Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

This article reports on the design and characteristics of substrate mimetics in protease-catalyzed reactions. Firstly, the basis of protease-catalyzed peptide synthesis and the general advantages of substrate mimetics over common acyl donor components are described. The binding behavior of these artificial substrates and the mechanism of catalysis are further discussed on the basis of hydrolysis, acyl transfer, protein-ligand docking, and molecular dynamics studies on the trypsin model. The general validity of the substrate mimetic concept is illustrated by the expansion of this strategy to trypsin-like, glutamic acid-specific, and hydrophobic amino acid-specific proteases. Finally, opportunities for the combination of the substrate mimetic strategy with the chemical solid-phase peptide synthesis and the use of substrate mimetics for non-peptide organic amide synthesis are presented.

Amifostine (WR-2721), a cytoprotective agent during high-dose cyclophosphamide treatment of non-Hodgkin's lymphomas: a phase II study

Clinical trials indicate that amifostine may confer protection on various normal tissues without attenuating anti-tumor response. When administered prior to chemotherapy or radiotherapy, it may provide a broad spectrum of cytoprotection including against alkylating drugs. The mechanism of protection resides in the metabolism at normal tissue site by membrane-bound alkaline phosphatase. Toxicity of this drug is moderate with hypotension, nausea and vomiting, and hypocalcemia being observed. We report a phase II study using amifostine as a protective drug against high-dose cyclophosphamide (HDCY) (7 g/m2), used to mobilize peripheral blood progenitor cells (PBPC) and to reduce tumor burden. We enrolled 29 patients, 22 (75.9%) affected by aggressive and 7 (24.1%) by indolent non-Hodgkin's lymphoma (NHL), who were submitted to 58 infusions of amifostine and compared them with a historical group (33 patients) affected by aggressive NHL and treated with VACOP-B followed by HDCY. The most important results in favor of amifostine were the reduction of intensity of cardiac, pulmonary and hepatic toxicity, and a significant reduction of frequency and severity of mucositis (P = 0.04). None of the 29 patients died in the protected group, while in the historical group 2/33 patients died because of cardiac or pulmonary toxicity and 2 patients stopped therapy due to toxicity. Amifostine did not prevent the aplastic phase following HDCY. PBPC collection and hematological recovery were adequate in both groups. The number of CFU-GM (colony-forming units-granulocyte/macrophage) colonies and mononuclear cells in the apheresis products was significantly higher in the amifostine group (P = 0.02 and 0.01, respectively). Side effects were mild and easily controlled. We conclude that amifostine protection should be useful in HDCY to protect normal tissues, with acceptable side effects.

The starch from Solanum lycocarpum St. Hill. fruit is not a hypoglycemic agent

We have investigated the hypoglycemic effect induced by the starch obtained from the unripe fruits of Solanum lycocarpum (Solanaceae). Per os administration of the starch (1000 or 2000 mg/kg, twice daily for 7 days, N = 6) did not change glycemia levels of nondiabetic female Swiss mice weighing 25-30 g. In streptozotocin-induced diabetic mice, similar treatment with the starch did not change the elevated glycemia 3 h after the last dose (diabetic treated with saline = 288 ± 17/309 ± 18; starch 1000 mg/kg = 295 ± 33; starch 2000 mg/kg = 258 ± 37; N = 5). In animals fasted for 15 h, per os administration of glucose (600 mg/kg) significantly increased glycemia 1 h later. Previous (-30 min) treatment of the animals with the starch (1000 or 2000 mg/kg; N = 5) did not change the increase of glycemia. Per os administration of the starch (1000 or 2000 mg kg-1 day-1, twice daily for 7 days) did not induce body weight gain or loss. The chemical analysis of the starch indicated the presence of glycoalkaloids, a finding that represents a reason for concern since many of these substances are generally toxic. In interviews with 56 diabetic patients, 29 medicinal plants were reported as useful in their treatment of diabetes and S. lycocarpum was the sixth most frequently mentioned. All patients interviewed reported that they also used insulin or oral hypoglycemic drugs. The results of the present study do not provide evidence for a hypoglycemic effect associated with the polysaccharide fraction of S. lycocarpum in either normal or hyperglycemic mice. These data demonstrate the need for adequate pharmacological investigation of the natural products widely used in folk medicine.

