Induction of glutathione S-transferase genes of Nicotiana benthamiana following infection by Colletotrichum destructivum and C. orbiculare and involvement of one in resistance.

Four glutathione S-transferase (GST) genes, NbGSTU1, NbGSTU2, NbGSTU3, and NbGSTF1, were amplified from cDNA of Nicotiana benthamiana leaves infected with Colletotrichum destructivum using primers based on conserved regions of N. tabacum GST sequences. Expression of NbGSTU1 and NbGSTU3 increased progressively during infection by either C. destructivum or Colletotrichum orbiculare, except for a slight decrease by NbGSTU1 late in the infection, whereas NbGSTU2 and NbGSTF1 expression remained relatively constant. Each of the four genes was cloned into a PVX vector for virus-induced gene silencing, and reduced expression of the four genes was detected by RT-PCR. A statistically significant increase in susceptibility of N. benthamiana to infection following gene silencing was found only for NbGSTU1-silenced plants, which had 130% more lesions and 67% more colonization by C. orbiculare compared with control plants. These results demonstrate that the different GST genes respond in different ways to fungal infection, and at least one plant GST gene has an important role in disease development.

A novel testosterone gel formulation normalizes androgen levels in hypogonadal men, with improvements in body composition and sexual function.

OBJECTIVE: To compare the safety and efficacy of two doses of a new testosterone gel formulation (Testim Auxilium Pharmaceuticals, Inc., Norristown, PA, USA) to a permeation-enhanced testosterone patch (Andropatch), GlaxoSmithKline, UK) for treating men with confirmed low serum testosterone levels, and associated signs and symptoms of hypogonadism. PATIENTS AND METHODS: In all, 208 men were randomized and treated at 29 centres in Denmark, Germany, Netherlands, Sweden and the UK. The men were treated for 90 days, and the pharmacokinetics and treatment effectiveness of Testim at two doses (50 and 100 mg/day, delivering a daily dose of 5 and 10 mg testosterone, respectively) and Andropatch (2 x 2.5 mg patches, each delivering 2.5 mg testosterone and containing 12.2 mg of testosterone) were compared. Pharmacokinetic profiles were obtained, body composition measured, and mood and sexual function data recorded. RESULTS: Testim produced dose-dependent improvements in all pharmacokinetic variables compared with Andropatch. The mean increases from baseline to 90 days in testosterone were 12.41, 6.54 and 3.82 nmol/L for Testim 100 and 50 mg/day and the Andropatch, respectively. Both doses of Testim significantly improved positive and negative mood over baseline; Andropatch did not. All three treatments increased lean body mass, and the higher dose of Testim produced a significant decrease in percentage body fat. At all sample times both doses of Testim significantly improved sexual performance, sexual motivation, sexual desire and spontaneous erections. Andropatch provided insignificant improvements from baseline at all sample times for sexual desire, an inconsistent improvement in sexual motivation, but no effect on spontaneous erections. These results are similar to those previously reported for testosterone replacement therapy in hypogonadal men, suggesting that normalization of serum testosterone restores sexual function. However, the present data suggest that higher serum testosterone levels may further improve sexual function. Gel treatment was well tolerated, while patch treatment produced higher rates of application-site reactions and study discontinuation. CONCLUSION: The favourable pharmacokinetic profile and treatment outcome, combined with the enhanced tolerability of Testim, suggest that this new gel formulation is a safe and effective treatment in men with low serum testosterone levels and associated signs and symptoms of hypogonadism.

Susceptibility of low- and high-density lipoproteins from diabetic subjects to in vitro oxidative modification.

AIMS: To investigate the hypothesis that lipid peroxidation of both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) is important in the development of atherosclerosis. METHODS: We have investigated whether LDL and HDL from patients with Type 1 diabetes mellitus (DM, n = 16) and Type 2 DM (n = 15) is more susceptible to Cu2+ -induced lipid peroxidation than LDL and HDL from a similar number of nondiabetic controls matched for age, gender and serum cholesterol. RESULTS: The vitamin E content of LDL and HDL from both groups of diabetic patients was not significantly different from controls. The LDL from Type 2 diabetic patients and HDL from both diabetic groups were significantly richer in triglyceride than controls. Phospholipid was decreased in LDL from Type 2 diabetic patients and protein was decreased in HDL in Type 1 DM, but otherwise the composition of LDL and HDL in diabetic subjects was similar to controls. No significant differences were observed in the generation of conjugated dienes or lipid peroxides in either LDL or HDL when the two groups were compared with each other or with their respective controls. CONCLUSIONS: Increased lipid peroxidation occurring in vivo in diabetes is unlikely to be the result of increased susceptibility of lipoproteins to lipid peroxidation, but rather to increased generation of free radicals, to oxidation of lipids other than those present in serum lipoproteins or to decreases in antioxidant systems other than the fat-soluble antioxidants present in lipoproteins.

