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.

Crystallization, data collection and phasing of black-eyed pea trypsin/chymotrypsin inhibitor in complex with bovine beta-trypsin.

The black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a Bowman-Birk-type inhibitor from Vigna unguiculata seeds. A complex of BTCI with bovine beta-trypsin was crystallized by the hanging-drop vapour-diffusion method with ammonium sulfate as precipitant. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 59.3, b = 61.8, c = 80.0 A. Diffraction data were collected to 2.36 A resolution and were processed to give an overall R(sym) of 0.137. The Matthews coefficient for one complex per asymmetric unit is 2.2 A(3) Da(-1), with a corresponding solvent content of 43%. After molecular replacement and initial refinement, the model gives an R(cryst) of 0.361 and an R(free) of 0.432.

Thermodynamics of the binding of chymotrypsin with the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI).

The binding of alpha-chymotrypsin to black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) has been studied using the inhibitory activity against the enzyme and the formation of the complex enzyme/inhibitor followed by measurements of fluorescence polarization. Apparent equilibrium constants were estimated for several temperatures and the values obtained range from 0.32 x 10(7) to 1.36 x 10(7) M(-1). The following values were found from van't Hoff plots: delta H(0)vh = 10.8 kcal mol(-1) (from inhibitory assays) and 11.1 kcal mol(-1) (from fluorescence polarization); delta S(0) = 67.9 and = 67.8 kcal K(-1) mol(-1), respectively. Calorimetric binding enthalpy was determined (corrected for the ionization heat of the buffer) and the resulting value was delta H(0)cal = 4.9 kcal mol(-1). These results indicate that the binding of chymotrypsin to BTCI is an entropically driven process.

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.

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.

Contribution of adrenergic mechanisms to glucose counterregulation in humans.

To assess the role of adrenergic mechanisms during prolonged hypoglycemia, eight normal subjects were studied on six occasions. In study 1, insulin was infused subcutaneously (15 mU.m-2.min-1 for 12 h), and plasma glucose concentration (PG) decreased from 89 +/- 2 to 50 +/- 1 mg/dl. In study 2 (insulin as in study 1 + propranolol and phentolamine + variable glucose to maintain PG as in study 1), the rate of hepatic glucose production (HGO, [3-3H]glucose) was approximately 30% lower after 1.5 h, and the rate of peripheral glucose utilization (GU) was approximately 15% greater after 5 h. To quantitate the effects of adrenergic mechanisms on glucose counterregulation, in a control study (study 3), glucoregulatory hormone secretion was blocked, and the hormones were reinfused to reproduce study 1. When alpha- and beta-blockade plus variable glucose were superimposed to study 3 (study 4), HGO was approximately 25% lower (after 2 h), and GU was approximately 10% greater (after 6 h) vs. study 3. When glucose was not infused to match PG of study 3 (study 5), severe hypoglycemia developed (PG at 7 h 36 +/- 2 vs. 62 +/- 3 mg/dl). Finally, when glucose was not infused during alpha- and beta-blockade of study 2 (study 6), PG was 49 +/- 3 mg/dl at 7 h vs. 65 +/- 3 mg/dl of the control study (study 1), despite greater secretion of glucagon, growth hormone, and cortisol. It is concluded that adrenergic mechanisms play a key counterregulatory role, even in the presence of appropriate responses of glucagon and that greater increases in glucagon (and other counterregulatory hormones) cannot compensate fully for absent contribution of adrenergic mechanisms to counterregulation.

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.

Evidence against important catecholamine compensation for absent glucagon counterregulation.

To assess the counterregulatory role of glucagon and to test the hypothesis that catecholamines can largely compensate for an impaired glucagon response, four studies were performed in seven normal volunteers. In all studies, insulin was infused subcutaneously (15 mU.m-2.min-1) and increased circulating insulin approximately twofold to levels (26 +/- 1 microU/ml) observed with intensive insulin therapy. In study 1, plasma glucose fluxes (D-[3-3H]glucose) and plasma substrate and counterregulatory hormone concentrations were simply monitored; plasma glucose decreased from 87 +/- 2 mg/dl and plateaued at 51 +/- 2 mg/dl for 3 h. In study 2 [pituitary-adrenal-pancreatic (PAP) clamp], secretion of insulin and counterregulatory hormones (except for catecholamines) was prevented by somatostatin (0.5 mg/h i.v.) and metyrapone (0.5 g/4 h per os), and glucagon, cortisol, and growth hormone were reinfused to reproduce the concentrations of study 1. In study 3 (lack of glucagon response), the PAP clamp was performed with maintenance of plasma glucagon at basal levels, and glucose was infused whenever needed to reproduce plasma glucose concentration of study 2. Study 4 was identical to study 3, but exogenous glucose was not infused. The PAP clamp (study 2) reproduced glucose concentrations and fluxes observed in study 1. In studies 3 and 4, isolated lack of glucagon response did not affect glucose utilization but caused an early and persistent decrease in hepatic glucose production (approximately 60%) that caused plasma glucose to decrease to 38 +/- 2 mg/dl (P less than 0.01 vs. control 62 +/- 2 mg/dl), despite compensatory increases in plasma epinephrine. We conclude that, in a model of clinical hypoglycemia, glucagon's effect on hepatic glucose production is a dominant counterregulatory factor in humans and that its absence cannot be compensated for by increased epinephrine secretion.

[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.

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.

Contribution of cortisol to glucose counterregulation in humans.

