Salt bridge impact on global rigidity and thermostability in thermophilic citrate synthase.

It has been suggested that structural rigidity is connected to thermostability, e.g. in enzymes from thermophilic microorganisms. We examine the importance of correctly handling salt bridges, and interactions which we term 'strong polars', when constructing the constraint network for global rigidity analysis in these systems. Through a comparison of rigidity in citrate synthases, we clarify the relationship between rigidity and thermostability. In particular, with our corrected handling of strong polar interactions, the difference in rigidity between mesophilic and thermophilic structures is detected more clearly than in previous studies. The increase in rigidity did not detract from the functional flexibility of the active site in all systems once their respective temperature range had been reached. We then examine the distribution of salt bridges in thermophiles that were previously unaccounted for in flexibility studies. We show that in hyperthermophiles these have stabilising roles in the active site; occuring in close proximity to key residues involved in catalysis and binding of the protein.

An assessment of the coverage of a district-wide diabetic retinopathy screening service.

AIMS: To assess the coverage of the diabetes retinopathy screening service (DRSS) in North Staffordshire, to identify patient characteristies associated with non-attendance and to assess the proportion of patients with diabetic retinopathy who achieved glycaemic and blood pressure (BP) control targets. METHODS: Data for all patients who underwent annual retinal screening between 1 May 2000 and 30 April 2001 were obtained from the North Staffordshire Diabetes Register. Age, gender, ethnicity, socio-economic status, type and duration of diabetes were compared between patients who underwent eye screening and those who did not. Frequencies of patients who achieved glycaemic and BP targets in these groups of patients were compared to the remaining patients. RESULTS: 5646 of the 11682 (48%) patients on the diabetes register underwent retinal screening during the year. Patients with Type 2 diabetes, older patients, patients belonging to ethnic minorities and those wholly managed in primary care were less likely to attend for eye screening (P < 0.05 for all groups) with ethnic minority or primary care management demonstrating independent influence (P < 0.001). The percentage of patients with retinopathy achieving HbA1c and systolic BP targets was significantly lower than in their unaffected counterparts (chi2 = 63, P < 0.001 and chi2 = 71, P < 0.001 respectively). CONCLUSIONS: The efficacy of the DRSS in North Staffordshire is low and might be improved by targeting specific patient groups. Glycaemic control and systolic BP control needs to be improved in patients with diabetic retinopathy.

An audit of 500 subcutaneous glucagon stimulation tests to assess growth hormone and ACTH secretion in patients with hypothalamic-pituitary disease.

OBJECTIVE: The insulin tolerance test (ITT) is usually regarded as the 'gold standard' for the assessment of the hypothalamic-pituitary axis (growth hormone (GH) and ACTH) but must be used with caution and is contra-indicated in certain groups of patients. The glucagon stimulation test (GST) has previously been shown to be a good alternative when the ITT is contra-indicated and like the ITT stimulates both GH and ACTH secretion. There is however limited data on the use of the GST in patients with hypothalamic-pituitary disease. DESIGN AND PATIENTS: An audit of 500 GST was performed in 374 patients with hypothalamic-pituitary disease. Glucagon was administered via the subcutaneous route and bloods were taken at times 0 90 120 150 180 210 and 240 minutes. RESULTS: In the vast majority peak GH (84.4%) and cortisol (93%) responses occurred between 120 and 180 minutes Little information was obtained from the 240 minute sample. The medical supervision required was minimal and the side-effects encountered during this test were mild; 20% of the tests were associated with nausea occasionally with vomiting sweating or headaches. Four patients fainted but recovered quickly. CONCLUSIONS: This large audit has shown that the glucose stimulation test is well tolerated and can easily be performed in an out-patient setting with minimal medical supervision. The 240 minute sample added little additional information and could be omitted.

Comparable vasorelaxant effects of 17alpha- and 17beta-oestradiol on rat mesenteric resistance arteries: an action independent of the oestrogen receptor.