Lack of association of angiotensin II type 1 receptor gene polymorphism with diabetic nephropathy in insulin-dependent diabetes mellitus.

Several observations suggest that inherited factors are influential in the development of nephropathy in patients with insulin-dependent diabetes mellitus (IDDM). Genetic components of the renin angiotensin system are possible candidate genes. The aim of this study was to determine the role of the hypertension associated angiotensin II type 1 receptor (AT1R) gene A1166C polymorphism in susceptibility to nephropathy in IDDM. We examined 264 Caucasoid patients with IDDM and overt nephropathy (as defined by persistent proteinuria in the absence of other causes, hypertension and retinopathy), 136 IDDM patients with long duration of diabetes and no nephropathy (LDNN group), 200 recently diagnosed IDDM patients (Sporadic Diabetic group), and 212 non-diabetic subjects. The AT1R gene polymorphism was assessed using the polymerase chain reaction and restriction isotyping. Genotype frequencies did not differ significantly between the sporadic diabetic group and the nephropathy group (p = 0.245), nor between the long duration non-nephropathy group and the nephropathy group (p = 0.250). Allele frequencies were not significantly different between the three groups (p = 0.753). We conclude that there is no significant association between the hypertension associated AT1R gene polymorphism and diabetic nephropathy in patients with IDDM in the UK.

Examination of two genetic polymorphisms within the renin-angiotensin system: no evidence for an association with nephropathy in IDDM.

Premature cardiovascular disease is common in insulin-dependent diabetic (IDDM) patients who develop diabetic nephropathy. Genetic polymorphism within the renin-angiotensin system has been implicated in the aetiology of a number of cardiovascular disorders; these loci are therefore candidate genes for susceptibility to diabetic renal disease. We have examined the angiotensin converting enzyme insertion/deletion polymorphism and angiotensinogen methionine 235 threonine polymorphism in a large cohort of Caucasian patients with IDDM and diabetic nephropathy. Patients were classified as having nephropathy by the presence of persistent dipstick positive proteinuria (in the absence of other causes), retinopathy and hypertension (n = 242). Three groups were examined for comparison: ethnically matched non-diabetic subjects (n = 187); a geographically defined cohort of newly diagnosed diabetic patients (n = 341); and IDDM patients with long duration of disease (> 15 years) and no evidence of overt nephropathy (n = 166). No significant difference was seen in distribution of angiotensin converting enzyme or angiotensinogen genotypes between IDDM patients with nephropathy and recently diagnosed diabetic subjects (p = 0.282 and 0.584, respectively), nor the long-duration non-nephropathy diabetic subjects (p = 0.701 and 0.190, respectively). We conclude that these genetic loci are unlikely to influence susceptibility to diabetic nephropathy in IDDM in the United Kingdom.

Treatment of dyslipoproteinaemia in diabetes mellitus.

Coronary heart disease (CHD) is the most common cause of premature death in diabetes. Hypercholesterolaemia occurs in diabetes with about the same frequency as in the general population, but it confers a greater risk of CHD in diabetes. Hypertriglyceridaemia and low serum high density lipoprotein (HDL) cholesterol levels are more common in diabetes, particularly non-insulin-dependent diabetes. Nephropathy increases the severity of dyslipoproteinaemia. There remains a reluctance to apply the results of cholesterol-lowering trials to diabetes. No trial has been specifically in diabetes, but this should not constrain the treatment of diabetic patients at clearly high CHD risk. It is suggested that fasting lipids should be measured in all diabetic patients aged less than 70 years with established CHD or whose non-fasting cholesterol is >6.00 mmol I(-1) or triglycerides >3.00 mmol I(-1). For those with raised lipids glycaemic control should be improved, if possible, and dietary therapy aimed at a decrease in fat intake, particularly saturated fat, and weight reduction in the obese. Lipid-lowering drugs are required in patients with CHD and serum cholesterol >5.5 mmol I(-1) with the aim of decreasing non-HDL to <4.00 mmol I(-1). In patients without CHD lipid-lowering drugs should be considered when serum cholesterol exceeds 6.5 mmol I(-1) and the risk of CHD is greater than 20 % over the next 10 years. There is no evidence that pursuing this policy beyond the age of 70 years is beneficial. Diabetic women with dyslipoproteinaemia should, however, be treated in the same way as men. The knowledge that hypertriglyceridaemia and low serum HDL cholesterol are present helps in the assessment of CHD risk and the choice of medication to decrease non-HDL cholesterol, but there is no evidence that their treatment in the absence of raised cholesterol is of benefit.

