Characterization of wax as a potential diffraction intensity standard for macromolecular crystallography beamlines.

A number of commercially available waxes in the form of thin disc samples have been investigated as possible diffraction intensity standards for macromolecular crystallography synchrotron beamlines. Synchrotron X-ray powder diffraction measurements show that beeswax offers the best performance of these waxes owing to its polycrystallinity. Crystallographic lattice parameters and diffraction intensities were examined between 281 and 309 K, and show stable and predictable thermal behaviour. Using an X-ray beam of known incident flux at lambda = 1 A, the diffraction power of two strong Bragg reflections for beeswax were quantified as a function of sample thickness and normalized to 10(10) photons s(-1). To demonstrate its feasibility as a diffraction intensity standard, test measurements were then performed on a new third-generation macromolecular crystallography synchrotron beamline.

Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A2.

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 A, and diffracted to 1.75 A resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 A, and diffracted to 2.6 A resolution. The crotapotin crystal diffracted to 2.3 A resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

Metabolic effects of glucagon on plasma and urine zinc, copper, calcium and magnesium levels.

The present study determines the effect of glucagon on the behavior of zinc, copper, calcium and magnesium ions in human plasma and urine. Five normal adults were submitted to intravenous infusion of 2.0 mg glucagon over a period of 120 min. A decrease in plasma magnesium and copper was observed with no significant change in urine ion concentrations. We related plasma magnesium mobilization to glucagon, and copper mobilization to plasma variation in free fatty acids and albumin.

Acute effects of human growth hormone on zinc, copper, calcium and magnesium metabolism in normal subjects.

The acute effects of human growth hormone (hGH) on the metabolism of zinc, copper, calcium and magnesium ions was studied. Seven normal subjects were perfused iv with doses of 0.2 or 1.0 mg hGH over a period of 60 min. An increase in plasma zinc, calcium and magnesium, and a decrease in plasma copper levels was noted for the 0.2 mg dose, and these effects were reversed for the 1.0 mg dose although no urine changes occurred. Plasma mobilization of these ions appears to be related to their roles as essential physiological modulators for the action of hGH.

Hormonal and metabolic study of a case of adiposis dolorosa (Dercum's disease).

A case of a 43-year-old nonobese woman with adiposis dolorosa (Dercum's disease) is reported. Muscle glucose uptake and oxidation before and after ingestion of 75 g of glucose were similar to control group values, although a greater insulin release (16,578 vs 6,242 +/- 1,136 microU/3 h) occurred simultaneously. In vitro studies of abdominal normal and painful subcutaneous adipose tissue of the patient revealed lower responsiveness to norepinephrine and lack of response to the antilipolytic effect of insulin in the painful adipose tissue (0.98 vs 1.43 microM FFA/10(6) cells at 5.0 microM of norepinephrine). The disease was not correlated with the HLA system and there were no alterations in hormonal secretion at the pituitary, adrenal, gonadal, and thyroid levels. These findings indicate the presence of peripheral insulin resistance in this patient with adiposis dolorosa.

Effect of copper on the secretion of human growth hormone.

The response of human growth hormone to the oral administration of 5.0 mg copper was studied in plasma from 12 normal adults. Blood samples were collected at 30 min intervals over a period of 240 min after two basal measurements taken at time = -30 and time = 0 min. Six subjects responded to the stimulus with increased growth hormone secretion, revealing a positive correlation with plasma copper levels. The other six subjects presented a similar rise in plasma copper levels but no increase in plasma growth hormone levels. These results suggest that acute, high blood copper levels can increase basal growth hormone secretion in normal individuals, presumably by acting on the hypothalamic center.

Endocrine interaction between zinc and prolactin. An interpretative review.

Zinc plays a very important role in animal and human metabolism. Nowadays, it is one of the most extensively studied trace element, since its sphere of action has been demonstrated to be very broad. From the biochemical standpoint, it controls more than 300 different enzymes, many of them involved with intermediary metabolism, DNA and RNA synthesis, gene expression, and immunocompetence. It also plays a significant role in hormonal homeostasis, since it can interact with almost all hormones. Zn2+ is closely related to the thyroid and steroid hormones, insulin, parathormone, and pituitary hormones, particularly prolactin (PRL). Zn2+ can inhibit PRL secretion within a range of physiologically and pharmacologically relevant concentrations. This property has raised the possibility of clinical applications of zinc. In this article, we review the literature on the subject in an attempt to provide a comprehensible general view.