17beta-Oestradiol (17betaE(2)) has vasorelaxant properties that may contribute to its beneficial cardiovascular effects. The mechanism of vasorelaxation remains controversial, but does not appear to involve interaction of 17betaE(2) with its nuclear receptor. The present study examined the effects on resistance arteries of 17betaE(2) and its isomer, 17alpha-oestradiol (17alphaE(2)), which does not bind to the classical oestrogen receptor. In arteries precontracted with either noradrenaline or KCl, 17betaE(2) and 17alphaE(2) caused comparable relaxation in a concentration-dependent manner over the concentration range 0.1-10 micromol/l, with no significant difference in the maximal effect obtained. Pre-incubation of the arteries with 17betaE(2) or 17alphaE(2) for 15 min reduced the magnitude and duration of the force generated with both noradrenaline and KCl to a comparable degree. Vasorelaxation induced by either 17betaE(2) or 17alphaE(2) was not blocked by an inhibitor of NO synthase or by protein synthesis inhibitors, indicating that vasodilatation is not dependent upon either NO generation or protein synthesis. In the absence of extracellular calcium, both oestradiols still relaxed arteries precontracted with NA, suggesting that they inhibit intracellular calcium release. Both 17betaE(2) and 17alphaE(2) therefore have important and comparable vasorelaxant properties that do not require interaction with the nuclear oestrogen receptor. Direct interactions with the cell membrane or with ion-channel proteins may be responsible.

The thiazolidinedione rosiglitazone (BRL-49653) lowers blood pressure and protects against impairment of endothelial function in Zucker fatty rats.

Human obesity is associated with insulin resistance, hyperinsulinemia, and a predisposition to hypertension and vascular disease, the origin of which may lie in impairment of endothelial function. We tested the effects of the thiazolidinedione rosiglitazone on blood pressure and endothelial function in insulin-resistant fatty Zucker rats, which display hypertension and abnormal endothelial cell function. We studied fatty Zucker rats given rosiglitazone maleate (50 micromol/kg diet; n = 8) for 9-12 weeks (treated fatty), untreated fatty rats (n = 8), and lean rats (n = 8) given diet alone. At the end of the study, systolic blood pressure was significantly higher in untreated fatty (147 +/- 5 mmHg) than in lean rats (125 +/- 2 mmHg; P < 0.05), but rosiglitazone treatment prevented the development of hypertension in fatty rats (123 +/- 1 mmHg). Fasting hyperinsulinemia in untreated fatty rats (28.7 +/- 6.0 ng/ml) was significantly lowered by rosiglitazone (7.0 +/- 1.4 ng/ml; P < 0.05 vs. untreated fatty), but remained significantly higher than the levels seen in lean rats (1.5 +/- 0.4 ng/ml; P < 0.01). Mesenteric arteries were studied in a myograph. Maximal acetylcholine chloride (1.1 micromol/l)-induced relaxation of norepinephrine hydrochloride (NE)-induced constriction was impaired in untreated fatty (62.4 +/- 3.4%) vs. lean (74.3 +/- 3.5%; P = 0.01) rats; this defect was partially prevented by rosiglitazone (66.5 +/- 3.0%; P = 0.01 vs. untreated fatty). Insulin (50 mU/l) significantly attenuated the contractile response to NE in lean rats (14.7 +/- 3.3%; P = 0.02); this vasodilator effect of insulin was absent in untreated fatty rats at concentrations of 50-5,000 mU/l, but was partially restored by rosiglitazone (9.7 +/- 2.5% attenuation; P = 0.02 vs. no insulin). Thus, rosiglitazone prevents the development of hypertension and partially protects against impaired endothelial function associated with insulin resistance. These latter effects may contribute to the drug's antihypertensive properties.

Differential vasoactive effects of the insulin sensitizers rosiglitazone (BRL 49653) and troglitazone on human small arteries in vitro.

BRL 49653 (rosiglitazone) and troglitazone are thiazolidinedione insulin-sensitizing agents, which are undergoing clinical evaluation as treatments for NIDDM. Potential side effects of thiazolidinediones include edema and hemodilution. Although the underlying mechanisms are presently unclear, animal and human studies have demonstrated a vasodilator action of troglitazone, which could in theory cause fluid retention. This in vitro study compared the direct vasodilator effects of troglitazone and BRL 49653 in small arteries (n = 44) from human subcutaneous fat. In arterial rings with a functioning endothelium and preconstricted with norepinephrine (NE; 6 micromol/l), troglitazone (n = 22 vessels), but not BRL 49653 (1-100 micromol/l), caused a concentration-related relaxation (69.4 +/- 5.2% at 100 micromol/l; P < 0.01). In the presence of indomethacin (IM; 10 micromol/l; n = 12), this vasorelaxant effect of troglitazone was abolished (P < 0.01 vs. troglitazone alone) and replaced by enhanced vasoconstriction (58.5 +/- 39.5% over the NE baseline) similar in magnitude to that produced by troglitazone vehicle (ethanol) alone (n = 16; NS vs. ethanol vehicle). By contrast, BRL 49653 (100 micromol/l; n = 22) and an equivalent volume of ethanol alone (n = 12) caused similar degrees of vasoconstriction (18.7 +/- 14.6 and 22.5 +/- 8.0%, respectively; NS). In the presence of IM (10 micromol/l; n = 10), the vasoconstrictor effect of BRL 49653 was enhanced (41.5 +/- 14.4%), although not significantly (NS vs. BRL 49653 alone or ethanol alone). Additional studies in Wistar rat arteries showed a similar vasodilator effect of troglitazone that was not inhibited by L-NAME (100 micromol/l). The alpha-tocopherol moiety alone had no vasorelaxant effect at concentrations up to 300 micromol/l. Thus, in human arterial resistance vessels in vitro, BRL 49653 does not possess the direct, IM-sensitive vasorelaxant action of troglitazone. This vasodilation could, in theory, permit transmission of systemic pressure to the capillary bed.

