Modest decrements in plasma glucose concentration cause early impairment in cognitive function and later activation of glucose counterregulation in the absence of hypoglycemic symptoms in normal man.

To establish the glycemic threshold for onset of neuroglycopenia (impaired cognitive function, measured by the latency of the P300 wave), activation of hormonal counterregulation and hypoglycemic symptoms, 12 normal subjects were studied either under conditions of insulin-induced, glucose-controlled plasma glucose decrements, or during maintenance of euglycemia. A decrement in plasma glucose concentration from 88 +/- 3 to 80 +/- 1 mg/dl for 150 min did not result in changes in the latency of the P300 wave nor in an activation of counterregulatory hormonal response. In contrast, a greater decrement in plasma glucose concentration from 87 +/- 3 to 72 +/- 1 mg/dl for 120 min caused an increase in the latency of the P300 wave (from 301 +/- 12 to 348 +/- 20 ms, P less than 0.01), a subsequent increase in all counterregulatory hormones but no hypoglycemic symptoms. Finally, when plasma glucose concentration was decreased in a stepwise manner from 88 +/- 2 to 50 +/- 1 mg/dl within 75 min, the increase in the latency of the P300 wave was correlated with the corresponding plasma glucose concentration (r = -0.76, P less than 0.001). The glycemic threshold for hypoglycemic symptoms was 49 +/- 2 mg/dl. Thus, in normal man the glycemic threshold for neuroglycopenia (72 +/- 1 mg/dl) is greater than currently thought; the hormonal counterregulation follows the onset of neuroglycopenia; the hypoglycemic symptoms are a late indicator of advanced neuroglycopenia.

A Bowman-Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition.

Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman-Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment.

Acute antihyperglycemic mechanisms of metformin in NIDDM. Evidence for suppression of lipid oxidation and hepatic glucose production.

To establish the antihyperglycemic mechanisms of metformin in non-insulin-dependent diabetes mellitus (NIDDM) independently of the long-term, aspecific effects of removal of glucotoxicity, 21 NIDDM subjects (14 obese, 7 nonobese) were studied on two separate occasions, with an isoglycemic (plasma glucose approximately 9 mM) hyperinsulinemic (two-step insulin infusion, 2 h each, at the rate of 4 and 40 mU.m-2.min-1) clamp combined with [3-3H]glucose infusion and indirect calorimetry, after administration of either metformin (500 mg per os, at -5 and -1 h before the clamp) or placebo. Compared with placebo, hepatic glucose production (HGP) decreased approximately 30% more after metformin (from 469 +/- 50 to 330 +/- 54 mumol/min), but glucose uptake did not increase. Metformin suppressed free fatty acids (FFAs) by approximately 17% (from 0.42 +/- 0.04 to 0.35 +/- 0.04 mM) and lipid oxidation by approximately 25% (from 4.5 +/- 0.4 to 3.4 +/- 0.4 mumol.kg-1.min-1) and increased glucose oxidation by approximately 16% (from 16.2 +/- 1.4 to 19.3 +/- 1.3 mumol.kg-1.min-1) compared with placebo (P < 0.05), but did not affect nonoxidative glucose metabolism, protein oxidation, or total energy expenditure. Suppression of FFA and lipid oxidation after metformin correlated with suppression of HGP (r = 0.70 and r = 0.51, P < 0.001). The effects of metformin in obese and nonobese subjects were no different. We conclude that the specific, antihyperglycemic effects of metformin in the clinical condition of hyperglycemia in NIDDM are primarily due to suppression of HGP, not stimulation of glucose uptake, and are mediated, at least in part, by suppression of FFA and lipid oxidation.

A reliable and reproducible test for adequate glucose counterregulation in type I diabetes mellitus.

The safety, reproducibility, and reliability of an insulin infusion test for assessment of adequate glucose counterregulation were evaluated in 18 patients with type I (insulin-dependent) diabetes mellitus. When the test (a 60-min, 30-mU/m2/min insulin infusion) was administered on three separate occasions at 3-4-wk intervals, coefficients of variation for plasma glucose and counterregulatory hormone (glucagon, epinephrine, cortisol, and growth hormone) responses averaged less than 8%. No patient experienced symptoms requiring discontinuation of the test and plasma glucose concentrations increased spontaneously after stopping the insulin infusion. Using objective criteria based on plasma glucose nadirs or postnadir rates of plasma glucose recovery, no patient judged to have adequate glucose counterregulation by the test (postnadir rates of plasma glucose recovery or plasma glucose nadir above 0.4 mg/dl/min and 45 mg/dl) developed severe hypoglycemia (plasma glucose less than 40 mg/dl) during up to 7 mo of intensive insulin therapy, whereas nearly all patients with inadequate counterregulation did. We conclude that this test, when performed in standardized conditions, is safe and reproducible and can reliably predict those patients with type I diabetes who are at risk of developing severe hypoglycemia during intensive insulin therapy.