Non-obese adult onset diabetes with oral hypoglycemic agent failure: islet cell autoantibodies or reversible beta cell refractoriness?

Pancreatic ß cell function and insulin sensitivity, analyzed by the homeostasis model assessment, before and after 24 weeks of insulin therapy were studied and correlated with the presence of autoantibodies against ß cells (islet cell and anti-glutamic acid decarboxylase antibodies), in a group of 18 Brazilian lean adult non-insulin-dependent diabetes mellitus (NIDDM) patients with oral hypoglycemic agent failure (OHAF). Median fasting plasma glucose before and after insulin treatment was 19.1 and 8.5 mmol/l, respectively (P < 0.001); median HbA1c was 11.7% before vs 7.2% after insulin treatment (P < 0.001). Forty-four percent of the patients were positive (Ab+) to at least one autoantibody. Fasting C-peptide levels were lower in Ab+ than Ab- patients, both before (Ab+: 0.16 ± 0.09 vs Ab-: 0.41 ± 0.35 nmol/l, P < 0.003) and after insulin treatment (Ab+: 0.22 ± 0.13 vs Ab-: 0.44 ± 0.24 nmol/l, P < 0.03). Improvement of Hß was seen in Ab- (median before: 7.3 vs after insulin therapy: 33.4%, P = 0.003) but not in Ab+ patients (median before: 6.6 vs after insulin therapy: 20.9%). These results show that the OHAF observed in the 18 NIDDM patients studied was due mainly to two major causes: autoantibodies and ß cell desensitization. Autoantibodies against ß cells could account for 44% of OHAF, but Ab- patients may still present ß cell function recovery, mainly after a period of ß cell rest with insulin therapy. However, the effects of ß cell function recovery on the restoration of the response to oral hypoglycemic agents need to be determined.

Assessment of tidal volume and thoracoabdominal motion using volume and flow-oriented incentive spirometers in healthy subjects

The objective of the present study was to evaluate incentive spirometers using volume- (Coach and Voldyne) and flow-oriented (Triflo II and Respirex) devices. Sixteen healthy subjects, 24 ± 4 years, 62 ± 12 kg, were studied. Respiratory variables were obtained by respiratory inductive plethysmography, with subjects in a semi-reclined position (45º). Tidal volume, respiratory frequency, minute ventilation, inspiratory duty cycle, mean inspiratory flow, and thoracoabdominal motion were measured. Statistical analysis was performed with Kolmogorov-Smirnov test, t-test and ANOVA. Comparison between the Coach and Voldyne devices showed that larger values of tidal volume (1035 ± 268 vs 947 ± 268 ml, P = 0.02) and minute ventilation (9.07 ± 3.61 vs 7.49 ± 2.58 l/min, P = 0.01) were reached with Voldyne, whereas no significant differences in respiratory frequency were observed (7.85 ± 1.24 vs 8.57 ± 1.89 bpm). Comparison between flow-oriented devices showed larger values of inspiratory duty cycle and lower mean inspiratory flow with Triflo II (0.35 ± 0.05 vs 0.32 ± 0.05 ml/s, P = 0.00, and 531 ± 137 vs 606 ± 167 ml/s, P = 0.00, respectively). Abdominal motion was larger (P < 0.05) during the use of volume-oriented devices compared to flow-oriented devices (52 ± 11% for Coach and 50 ± 9% for Voldyne; 43 ± 13% for Triflo II and 44 ± 14% for Respirex). We observed that significantly higher tidal volume associated with low respiratory frequency was reached with Voldyne, and that there was a larger abdominal displacement with volume-oriented devices.