Impaired insulin-induced attenuation of noradrenaline-mediated vasoconstriction in insulin-resistant obese Zucker rats.

1. Insulin resistance is associated with hypertension but the underlying mechanism is unclear. We tested the hypothesis that insulin-induced vasodilatation is impaired in insulin-resistant obese Zucker rats. We studied mesenteric artery (approximately 220 microns diameter) function before the development of hypertension in 3-month old obese Zucker rats and age-matched lean rats. 2. In vessels from lean rats, insulin at concentrations of 50, 500 and 5000 m-units/l attenuated the constriction in response to noradrenaline (50 m-units/l: 8 +/- 3%, P < 0.05; 500 m-units/l: 13 +/- 3%, P < 0.02; 5000 m-units/l: 13 +/- 2%, P < 0.02). 3. Vessels from obese rats failed to show any such response to insulin (2 +/- 6% increase in maximal tension with 5000 m-units/l; not significant), both in the presence and absence of L-arginine (3 mmol/l). 4. Vessels from obese rats showed slight but significant impairment in the vasodilator response to acetylcholine (5 x 10(-8)-10(-4) mol/l) (obese: 64.1 +/- 3.7% relaxation; lean: 77.3 +/- 3.7% relaxation; P < 0.05); however, relaxation in response to A23187 was not significantly different between the phenotypes (obese: 81.3 +/- 10.6% relaxation; lean: 79.1 +/- 9.7% relaxation; not significant). 5. Systolic blood pressure was not significantly different in lean (126 +/- 8 mmHg) and obese (127 +/- 7 mmHg) rats at the time of study (not significant). 6. We conclude that insulin-induced attenuation of noradrenaline-mediated vasoconstriction is impaired in the obese Zucker rat and that this defect precedes and therefore could contribute to the development of hypertension in this insulin-resistant model. The defect in insulin action could reside in the endothelial generation of nitric oxide, as endothelial function is also abnormal.

Diagnostic difficulties in periodic Cushing's syndrome.

A 22-year-old black woman presented with symptoms suggestive of Cushing's syndrome three years after chemotherapy for a presumed teratoma with cervical lymphadenopathy. Initially, the absence of clinical signs and the demonstration of two normal 24 h urinary free cortisols appeared to exclude the diagnosis, but an ectopic adrenocorticotropin-producing thymic carcinoid was subsequently removed surgically. Cushing's syndrome due to ectopic adrenocorticotropin production can be difficult to diagnose, particularly if there is periodic hormonogenesis.

Insulin-induced attenuation of noradrenaline-mediated vasoconstriction in resistance arteries from Wistar rats is nitric oxide dependent.

1. We studied the mechanism of insulin-mediated attenuation of noradrenaline-induced vasoconstriction in mesenteric resistance arteries (approximately 210 microns diameter) from 10-week-old male Wistar rats (n = 10; weight 321 +/- 11 g). Exposure to physiological concentrations of insulin (50 m-units/l) significantly blunted the contractile response to noradrenaline over the concentration range 3 x 10(-6) to 3 x 10(-5) mol/l (16 vessels; 13.1 +/- 4.3% reduction in maximum tension at 3 x 10(-5) mol/l noradrenaline; P < 0.01 versus no insulin). 2. This effect of insulin was prevented by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (10(-4) mol/l; 16 vessels; 3.3 +/- 9.1% reduction in maximum tension; P = 0.8 versus no insulin). There was no evidence of fatigue in four noradrenaline dose-response curves for 16 control vessels in the absence of insulin and NG-nitro-L-arginine methyl ester (P = 0.8; first versus second dose-response curve). With L-arginine present in the incubation medium, insulin again attenuated the noradrenaline-induced vasoconstriction (10.7 +/- 3.2% reduction in tension; P = 0.02 versus L-arginine and no insulin; P = not significant versus insulin and no L-arginine). 3. Endothelium-dependent relaxation was initially confirmed in all vessels by demonstrating normal acetylcholine- (5.4 x 10(-7) to 1.1 x 10(-4) mol/l) induced vasodilatation in vessels preconstricted with noradrenaline (6 x 10(-6) mol/l) in the absence of NG-nitro-L-arginine methyl ester, L-arginine and insulin (P = not significant between the different groups of vessels). 4. We conclude that insulin attenuates noradrenaline-induced vasoconstriction in resistance arteries by stimulation of nitric oxide release. Abnormal insulin-stimulated nitric oxide release could be of relevance in the pathogenesis of hypertension and diabetic microvascular disease.