Demonstration of a dawn phenomenon in normal human volunteers.

To ascertain whether the dawn phenomenon occurs in nondiabetic individuals and, if so, whether it is due to an increase in glucose production or a decrease in glucose utilization, we determined plasma concentrations of glucose, insulin, C-peptide, and counterregulatory hormones, as well as rates of glucose production, glucose utilization, and insulin secretion at one-half-hourly intervals between 1:00 and 9:00 a.m. in eight normal volunteers. After 5:30 a.m., plasma glucose, insulin, and C-peptide concentrations all increased significantly; rates of glucose production, glucose utilization, and insulin secretion also increased (all P less than 0.05). Plasma cortisol, epinephrine, and norepinephrine increased significantly from nocturnal nadirs between 4:00 and 6:30 a.m. Plasma growth hormone, which had increased episodically between 1:00 and 4:30 a.m., decreased thereafter nearly 50% (P less than 0.05). Plasma glucagon did not change significantly throughout the period of observation. These results indicate that a dawn-like phenomenon, initiated by an increase in glucose production, occurs in nondiabetic individuals. Thus, early morning increases in plasma glucose concentrations and insulin requirements observed in IDDM and NIDDM may be an exaggeration of a physiologic circadian variation in hepatic insulin sensitivity induced by antecedent changes in catecholamine and/or growth hormone secretion.

Comparison of glucose counterregulation during short-term and prolonged hypoglycemia in normal humans.

To compare glucose counterregulatory mechanisms during short-term hypoglycemia and prolonged hypoglycemia, insulin was infused either intravenously (160 mU X M-2 X min) for 10 min or subcutaneously (15 mU X M-2 X min) for 12 h in normal volunteers. With each type of insulin infusion, hypoglycemia (approximately 50 mg/dl) was either allowed to develop or was prevented (control experiments) by the glucose-clamp technique. During prolonged hypoglycemia, both increased glucose production (1.55 +/- 0.05 versus 0.33 +/- 0.14 mg X kg-1 X min in control experiments at 12 h, P less than 0.01) and suppressed glucose utilization (1.55 +/- 0.06 versus 3.17 +/- 0.15 mg X kg-1 X min in control studies at 12 h, P less than 0.01) were involved in counterregulation. During short-term hypoglycemia, only increased glucose production (3.23 +/- 0.33 versus 0.06 +/- 0.03 mg X kg-1 X min in control experiments at 60 min) was involved, since glucose clearance actually increased (3.99 +/- 0.20 versus 2.88 +/- 0.02 ml X kg-1 X min in control experiments at 60 min, P less than 0.01). Estimated portal venous insulin concentrations decreased 40% (basal 24 +/- 3 versus 14 +/- 1 mU/ml at 60 min, P less than 0.01) in the short-term hypoglycemia experiments but remained at basal levels (basal 25 +/- 1 versus approximately 26 microU/min between 1 and 12 h) during prolonged hypoglycemia. Despite the fact that hypoglycemia was more gradually induced in the prolonged hypoglycemia model, peak counterregulatory hormone responses were at least as great as those during short-term hypoglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of new computerized method for recording 7-day food intake in IDDM patients.

OBJECTIVE: To evaluate a new computerized method for recording 7-day food intake. RESEARCH DESIGN AND METHODS: Randomized crossover trial was conducted with patients recording the amount and type of every food and drink consumed during a week by either a computerized device (Food-meter) or recording the data in a diary. Each method was applied twice. Twenty-one insulin-dependent diabetic patients (mean +/- SD age 25 +/- 9 yr) were studied. RESULTS: The two methods showed very good agreement in the evaluation of the patients' diets (1792 +/- 408 vs. 1764 +/- 436 kcal/day, 84 +/- 19 vs. 82 +/- 21 g/day protein, 68 +/- 22 vs. 67 +/- 23 g/day fat, 210 +/- 60 vs. 207 +/- 58 g/day carbohydrate with the conventional and computerized methods, respectively). The variability between the methods and the variability within each method were of similar magnitude. CONCLUSIONS: The Food-meter represents a useful tool for computerizing the 7-day food record. The method is easy, reliable, and time saving. Moreover, it minimizes the risk of transcriptional errors.

Analysis of the black-eyed pea trypsin and chymotrypsin inhibitor-alpha-chymotrypsin complex.

The black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a member of the Bowman-Birk protease inhibitor (BBI) family. The three-dimensional model of the BTCI-chymotrypsin complex was built based on the homology to Bowman-Birk inhibitors with known structures. An extensive theoretical and experimental study of these known structures has been performed. The model confirms the ideas about Bowman-Birk inhibitor structure-function relations and agrees well with our experimental data (circular dichroism, IR and light scattering). The electrostatic potentials at the enzyme-inhibitor contact surface reveal a pattern of complementary electrostatic potentials from which mutations can be inferred that could give these inhibitors an altered specificity.