To test the hypothesis that cortisol secretion plays a counterregulatory role in hypoglycemia in humans, four studies were performed in eight normal subjects. In all studies, insulin (15 mU.m-2.min-1) was infused subcutaneously (plasma insulin 27 +/- 1 microU/ml). In study 1, plasma glucose concentration and glucose fluxes [( 3-3H]glucose), substrate, and counterregulatory hormone concentrations were simply monitored, and plasma glucose decreased from 89 +/- 2 to 52 +/- 2 mg/dl for 12 h. In study 2, (pituitary-adrenal-pancreatic clamp), insulin and counterregulatory hormone secretion (except for catecholamines) was prevented by somatostatin (0.5 mg/h, iv) and metyrapone (0.5 g/4 h, per os), and glucagon, cortisol, and growth hormone were infused to reproduce the concentrations of study 1. In study 3 (lack of cortisol increase), the pituitary-adrenal-pancreatic clamp was performed with maintenance of plasma cortisol at basal levels, and glucose was infused, whenever needed, to reproduce plasma glucose concentration of study 2. Study 4 was identical to study 3, but exogenous glucose was not infused. Isolated lack of cortisol increase caused a approximately 22% decrease in hepatic glucose production (P less than 0.01) and a approximately 15% increase in peripheral glucose utilization (P less than 0.01), which resulted in greater hypoglycemia (37 +/- 2 vs. 52 +/- 2 mg/dl, P less than 0.01) despite compensatory increases in plasma epinephrine. Lack of cortisol response also reduced plasma free fatty acid, beta-hydroxybutyrate, and glycerol concentrations approximately 50%. We conclude that cortisol normally plays an important counterregulatory role during hypoglycemia by augmenting glucose production, decreasing glucose utilization, and accelerating lipolysis.

Demonstration of a role for growth hormone in glucose counterregulation.

To test the hypothesis that growth hormone secretion plays a counterregulatory role in prolonged hypoglycemia in humans, four studies were performed in nine normal subjects. Insulin (15 mU.M-2.min-1) was infused subcutaneously (plasma insulin 27 +/- 2 microU/ml), and plasma glucose decreased from 88 +/- 2 to 53 +/- 1 mg/dl for 12 h. In study 1, plasma glucose, glucose fluxes (D-[3-3H]glucose), substrate, and counterregulatory hormone concentrations were simply monitored. In study 2 (pituitary-adrenal-pancreatic clamp), insulin and counterregulatory hormone secretions (except for catecholamines) were prevented by somatostatin (0.5 mg/h iv) and metyrapone (0.5 g/4 h po), and glucagon, cortisol, and growth hormone were reinfused to reproduce the concentrations of study 1. In study 3 (lack of growth hormone increase), the pituitary-adrenal-pancreatic clamp was performed with maintenance of plasma growth hormone at basal levels, and glucose was infused whenever needed to reproduce plasma glucose concentration of study 2. Study 4 was identical to study 3, but exogenous glucose was not infused. Isolated lack of a growth hormone response caused a decrease in hepatic glucose production and an increase in glucose utilization that resulted in an approximately 25% greater hypoglycemia despite compensatory increases in plasma catecholamines. Plasma free fatty acid, 3-beta-hydroxybutyrate, and glycerol concentrations were reduced approximately 50%. It is concluded that growth hormone normally plays an important counterregulatory role during hypoglycemia by augmenting glucose production, decreasing glucose utilization, and accelerating lipolysis.

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.

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).

The effect of asymptomatic nocturnal hypoglycemia on glycemic control in diabetes mellitus.

To assess the effect of asymptomatic nocturnal hypoglycemia on glycemic control in insulin-dependent diabetes mellitus, we studied, on three nights, 10 patients receiving their usual regimens of continuous subcutaneous insulin infusion. During a control night, the patients' mean (+/- SE) plasma glucose level reached a nadir of 4.5 +/- 0.2 mmol per liter at 3 a.m.; the fasting glucose level was 5.9 +/- 0.3 mmol per liter at 7:30 a.m., and a peak glucose level of 8.6 +/- 0.3 mmol per liter was reached at 10 a.m., after breakfast. During nights two and three, supplemental insulin was infused intravenously from 10 p.m. to 2 a.m. to simulate a clinical overdose of insulin. On these nights, either hypoglycemia (2.4 +/- 0.2 mmol per liter) was permitted to occur or a nearly normal glucose level (5.5 mmol per liter) was maintained by infusion of glucose. The subjects were asymptomatic on all three nights. Despite comparable plasma free insulin levels from 4 to 11 a.m., both fasting (7.3 +/- 0.2 mmol per liter) and postbreakfast (12.5 +/- 0.4 mmol per liter) plasma glucose levels were significantly higher after hypoglycemia than when hypoglycemia was prevented (6.2 +/- 0.2 mmol per liter and 8.7 +/- 0.4 mmol per liter, respectively; P less than 0.001 in both cases). Fasting levels of plasma glucose correlated directly with overnight plasma levels of epinephrine (r = 0.78, P less than 0.001), growth hormone (r = 0.57, P less than 0.009), and cortisol (r = 0.52, P less than 0.02) but correlated inversely with the overnight nadir of plasma glucose (r = -0.62, P less than 0.005). We conclude that asymptomatic nocturnal hypoglycemia can cause clinically important deterioration in glycemic control (the Somogyi phenomenon) in patients receiving intensive insulin therapy, and should therefore be considered in the differential diagnosis of unexplained morning hyperglycemia.

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.

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.

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.

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.