Zinc acutely and temporarily inhibits adrenal cortisol secretion in humans. A preliminary report.

Hypo- and hyperzincemia has been reported to cause alterations in the adrenal secretion. To determine the acute effect of zinc on cortisol levels, we studied 27 normal individuals of both sexes aged 20-27 y after a 12-h fast. The tests were initiated at 7:00 AM when an antecubital vein was punctured and a device for infusion was installed and maintained with physiological saline. Zinc was administered orally at 8:00 AM. Subjects were divided into an experimental group of 13 individuals who received doses of 25, 37.5, and 50 mg of zinc and a control group of 14 individual who received 20 mL of physiological saline. Serial blood samples were collected over a period of 240 min after basal samples (-30 and 0 min). We detected an acute inhibitory effect of zinc on cortisol secretion during 240 min of the study period in the experimental group.

Interrelationships of zinc with glucose and insulin metabolism in humans.

Hyperzincemia has been reported to cause alterations in the homeostasis of glycid metabolism. To determine this effect on plasma glucose and insulin levels, we studied 36 normal individuals of both sexes aged 22-26 y after a 12-h fast. The tests were initiated at 7:00 AM when an antecubital vein was punctured and a device for infusion was installed and maintained with physiological saline. Zinc was administered orally at 8:00 AM. Subjects were divided into an experimental group of 22 individuals who received doses of 25, 37.5, and 50 mg of zinc and a control group of 14 individuals. Blood samples were collected over a period of 240 min after the basal samples (-30 and 0 min). We did not detect any change in plasma glucose or insulin levels, a fact that we attribute either to the ineffectiveness of the 50 mg dose of zinc or to the lack of human response to the acute action of this trace element. The individuals who ingested zinc showed a significant fall in plasma cortisol, probably caused by the action of this trace element.

Interaction among zinc, glucose, and insulin in normal individuals during glucose and tolbutamid perfusion.

Reports in the literature have shown that acute or chronic zinc administration may cause hyperglycemia, with a fall in serum or insular insulin occurring in experimental animals. On the other hand, under conditions of both acute and chronic hyperglycemia, an increase, a decrease, or a normal level of blood zinc has been observed in studies conducted on humans. Thus, the objective of the investigation described here was to determine the relationship existing among zinc, glucose, and insulin under acute conditions. Thirty-six subjects of both sexes (mean age, 23 yr) were tested at 7:00 A.M. after a 12-h fast. Two antecubital veins of both forearms were punctured and maintained with physiological saline. Three experiments were performed in which zinc was administered orally, and hypertonic glucose and tolbutamid were administered intravenously. Blood samples were then collected over a period ranging from 93 to 240 min after the basal times of -30 and 0 min. Hyperzincemia did not cause changes in plasma glucose or insulin either in the absence of or during perfusion of glucose. Hyperglycemia, hypoglycemia, and hyperinsulinemia did not modify serum zinc levels. These results demonstrate that acute zinc administration did not change carbohydrate metabolism and that sudden variations in glucose and insulin levels did not modify the serum profile of zinc.

[Arterial hypertension in obese patients with heart hypertrophy. Effects of captopril on insulin sensitivity and growth hormone]. / Hipertensão arterial em pacientes obesos com hipertrofia cardíaca. Efeitos do captopril sobre a sensibilidade à insulina e hormônio de crescimento.