[A new proposal for nutritional education in a group of obese adolescents]. / Una nuova proposta di educazione alimentare in un gruppo di adolescenti obesi.

A new "active" system of monitoring and dietary education was experimented in a group of 20 obese adolescents waiting to start diet therapy. Each subject was requested to record all food and drink consumed during two separate periods, each lasting 7 days, at a distance of 15 days from each other, at home using an optic reader and book of bar codes. On the basis of answers to a questionnaire which was completed at the beginning and end of the study, aimed at assessing the level of knowledge of basic food hygiene, the majority of participants considered the experience useful and amusing and were willing to repeat it; in addition, a greater knowledge of nutritional principles and of the rations consumed was shown at the end of the study. The results of the study were analysed using the Food Meter-Miles computerised system and showed daily calorie intakes of 1514 +/- 524.0, M +/- DS (protein 18.3 +/- 5.1%, lipids 33.7 +/- 6.2%, glucose 47.9 +/- 8.6%, total fibre 14.4 +/- 4.6 g) in female subjects, and 2096.1 +/- 80.8 (protein 16.1 +/- 3.6%, lipids 38.2 +/- 2.9%, glucose 45.7 +/- 3.9%, total fibre 18.1 +/- 2.0 g) in male subjects. The number of foods chosen was very low considering the range of foods available (277) and the length of time studied: 35.7 +/- 11.8 and 35.0 +/- 6.0 respectively for female and male subjects. With regard to the number and type of meals eaten, a high number of snacks was observed which supplemented or replaced main meals.

Phylogenetic relationships of snake venom toxic proteins.

Two kinds of distance matrices have been formed from minimum mutational distances and absolute hydrophobicity differences obtained by comparison of aligned homologous sequences of 56 toxins from venom of snakes belonging to 7 genera. Phylogenetic trees were constructed from these distance matrices, employing the unweighted pair-group method using arithmetic averages (UPGMA). The Pearson product-moment correlation coefficient has been used to estimate the agreement between the original distance matrix and that obtained directly from the dendrogram. For all these procedures the set of computer programs PHYTREE (written in BASIC for micro-computer, and available from the author) has been used.

Structural similarity in Bowman-Birk type protease inhibitors based on hydrophobicity.

The degree of similarity in the three-dimensional structures of thirteen legume Bowman-Birk type protease inhibitors has been examined on the basis of the patterns of hydrophobicity found in their amino acid sequences, following the procedure described by Sweet & Eisenberg (1983). In the group of such double-headed protease inhibitors two sub-groups are distinguished presenting high structural similarity among their respective members and low similarity between them. Phylogenetic trees have been constructed from hydrophobicity difference and minimum mutational distance matrices, respectively.

Do snake venom cytotoxins exhibit amphiphilicity?

It is suggested, on the basis of the structural information available from the literature, that the molecules of cobramine B and homologous cytotoxins, in contrast to snake venom neurotoxins, are amphiphilic in the sense that they are composed of a predominantly hydrophobic multi-stranded beta-sheet and other regions sharply hydrophilic. It is possible that the direct lytic activity of snake venom cytotoxins is due, at least in part, to their amphiphathy.

Secondary structure of soybean trypsin inhibitor (Kunitz): a study by infrared spectroscopy.

The infrared spectrum (amide I' region) of Kunitz soybean trypsin inhibitor (SBTI) was obtained in D2O solution and resolved into Gaussian components. A prominent broad band centered at 1643 cm-1 is shown on the unresolved spectrum, which is usually assigned to N-deuterated peptide groups in an unordered structure, since SBTI is known to be devoid of alpha-helix by CD and X-ray crystallographic studies. In addition, shoulders are evident at 1632 cm-1 and 1676 cm-1, which correspond probably to the v(pi, O) and v(O, pi) components assigned to an antiparallel-chain beta-pleated sheet structure. Parameters (maximum absorptivity, wavenumber at the maximum of the band, and half-width of the band at half-height) for the four Gaussian component bands (in which the amide I' band was resolved) are given. A crude estimation of 4% is obtained for antiparallel beta-sheet in SBTI, i.e., this protein would be practically devoid of such a beta-structure. Notwithstanding the fact that this result is apparently in agreement with the far-UV CD spectrum (data reported in the literature), the predominant conformation class found in SBTI has been demonstrated to be approximate beta-sheet structures, with a small amount of regular sheet (Sweet et al., (1974) Biochemistry 13: 4212-4228).

Bowman-Birk type protease inhibitors: two-dimensional vector representation for their sequences.