Balance between education- and research-oriented publications from a Brazilian University Hospital

We analyzed the trends of scientific output of the University Hospital, Federal University of Rio de Janeiro. A total of 1420 publications were classified according to pattern and visibility. Most were non-research publications with domestic visibility. With time, there was a tendency to shift from non-research (or education-oriented) publications with domestic visibility to research publications with international visibility. This change may reflect new academic attitudes within the institution concerning the objectives of the hospital and the establishment of scientific research activities. The emphasis of this University Hospital had been on the training of new physicians. However, more recently, the production of new knowledge has been incorporated as a new objective. The analysis of the scientific production of the most productive sectors of the hospital also showed that most are developing non-research studies devoted to the local public while a few of the sectors are carrying out research studies published in journals with international status. The dilemma of quality versus quantity and of education versus research-oriented publication seems, however, to continue to exist within the specialized sectors. The methodology described here to analyze the scientific production of a university hospital can be used as a tool to better understand the evolution of medical research in Brazil and also to help formulate public policies and new strategies to include research among the major objectives of University Hospitals.

Evasion of immune responses by Trypanosoma cruzi, the etiological agent of Chagas disease

Infection with the protozoan parasite Trypanosoma cruzi leads to Chagas disease, which affects millions of people in Latin America. Infection with T. cruzi cannot be eliminated by the immune system. A better understanding of immune evasion mechanisms is required in order to develop more effective vaccines. During the acute phase, parasites replicate extensively and release immunomodulatory molecules that delay parasite-specific responses mediated by T cells. This immune evasion allows the parasite to spread in the host. In the chronic phase, parasite evasion relies on its replication strategy of hijacking the TGF-β signaling pathway involved in inflammation and tissue regeneration. In this article, the mechanisms of immune evasion described for T. cruzi are reviewed.

Evaluation of 99mTc-HYNIC-βAla-Bombesin(7-14) as an agent for pancreas tumor detection in mice

Pancreatic adenocarcinoma is important in oncology because of its high mortality rate. Deaths may be avoided if an early diagnosis could be achieved. Several types of tumors overexpress gastrin-releasing peptide receptors (GRPr), including pancreatic cancer cells. Thus, a radiolabeled peptide derivative of gastrin-releasing peptide (GRP) may be useful as a specific imaging probe. The purpose of the present study was to evaluate the feasibility of using99mTc-HYNIC-βAla-Bombesin(7-14)as an imaging probe for Capan-1 pancreatic adenocarcinoma. Xenographic pancreatic tumor was developed in nude mice and characterized by histopathological analysis. Biodistribution studies and scintigraphic images were carried out in tumor-bearing nude mice. The two methods showed higher uptake by pancreatic tumor when compared to muscle (used as control), and the tumor-to-muscle ratio indicated that99mTc-HYNIC-βAla-Bombesin(7-14)uptake was four-fold higher in tumor cells than in other tissues. Scintigraphic images also showed a clear signal at the tumor site. The present data indicate that99mTc-HYNIC-βAla-Bombesin(7-14)may be useful for the detection of pancreatic adenocarcinoma.

Potential of quixaba (Sideroxylon obtusifolium) latex as a milk-clotting agent

There are several obstacles to the use of chymosin in cheese production. Consequently, plant proteases have been studied as possible rennet substitutes, but most of these enzymes are unsuitable for the manufacture of cheese. The aim of this study was to evaluate the potential of latex from Sideroxylon obtusifolium as a source of milk-clotting proteases and to partially characterize the enzyme. The enzyme extract showed high protease and coagulant activities, with an optimal pH of 8.0 and temperature of 55 °C. The enzyme was stable in wide ranges of temperature and pH. Its activity was not affected by any metal ions tested; but was inhibited by phenylmethanesulfonyl fluoride and pepstatin. For the coagulant activity, the optimal concentration of CaCl2 was 10 µmol L- 1. Polyacrylamide gel electrophoresis showed four bands, with molecular weights between 17 and 64 kDa. These results indicate that the enzyme can be applied to the cheese industry.

Social control of public expenditures in a multilevel principal-agent approach

ABSTRACTThis study enhances the principal-agent model by incorporating a multilevel perspective and differences among agency situations. A theoretical discussion is developed using a proposed intersection of methodological focuses and a descriptive-exemplificative hypothetical analysis. The analysis is applied to public expenditure social control in representative democracies, and as a result, a principal-agent model unfolds that incorporates a decision-making perspective and focuses on formulation, negotiation, articulation, and implementation competencies. Thus, it is possible to incorporate elements into the principal-agent model to make it more permeable to individual, group, and societal idiosyncrasies with respect to public expenditure social control.