralized the virus under in vitro conditions. The significance of these results in relation to a potential rotavirus synthetic peptide-based vaccine is discussed.

Studies on the rate of selective uptake of amino acids by Trichinella larvae in vivo.

Groups of C57BL/6J mice, orally infected with 300 larvae each of Trichinella spiralis or T. pseudospiralis were injected with [3H]-alanine, tyrosine, tryptophan or glycine. The incorporation of isotope labelled amino acids into larval proteins was measured at 2, 6, and 12 months post-infection. It was shown that there is a significant increase in the in vivo uptake of isotope labelled amino acids with time by the larvae of T. spiralis and T. pseudospiralis. The level of uptake was highest for tyrosine followed by tryptophan, alanine and then glycine, for both species. The in vivo uptake of amino acids by T. pseudospiralis larvae was always higher than T. spiralis or the host at 6 and 12 months post-infection. At 2 months post-infection, T. spiralis uptake of these amino acids was higher, except for tyrosine. This may be related to the special needs of these larvae during the process of encystation. The higher metabolic requirements of T. pseudospiralis may be related to the higher energy needs of these non encapsulated, highly motile and mobile muscle larvae.

Contractile characteristics of the flexor muscle of mice infected with Trichinella spiralis, T. nativa or T. pseudospiralis.

A comparative analysis of skeletal muscle isometric contractile characteristics was performed in vivo on the flexor muscle of mice infected 6 mo earlier with 400 larvae of Trichinella pseudospiralis, Trichinella spiralis, or Trichinella nativa. The control group consisted of age- and sex-matched uninfected mice. The mice were injected with 0.1 ml of 50% urethane in saline, and the skin of the left hind limb was cut open longitudinally. The exposed flexor muscle was freed from the adjacent tissue and left attached freely to the knee joint while the tendon was hooked to a transducer. The signals were amplified with an amplifier connected to a chart recorder. The sciatic nerve was exposed and attached to an electrode. Impulses were generated and muscle contraction recorded. The exposed muscle and nerve were bathed in normal Krebs solution at all times and the animals were kept alive during the experiment. The normal muscle twitch tension of uninfected mice reached an average of 2.26 +/- 0.24 (SD) g. Tetany was achieved at 15 Hz. Low-Ca2+ Krebs depressed the twitch tension to 2.0 +/- 0.08 g while tetany remained at 15 Hz. Muscle twitch tension in mice infected with T. pseudospiralis reached 2.47 +/- 0.17 g and tetany at 15 Hz. Low Ca2+ depressed twitch tension to 1.14 +/- 0.12 g. Tetany was achieved at 20 Hz. In contrast, the muscle twitch of mice infected with T. nativa was significantly reduced to 1.4 +/- 0.09 g and tetany at 15 Hz. Low Ca2+ depressed twitch tension to 0.9 +/- 0.16 g and tetany at 15 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)