Hypogonadotrophic hypogonadism and primary amenorrhoea associated with increased melatonin secretion from a cystic pineal lesion.

A 17-year-old girl presented with primary amenorrhoea and failure to develop secondary sexual characteristics, although her height was above the 90th centile. Endocrine investigations revealed hypogonadotrophic hypogonadism (basal LH and FSH levels < 0.5 U/l; FSH rose to 2.0 U/l and LH to 1.0 U/l after GnRH). ACTH, GH, TSH and PRL secretion were normal. A magnetic resonance scan revealed no abnormality in the pituitary, pituitary stalk or hypothalamus but demonstrated a partly cystic enhancing lesion in the pineal region. Melatonin production (assessed as urinary 6-sulphatoxymelatonin: aMT6s) at baseline was markedly increased: 459-530 ng/kg/24 h compared with aged-matched controls: 136 +/- 69 (P = 0.01). However, melatonin production retained a largely normal rhythm with increased production during the night. Treatment with ethinyloestradiol 100 micrograms daily had no apparent effect on the production of melatonin. Treatment with atenolol, 100 mg daily at 1600 h, was associated with suppression of nocturnal melatonin secretion but a brisk rebound in the morning and a considerably delayed peak excretion time (10.2 h) compared with controls (3.9 h). It is likely the pineal lesion, which may be hyperplasia or possibly even tumour, was responsible for the increased melatonin secretion. These data support the hypothesis that melatonin may have a causal role in hypogonadotrophic hypogonadism.

Clomiphene-induced pituitary apoplexy in a patient with acromegaly.

We describe the case of an acromegalic woman with primary infertility who was treated with clomiphene, and subsequently developed pituitary apoplexy.

Vascular reactivity to noradrenaline and neuropeptide Y in the streptozotocin-induced diabetic rat.

The study aimed to assess vascular reactivity to noradrenaline with and without neuropeptide Y in diabetic rats, and to determine whether any abnormality could be attributed to insulin deficiency or to hyperglycaemia per se. The authors compared non-diabetic rats (n = 9) and rats with streptozotocin-induced diabetes that were either untreated (n = 10), or treated with insulin (n = 9) or food restriction (n = 8) to restore near-normoglycaemia. After 4 weeks of diabetes, contractile responses to noradrenaline (0.24-48 mumol L-1), without and with neuropeptide Y (0.1 mumol L-1), were assessed using an isometric myograph in two mesenteric arteries from each rat. Vessels from untreated diabetic rats were significantly more reactive to noradrenaline than the control vessels when tested without (P < 0.0001) but not with (P = NS) neuropeptide Y. Diabetic rats rendered nearly normoglycaemic through food restriction showed dose-response curves that were very similar to the untreated diabetic group (P = NS). By contrast, insulin-treated diabetic vessels showed reduced sensitivity to noradrenaline, with and without neuropeptide Y, compared with both the diet-restricted and untreated vessels (both P < 0.0001). The authors conclude that vascular sensitivity to noradrenaline, without or with neuropeptide Y, is reduced over a wide dose range in vessels taken from rats treated in vivo with insulin; furthermore, vessels taken from diabetic rats not treated with insulin (hypoinsulinaemic) tended to be more reactive than either control vessels or those taken from the insulin-treated rats. The latter group of rats were probably hyperinsulinaemic for much of the time; the results may therefore support the hypothesis that insulin acts as a vasodilator.

Design of a normal incidence multilayer imaging x-ray microscope.