Cholesterol rich apo B containing lipoproteins and smoking are independently associated with macrovascular disease in normotensive NIDDM patients.

A cross-sectional study of macrovascular disease (MVD) and associated metabolic and other risk factors was conducted in 87 normotensive NIDDM patients. MVD was assessed by Rose questionnaire, 12 lead resting ECG, duplex scanning of carotid and peripheral vessels, and ankle:brachial systolic blood pressure ratio. Fasting serum total cholesterol, total triglycerides, LDL cholesterol, HDL cholesterol, apolipoproteins AI and B, lipoprotein (a), HbA1, plasma glucose, insulin, and C-peptide responses to a carbohydrate rich meal, body mass index (BMI), waist-hip ratio, urinary albumin excretion rate, blood pressure, smoking and family history were assessed as possible 'risk factors'. Apolipoprotein:lipid ratios were calculated to estimate lipoprotein composition. Thirty-six patients had demonstrable MVD. The presence of MVD was associated with higher total triglycerides (p < 0.05), BMI (p < 0.05), systolic blood pressure (p < 0.01), a lower apo B:non HDL cholesterol ratio (p < 0.001), and smoking (p < 0.005) but no other measures. Multiple regression analysis revealed smoking and a low apo B:non HDL cholesterol to be independently associated with MVD. The low apo B:non HDL cholesterol suggests a high cholesterol content of apo B containing lipoproteins. This lipoprotein abnormality is not a feature of NIDDM, but when present in these patients may be particularly atherogenic.

Epstein-Barr virus-associated haemophagocytic syndrome in a patient with juvenile chronic arthritis.

We describe a case of reactive haemophagocytic syndrome (RHS) in a patient with previous polyarticular juvenile chronic arthritis (JCA). This is the first reported association of these conditions and may be indicative of defective immunological responses to viral infections in this form of JCA.

The pharmacokinetics of five pre-mixed combinations of 'short-' and 'intermediate-acting' (NPH) insulins in healthy subjects following subcutaneous administration.

The pharmacokinetics of five pre-mixed insulin preparations in the ratio of soluble to NPH insulin of 10:90, 20:80, 30:70, 40:60 and 50:50, were examined in a two part study in fasting healthy subjects. Each received by bolus subcutaneous injection into the anterior abdominal wall, on separate occasions one to two weeks apart, 20U of each of three pre-mixed insulin preparations in random order. In Part 1, nine subjects received Penmix 10:90, Penmix 20:80 and Penmix 30:70 and were observed over a period of 24 hours. In Part 2, eight subjects received Penmix 30:70, Penmix 40:60 and Penmix 50:50 and were observed over an 8 hr post-injection period. Three subjects were common to both parts of the study. Plasma glucose, C-peptide and insulin levels were measured frequently throughout both study periods. Increasing soluble insulin content in the pre-mixtures was reflected in increasing peak plasma insulin concentrations and a greater hypoglycaemic response. There were highly significant differences between the five premixtures and preparations in the 0-4 and 0-8 hours area under the curve (AUC) values for plasma glucose, C-peptide and immunoreactive insulin concentration (p < 0.01). Whereas a gradual difference between the premixtures was seen no two adjacent ones were significantly different, however an overall highly significant difference between the five preparations tested was observed.

The effect of acipimox in patients with type 2 diabetes and persistent hyperlipidaemia.