PURPOSE: To evaluate the effects of captopril (Cpt).on carbohydrate metabolism and growth hormone (GH) in adults hypertensive obese patients with normal (NGT) or impaired (IGT) glucose tolerance and left ventricular hypertrophy. METHODS: Ten patients (53 +/- 8 years), 8 women and 2 men, white, body mass index (BMI) > or = 26kg/m2, left ventricular mass index (LVMI) > 135g/m2 in man and > 110g/m2 in woman, with diastolic blood pressure (DBP) 95-115mmHg after 3 weeks of placebo, were identified by oral glucose tolerance test (OGTT-75g) as either with NGT or IGT, and treated with Cpt 25mg t.i.d. for 8 weeks. At the 8 weeks, dosage was increased to 50mg b.i.d. if DBP > 90mmHg or the decrease of the DBP < 10%, during the next 8 weeks. OGTT and clonidine tests (0,04mg/kg) with determinations, every 30 minutes of glucose, insulin, and GH during 2 hours, were performed. RESULTS: Cpt lowered SBP and DBP in the NGT group and IGT group. The LVMI and the left ventricular mass (LVM) decreased in the IGT group with no significant change in the NGT group. Cpt promoted in the IGT group decrease in the area under the curve (AUC) of glucose, and AUC of insulin, with increase of the AUC of the percent of the beta cell function, AUC of HC, and insulin sensitivity index with no significantly change in the NGT group. CONCLUSION: Adults hypertensive obese patients with IGT had decreased significantly in mean fasting level of GH concentrations compared to age, race, and BMI matched hypertensive patients with NGT. Treatment with Cpt induced a significant increased of the GH, with improvement of the metabolism in patients with IGT.

Zinc pharmacokinetic parameters in the determination of body zinc status in children.

BACKGROUND/OBJECTIVES: Serum or tissue zinc concentrations are often used to assess body zinc status. However, all of these methods are relatively inaccurate. Thus, we investigated three different kinetic methods for the determination of zinc clearance to establish which of these could detect small changes in the body zinc status of children. SUBJECTS/METHODS: Forty apparently healthy children were studied. Renal handling of zinc was investigated during intravenous zinc administration (0.06537 mg Zn/kg of body weight), both before and after oral zinc supplementation (5 mg Zn/day for 3 months). Three kinetic methods were used to determine zinc clearance: CZn-Formula A and CZn-Formula B were both used to calculate systemic clearance; the first is a general formula and the second is used for the specific analysis of a single-compartment model; CZn-Formula C is widely used in medical practices to analyze kinetic routine. RESULTS: Basal serum zinc values, which were within the reference range for healthy children, increased significantly after oral zinc supplementation. The three formulas used gave different results for zinc clearance both before and after oral zinc supplementation. CZn-Formula B showed a positive correlation with basal serum zinc concentration after oral supplementation (R2=0.1172, P=0.0306). In addition, CZn-Formula B (P=0.0002) was more effective than CZn-Formula A (P=0.6028) and CZn-Formula C (P=0.0732) in detecting small variations in body zinc status. CONCLUSIONS: All three of the formulas used are suitable for studying zinc kinetics; however, CZn-Formula B is particularly effective at detecting small changes in body zinc status in healthy children.

Characteristics of zinc binding to human red blood cell membranes.

The objective of the present study was to standardize the analysis of zinc binding on human red blood cell (RBC) membranes in 20 normal adults. The displacement studies revealed that at the maximal stable zinc concentration tested (600 microM), 57% (mean) of the bound 65Zn was displaced and to displace half maximal 65Zn, the stable zinc concentration was 300 microM. Scatchard plots revealed two classes of binding sites for zinc on RBC membranes: one with higher affinity, Kd = 1.20 x 10(-5) M (site I), and the other with lower affinity, Kd = 2.77 x 10(-4) M (site II). Binding sites occupancy was 97% means and 58.5% means for sites I and II, respectively. The displacement was affected by temperature, membrane protein concentration, freezing, thawing, and dialysis. Other metal cations, including Co++, Fe++, and Mn++, had very little effect on 65Zn displacement, in contrast copper displaced 65Zn from its binding sites on RBC membranes. Zinc binding to RBC membranes was rapid and readily reversible in a dynamic equilibrium with its binding sites. It is anticipated that this method will be applicable to studies of a wide variety of diseases specifically related to zinc metabolism in humans as well as in animals.

Effects of a single venous dose of zinc on thyroid status in healthy individuals and patients with graves' disease.

Zinc metabolism may regulate thyroid function acting at TRH (thyrotropin-releasing hormone) synthesis, peripheral deiodination of T4 (tetraiodothyronine), and binding of thyroid hormones to nuclear receptors. The aim of this study was to investigate the effect of acute zinc administration on TSH (thyroid-stimulating hormone), FT3 (free triiodothyronine), and FT4 (free tetraiodothyronine) in 10 healthy individuals and 12 hyperthyroid patients with Graves' disease. All these individuals were studied following 25 mg Zn(++) administered intravenously, at 7:00 a.m. after 12 h fast. Blood samples collected at 0, 3, 30, 60, 90, and 120 min after zinc administration showed no significant alteration in the plasma levels of TSH, FT3, and FT4 in hyperthyroid patients. There were no changes in the plasma levels of FT3 and FT4 in the control subjects, but TSH levels were acutely depressed by zinc administration. This study suggests that zinc given acutely and in pharmacological doses does not affect thyroid function in hyperthyroid subjects, but affect plasma TSH levels in healthy individuals.