Normal incidence multilayer Cassegrain x-ray telescopes were flown on the Stanford/MSFC Rocket X-Ray Spectroheliograph. These instruments produced high spatial resolution images of the Sun and conclusively demonstrated that doubly reflecting multilayer x-ray optical systems are feasible. The images indicated that aplanatic imaging soft x-ray /EUV microscopes should be achievable using multilayer optics technology. We have designed a doubly reflecting normal incidence multilayer imaging x-ray microscope based on the Schwarzschild configuration. The Schwarzschild microscope utilizes two spherical mirrors with concentric radii of curvature which are chosen such that the third-order spherical aberration and coma are minimized. We discuss the design of the microscope and the results of the optical system ray trace analysis which indicates that diffraction-limited performance with 600 Å spatial resolution should be obtainable over a 1 mm field of view at a wavelength of 100 Å. Fabrication of several imaging soft x-ray microscopes based upon these designs, for use in conjunction with x-ray telescopes and laser fusion research, is now in progress. High resolution aplanatic imaging x-ray microscopes using normal incidence multilayer x-ray mirrors should have many important applications in advanced x-ray astronomical instrumentation, x-ray lithography, biological, biomedical, metallurgical, and laser fusion research.

Soft X-ray Images of the Solar Corona with a Normal-Incidence Cassegrain Multilayer Telescope.

High-resolution images of the sun in the soft x-ray to extreme ultraviolet(EUV) regime have been obtained with normal-incidence Cassegrain multilayer telescopes operated from a sounding rocket in space. The inherent energy-selective property of multilayer-coated optics allowed distinct groups of emission lines to be isolated in the solar corona and the transition region. The Cassegrain telescopes provided images in bands centered at 173 and 256 angstroms. The bandpass centered at 173 angstroms is dominated by emission from the ions Fe IX Fe X. This emission is from coronal plasma in the temperature range 0.8 x 10(6) to 1.4 x 10(6)K. The images have angular resolution of about 1.0 to 1.5 arc seconds, and show no degradation because of x-ray scattering. Many features of coronal structure, including magnetically confined loops of hot plasma, coronal plumes, polar coronal holes, faint structures on the size scale of supergranulation and smaller, and features due to overlying cool prominences are visible in the images. The density structure of polar plumes, which are thought to contribute to the solar wind, has been derived from the observations out to 1.7 solar radii.

Complications of posterior sagittal anorectoplasty.

From 1982 to 1985, 23 patients underwent posterior sagittal anorectoplasty procedures: 12 as primary treatment for congenital anorectal malformations, 9 for treatment of fecal incontinence following a prior pull-through procedure, and 2 for treatment of fecal incontinence following trauma. Six patients (26%) developed seven complications specifically related to the procedure. One patient with a cloacal anomaly had partial dehiscence of the sacroperineal incision following total reconstruction. This resulted in retraction of vaginal and anal openings, which, however, have remained separate and patent. Two patients developed temporary femoral nerve palsies, unilateral in one patient lasting one week, and bilateral in one patient lasting four months. Four patients developed leaks from the suture line of the tailored ectatic rectum, which was pulled through to the perineum. In one male patient, the suture line was placed anteriorly, resulting in a rectourethral fistula, which required a repeat posterior sagittal dissection. One male, who had a redo procedure, developed a posterior diverticulum comparable to a large anal crypt. This was repaired prior to closure of the colostomy. One seven-year-old girl developed multiple rectocutaneous fistulae, which closed with conservative management in five months. One male infant developed a single supralevator rectocutaneous fistula, which closed after rediversion of feces with a colostomy and has remained so after colostomy closure. The majority of the complications encountered were probably preventable if careful attention to certain details of technique had been observed: careful padding of the groin areas when patients are prone, especially in older patients.(ABSTRACT TRUNCATED AT 250 WORDS)

A long-term follow-up on a duplication of the entire small intestine treated by gastroduplication.

Duplication of the entire small intestine resulting in gastrointestinal hemorrhage was managed by excision of the ulcerogenic lesion, ileo-ileostomy, and diversion of the gastric-lined intestinal duplication into the stomach. A 25 yr followup study reveals no evidence of recurrent bleeding or malabsorption secondary to bind-loop syndrome. This operative procedure is worthy of consideration in patients with this congenital anomaly.

Use of fresh amnion as a burn dressing.

Thirty-seven children with second and third degree burns dressed with amnion were compared to seventy-three children treated with Furacin (Eaton Labs, Norwich, N.Y.) dressing. Amnion was found to be as easy to use as Furacin. Fewer split thickness skin grafts were needed in amnion treated children and these patients required fewer days of hospitalization. Bacterial culture data suggests that amnion is as good as and possibly superior to nitrofurazone in decreasing the number of organisms on the burn wound. No adverse reactions to amnion were noted. The use of amnion is supported by this preliminary study and is deserving of further investigation and clinical use.