The degree of structural similarity in the legume Bowman-Birk type protease inhibitors has been examined on the basis of two-dimensional vector representation of the respective amino acid sequences. Amino acid residue size and hydrophobicity are the two dimensions used (Swanson, R. (1984) Bull. Math. Biol., 46: 623-639). For the set of such homologous proteins a consensus sequence is generated. A non-negative real-valued function on the set of compared sequences is proposed as a measure of dissimilarity between compared sequences. In the group of those double-headed protease inhibitors sub-groups are distinguished presenting high structural similarity among their respective members and lower similarity among them.

Prediction of the secondary structure of Bowman-Birk soybean protease inhibitor from LTS sequence.

The secondary structure of Bowman-Birk soybean inhibitor (BBI) has been predicted from its amino acid sequence (71 residues) using the statistical method of Chou and Fasman (1974) as well as the informational method of Garnier et al. (1978). According to both methods, no alpha-helical region is predicted in BBI. Short beta-strands at 11-15, 39-43, 48-52 and 57-59 are predicted by Chou and Fasman's method, and at 11-14, 39-43, 48-52 and 57-59 by the method of Garnier et al. Predicted beta-turn tetrapeptides are residues at 1-4, 6-9, 14-17, 18-21, 24-27, 30-33, 36-39, 60-63, 63-66 and 68-71, according to the method of Chou and Fasman. These predictive results indicate that BBI consists essentially of beta-turn, beta-strand, and unordered structures, which represent about 56, 25 and 19% (by difference) of its secondary structure, respectively. The predicted reverse turn amount in BBI is very high, in comparison with that found usually in proteins. No fitting of the experimental circular dichroism spectrum (in the 195-240 nm region), which was reported by Kay (1976), has proved satisfactory when using the parameters for helical, beta-sheet, beta-turn and unordered conformations proposed by several authors.

Phospholipases A2: a dendrogram from a distance matrix based on size and hydrophobicity of the residues in their homologous sequences.

A distance matrix was obtained from aligned homologous sequences of 32 phospholipases A2 (EC 3.1.1.4) (24 from Elapid and 5 from Viperid venoms, and 3 from mammals), on the basis of the quantities Dij which are defined from a two-dimensional vector representation of the amino acid residues (dimensions: size and hydrophobicity). These Dij quantities were proposed in a previous paper (Ventura, M. M., (1989), An. Acad. brasil. Ci., 61: 215). A dendrogram was constructed from this distance matrix employing, for cluster analysis, the unweighted pair-group using arithmetic averages. Two groups of phospholipases A2: a) Elapid venom enzymes together with the three mammalian pancreatic enzymes (bovine, equine and porcine), and b) Viper venom enzymes (Crotalus, Trimeresurus and Bitis enzymes) can be well distinguished in the topology of the dendrogram. The Elapid group of enzymes is divided into two subgroups: a) Naja, Hemachatus and Bungarus venom enzymes, and b) Notechis, Laticauda, Enhydrina and Oxyuranus venom enzymes. It is observed that there is a close similarity between the mammalian pancreatic phospholipases A2 and the enzymes from Naja, Hemachatus and Bungarus. These results are similar to those reported by Dufton and Hider (Eur. J. Biochem., 137:545 (1983] which were obtained from the distance matrix based on minimum mutation distance between 25 selected residue positions in the pairwise compared sequences.

Isolation and some properties of a trypsin inhibitor from seeds of Schizolobium parahyba (Vell.) Toledo.

A trypsin inhibitor was isolated as a homogenous protein from the seeds of guapuruvu-tree (Schizolobium parahyba (Vell.) Toledo). In addition to its strong inhibitory activity against trypsin the purified inhibitor presented a lesser activity against alpha-chymotrypsin. The purification of the protein inhibitor was achieved from the crude extract of deffated seeds through ammonium sulphate salting-out, successive chromatographies on Sephadex G-75 and DEAE-Sephadex A-50 columns followed by preparative polyacrylamide-slab electrophoresis. The following properties were presented by the purified inhibitor: molecular weight of 12,000 daltons, as estimated by gel filtration; isoelectric point at pH 5.0 - 5.2, by electrofocusing; combining molar ratio of 1:1 (mole trypsin/mole inhibitor), on the basis of inhibition assay and the molecular weight of 29,800 daltons found for the trypsin-inhibitor complex; A1%1-cm = 4.35, at 275 nm and pH 7.0. The inhibitor presents a high content of cystine (14 cystinyl residues per molecule) and is entirely devoid of methionine, tryptophan and free sulhydryl groups. The fluorescence spectra are typical for tyrosine with a strong quenching of emission indicated by the quantum yield. The circular dichroism spectra suggest a predominantly unordered structure for the inhibitor molecule.