Renal handling of zinc in insulin-dependent diabetes mellitus patients.

Hyperzincuria is a common feature in diabetic patients, which is still not understood. Based on the above consideration, the aim of the present study was to investigate the renal handling of zinc in insulin-dependent diabetes mellitus (IDDM) patients. The glomerular filtration rate, urinary zinc excretion, zinc clearance, zinc clearance/creatinine clearance ratio, zinc tubular reabsorption, glycosuria, plasma glucose, C-peptide, glucagon, and cortisol were investigated in 10 normal individuals (Group C1 and Group C2, respectively) and 10 IDDM patients (Group E1: hyperglycemic and glycosuric and Group E2: normoglycemic and aglycosuric) during placebo or venous zinc tolerance test. The results showed that urinary zinc excretion and renal zinc clearance were increased after zinc injection in normal individuals (Group C2) and IDDM patients (Groups E1 and E2) when compared with normal individuals-placebo (Group C1). However, these renal parameters were statistically more significant in the hyperglycemic and glycosuric diabetics (Group E1). Because patients in Group E1 had the lowest plasma C-peptide levels and showed a strong negative correlation between CZn++/Ccr ratio and this hormone, we suggest that in this setting insulin inhibits urinary zinc excretion.

Effect of acute and chronic oral zinc administration in hyperprolactinemic patients.

The inverse relationship between zinc (Zn(++)) and prolactin (PRL) was detected in in vitro studies, whereas in vivo results are contradictory. In order to evaluate this controversial subject we studied patients with hyperprolactinemia. Basal serum Zn(++) levels and serum PRL response to acute and chronic oral Zn(++) administration were evaluated in seven patients with prolactinomas and one with idiopathic hyperprolactinemia. Serum PRL levels did not change after acute oral Zn(++) administration (37.5 mg), although Zn(++) levels increased from 1.11+/-0.15 to 2.44+/-0.39 mug/mL (P<0.05). ZnZn(++) administration (47.7 mg daily) during 60 days increased serum Zn(++) levels from 1.11 +/- 0.15 to 1.59 +/- 0.58 mug/mL (p < 0.05) but caused no change in serum PRL levels. The TRH tolerance test (200 mug) was performed before and after 60 days of Zn(++) administration, and PRL response to TRH was unchangeable and similar in both tests. We concluded that acute or chronic Zn(++) administration does not inhibit PRL secretion in basal condition or by TRH effect in hyperprolactinemic patients.

Effect of zinc administration on thyrotropin releasing hormone-stimulated prolactinemia in healthy men.

Previous in vitro studies have demonstrated zinc (Zn++) inhibition of basal and of potassium (K+) or thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion, in a selective, reversible, and dose-dependent manner. Thus, Zn++ may regulate physiologically pituitary PRL secretion. Furthermore, studies with patients with uremia, cirrhosis or prolactinoma, have shown the coexistence of hypozincemia and hyperprolactinemia and zinc supplementation did not correct hyperprolactinemia in these patients. In normal individuals Zn++ administration produced controversial results on PRL secretion. Here, we investigated whether zinc administration affects TRH-stimulated PRL in healthy men. We found that Zn++ administration does not change the TRH-stimulated PRL. Therefore, in normal conditions, Zn++ does not inhibit TRH-stimulated prolactinemia. In addition, we found that acute increases of blood PRL and TRH do not alter blood Zn++ levels.

Zinc kinetics in insulin-dependent diabetes mellitus patients.

Zinc has an important role in the control of carbohydrate metabolism, and diabetic patients are at risk for zinc deficiency. However, there are conflicting data concerning nutritional zinc status. In order to investigate this topic, 10 normal and 10 insulin-dependent diabetic patients were studied following venous zinc tolerance test. Our results found no evidence of zinc deficiency or of changes on the kinetic parameters of zinc in patients with insulin-dependent diabetes mellitus following a venous zinc tolerance test.