A placebo-controlled, double-blind study was performed to assess the effect of 12 weeks treatment with acipimox (250 mg three times per day) on lipoproteins and glycaemic control in patients with Type 2 diabetes. All patients studied had persistent hyperlipidaemia despite acceptable glycaemic control on treatment with diet alone or diet and oral hypoglycaemic agents, achieving glycosylated haemoglobin (HbA1) of less than 10.5% but with fasting total triglycerides greater than 2.5 mmol l-1 or total cholesterol greater than 6.5 mmol l-1. Forty-eight patients were randomized to treatment, 21 to acipimox and 27 to placebo; 43 completed the trial. All patients had been diabetic for at least 1 year. Total cholesterol fell by 6% and total triglycerides by 19% following 12 weeks of acipimox, compared to rises in the placebo group of 1% and 16%, respectively (p less than 0.05). There were no significant differences between acipimox and placebo in the change in low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, apolipoproteins AI, AII, or B, or in glycaemic control during the treatment period. Acipimox is effective in reducing fasting total cholesterol and total triglycerides in patients with Type 2 diabetes with acceptable blood glucose control but persistent hyperlipidaemia. Acipimox does not adversely affect glucose tolerance.

Renal hemodynamics in newly presenting non-insulin dependent diabetes mellitus.

Little information is available regarding renal hemodynamics in non-insulin dependent diabetics (NIDDMs), despite their numerical significance to renal support programs. Therefore, simultaneous determination of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) (expressed to 1.73 m2 surface area) was performed in 110 newly presenting normotensive NIDDMs [mean (SD) age: 52.5 (10.1) years] and compared with 32 normal subjects of similar age range [age: 52.2 (11.3) years]. Mean (SD) GFR, ERPF and filtration fraction (FF) for the NIDDMs were: 117 (22) ml/min (range: 74 to 178), 534 (123) ml/min and 22.4 (3.0)%, and for the normals: 95 (12) ml/min (range: 75 to 119), 472 (70) ml/min and 20.2 (2.2%), respectively. As a group, NIDDMs demonstrated significantly greater GFR (P less than 0.001), ERPF (P less than 0.005) and FF (P less than 0.001). GFR values above 140 ml/min were observed in 16% of the NIDDMs, while 45% had GFRs in excess of mean +/- 2 SD of the normals. NIDDMs demonstrated a positive relationship for GFR with ERPF (P less than 0.001) and an inverse association for both GFR and ERPF with age (P less than 0.001). Multivariable regression analysis revealed ERPF as the strongest explanatory variable for GFR in the NIDDMs (P less than 0.001), followed by age (P less than 0.01). Significant contributions to the regression analysis were not observed for body mass index, systolic or diastolic blood pressures, glycosylated hemoglobin nor fasting levels of plasma glucose, cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides. Urinary albumin excretion rates were not correlated with GFR, ERPF or FF. Microalbuminuria was detected in 7% of the NIDDMs in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperinsulinaemia and microvascular angina ("syndrome X")

Glucose and insulin responses to a glucose load in 11 patients with angina attributed to microvascular coronary dysfunction were compared with those in 11 healthy subjects matched for age, sex, and body mass. Stimulated hyperinsulinaemia was demonstrated in the microvascular angina group. The findings suggest a role for increased concentrations of insulin in coronary microvascular dysfunction.

Apolipoprotein and lipid ratios in treated non-insulin dependent diabetics.

Fasting total cholesterol (TC), triglycerides (TG), HDL cholesterol (HDL C), apolipoprotein A1 (apo A1) and apolipoprotein B (apo B) were measured in 35 non-insulin dependent diabetic patients treated by diet with or without sulphonylureas and 35 control subjects matched for age, sex, and body mass index. Ratios of apolipoprotein and lipid were calculated. The diabetics were well controlled with a mean (+/- SD) glycosylated haemoglobin (HbA1) of 8.5 +/- 1.3% (normal range less than 8%). Compared to non-diabetic control subjects apo A1: HDL C, apo B: TC, and apo B: calculated LDL C were significantly higher in the NIDDM patients, (112.9 +/- 26.3 vs 83.0 +/- 28.7, p less than 0.001, 15.89 +/- 1.68 vs 14.22 +/- 3.48, p less than 0.01, and 24.32 +/- 3.19 vs 22.33 +/- 5.49, p less than 0.05 respectively). These findings reflect differences in cholesterol content in the absence of differences in apolipoprotein concentrations between the NIDDM and control groups. The cardiovascular risk ratio HDL C: non HDL C was significantly lower in the NIDDM patients (0.25 +/- 0.09 vs 0.31 +/- 0.15, p less than 0.01), but there was no difference in apo A1:apo B (1.42 +/- 0.42 vs 1.43 +/- 0.52, NS). Although apo A1: apo B correlated well with HDL C:non HDL C in both NIDDM and controls (r = 0.88, 0.72, p less than 0.001 respectively) the slope of the relationships differed b = 4.01 NIDDM vs 2.50 controls (95% confidence intervals for difference is 0.22-2.78). Simple widely available methods can identify abnormalities of lipoprotein content in treated NIDDM patients. Both HDL and LDL contain less cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Model for simulating the effects of management practices on pesticide concentrations in groundwater.

This manuscript describes the RUSTIC model and presents the results of an example application. RUSTIC links three subordinate models in order to predict pesticide fate and transport through the crop root zone, unsaturated zone, and saturated zone to drinking water wells: PRZM, VADOFT, and SAFTMOD. PRZM is a one-dimensional finite-difference model which accounts for pesticide fate and transport in the crop root zone. This release of PRZM incorporates several features in addition to those simulated in the original PRZM code: specifically, soil temperature simulation, volatilization and vapor phase transport in soils, irrigation simulation and a method of characteristics (MOC) algorithm to eliminate numerical dispersion. PRZM is now capable of simulating fate and transport of the parent compound and up to two daughter species. VADOFT is a one-dimensional finite-element code which solves the Richard's equation for flows in the unsaturated zone. The user may make use of constitutive relationships between pressure, water content, and hydraulic conductivity to solve the flow equations. VADOFT may also simulate the fate and transport of two parent and two daughter products. SAFTMOD is a two-dimensional finite-element model which simulates saturated solute flow and transport in either an X-Y or X-Z configuration. The codes are linked together with the aid of a flexible execution supervisor which allows the user to build models which are tailored to site-specific situations. In order to perform exposure assessments, the code is equipped with a Monte Carlo pre- and post-processor. The application of the model is to a potato field on Long Island, New York. The results indicate that RUSTIC is capable of realistically matching data observed in downgradient monitoring wells following 3 years of aldicarb application.

Recombinant DNA derived monomeric insulin analogue: comparison with soluble human insulin in normal subjects.

OBJECTIVE: To compare the rate of absorption from subcutaneous tissue and the resulting hypoglycaemic effect of iodine-125 labelled soluble human insulin and a monomeric insulin analogue derived by recombinant DNA technology. DESIGN: Single blind randomised comparison of equimolar doses of 125I labelled soluble human insulin and insulin analogue. SETTING: Study in normal people at a diabetes research unit and a university department of medical physics. SUBJECTS: Seven healthy male volunteers aged 20-39 not receiving any other drugs. INTERVENTIONS: After an overnight fast and a basal period of one hour two doses (0.05 and 0.1 U/kg) of 125I labelled soluble human insulin and insulin analogue were injected subcutaneously into the anterior abdominal wall on four separate days. END POINT: To find a fast acting insulin for meal related requirements in insulin dependent diabetics. MEASUREMENTS and main results--Residual radioactivity at the injection site was measured continuously for the first two hours after injection of the 125I labelled preparations and thereafter for five minutes simultaneously with blood sampling. Frequent venous blood samples were obtained over six hours for determination of plasma immunoreactive insulin, insulin analogue, glucose, and glucagon values. Time to 50% of initial radioactivity at the injection site for the insulin analogue compared with soluble insulin was 61 v 135 minutes (p less than 0.05) with 0.05 U/kg and 67 v 145 minutes (p less than 0.001) with 0.1 U/kg. Concentrations in plasma increased faster after the insulin analogue compared with soluble insulin, resulting in higher plasma concentrations between 10 and 150 minutes (0.001 less than p less than 0.05) after 0.05 U/kg and between 40 and 360 minutes (0.001 less than p less than 0.05) after 0.1 U/kg. The hypoglycaemic response to insulin analogue was a plasma glucose nadir at 60 minutes with both doses compared with 90 and 120 minutes with soluble insulin at 0.5 and 0.1 U/kg respectively. The response of glucagon substantiated the earlier and more dramatic hypoglycaemic effect with the insulin analogue. CONCLUSIONS: The much faster absorption from subcutaneous tissue of the disubstituted monomeric insulin analogue compared with soluble insulin suggests that the analogue may be a potential candidate for rapid insulin delivery after subcutaneous bolus injection.

Another example of the rare Ay phenotype.

The red cells of a patient typed as group O, but her serum lacked anti-A. Upon further testing, it appeared that the ABO phenotype was Am. Saliva studies demonstrated a normal amount of H substance but reduced A substance. Family studies established the ABO genotype of the proposita as A1O. The results suggest that modification of the cellular and salivary expression of the A antigen in this patient was the result of a double dose of the modifier gene y, and that the ABO phenotype, while having characteristics similar to that of an Am, was actually Ay.