The Kunitz chymotrypsin inhibitor from Erythrina velutina seeds displays activity against HeLa cells through arrest in cell cycle.

Erythrina velutina is a species of arboreal leguminous that occurs spontaneously in the northeastern states of Brazil. Leguminous seeds represent an abundant source of peptidase inhibitors, which play an important role in controlling peptidases involved in essential biological processes. The aim of this study was to purify and characterize a novel Kunitz-type peptidase inhibitor from Erythrina velutina seeds and evaluate its anti-proliferative effects against cancer cell lines. The Kunitz-type chymotrypsin inhibitor was purified from Erythrina velutina seeds (EvCI) by ammonium sulphate fractionation, trypsin- and chymotrypsin-sepharose affinity chromatographies and Resource Q anion-exchange column. The purified EvCI has a molecular mass of 18 kDa with homology to a Kunitz-type inhibitor. Inhibition assays revealed that EvCI is a competitive inhibitor of chymotrypsin (with K i of 4 × 10-8 M), with weak inhibitory activity against human elastase and without inhibition against trypsin, elastase, bromelain or papain. In addition, the inhibitory activity of EvCI was stable over a wide range of pH and temperature. Disulfide bridges are involved in stabilization of the reactive site in EvCI, since the reduction of disulfide bridges with DTT 100 mM abolished ~ 50% of its inhibitory activity. The inhibitor exhibited selective anti-proliferative properties against HeLa cells. The incubation of EvCI with HeLa cells triggered arrest in the cell cycle, suggesting that apoptosis is the mechanism of death induced by the inhibitor. EvCI constitutes an interesting anti-carcinogenic candidate for conventional cervical cancer treatments employed currently. The EvCI cytostatic effect on Hela cells indicates a promised compound to be used as anti-carcinogenic complement for conventional cervical treatments employed currently.

Translation and linguistic validation of the EORTC QLQ-PAN26 questionnaire for assessment of health-related quality of life in patients with pancreatic cancer and chronic pancreatitis into isiXhosa and Afrikaans.

BACKGROUND: Health-related quality of life (HRQOL) parameters have become important components in the holistic management of pancreatic ductal adenocarcinoma (PDAC) and are now increasingly incorporated in treatment protocols. The European Organisation for Research and Treatment of Cancer (EORTC) pancreatic cancer specific questionnaire (QLQ-PAN26) has also been validated for chronic pancreatitis (CP). The objective was to translate the EORTC QLQPAN26 questionnaire into and validate it for isiXhosa and Afrikaans. METHODS: Following the EORTC translation procedure, two forward translations of the English version into isiXhosa and Afrikaans were performed independently by two language practitioners for each language, followed by reconciliation of disagreements. A back translation of the reconciled version into English by a second pair of language practitioners was done. The results of all the steps were summarised with comments in a report for review by the EORTC translation unit. After proofreading by an external proof-reader chosen by the translation unit, pilot testing was performed on a cohort of ten isiXhosa patients and ten Afrikaans patients with PDAC or chronic pancreatitis. Results were summarised in a pilottesting report, and the final version approved by the translation unit. RESULTS: Thirteen patients diagnosed with PDAC and seven with CP were included in the study. The questionnaire was completed electronically (n = 12) or on paper (n = 8). Median age in the isiXhosa group was 53.7 (range 41-63) and in the Afrikaans group 60.9 (range 35-79). Questions 31-54 had a 100% completion rate, while 35% of respondents did not complete Q55 and Q56. Internal consistency was satisfactory in isiXhosa (alpha = 0.88) and Afrikaans (alpha = 0.89). CONCLUSION: The EORTC QLQ-PAN26 used in patients with PDAC and CP has been translated and linguistic validation performed in isiXhosa and Afrikaans. Availability of a questionnaire in patients' mother tongue should increase the validity of results.

CT colonography versus colonoscopy in the follow-up of patients after diverticulitis - a prospective, comparative study.

AIM: To assess whether computed tomography colonography (CTC) is a viable alternative to colonoscopy or double contrast barium enema in the follow-up of patients after diverticulitis. MATERIAL AND METHODS: Fifty patients underwent CTC followed immediately by colonoscopy. Results were blinded to the examiners. Findings of diverticular disease and patient acceptance were evaluated. RESULTS: Bowel preparation and distension were good in the majority of CTC and colonoscopy examinations. Diverticular disease was found in 96% of patients at CTC and in 90% at colonoscopy. The rate of agreement between CTC and colonoscopy for diverticular findings in the sigmoid colon was good (kappa=0.64). No complications were seen. Patients found colonoscopy more uncomfortable (p<0.03), more painful (p<0.001), and more difficult (p<0.01) than CTC. Of the patients favouring one examination, 74% preferred CTC. CONCLUSION: CTC appears to have a better diagnostic potential for imaging of diverticular disease-specific findings, when compared with colonoscopy. Also, CTC was less uncomfortable and was preferred by a majority of patients. CTC seems to be a reasonable alternative in follow-up of patients with symptomatic diverticular disease.

[Confirmatory information search in simultaneous vs. sequential information presentation]. / Konfirmatorische Informationssuche bei simultaner vs. sequentieller Informationsvorgabe.

After having made a preliminary or final decision, people prefer information that supports their chosen alternative to information that conflicts with their choice. Jonas, Schulz-Hardt, Frey, and Thelen (in press) found that sequential presentation of information leads to an even stronger preference for supporting information than the traditional form of simultaneous presentation. Their proposed explanation for this effect was that sequential presentation induces a focus on the prior decision, thereby increasing commitment to this decision. The present experiment was designed to rule out an alternative explanation: Being repeatedly confronted with pieces of information to select from could induce the participants to search for more information than they consider to be necessary, and because less effort is required to process supporting information the additional information requests are predominantly for these supporting pieces of information. To test this alternative explanation, in the present experiment--as in the Jonas et al. (in press) experiments--simultaneous vs. sequential information presentation following a preliminary decision was manipulated. In contrast to the former experiments, this time the number of information requests was fixed: Participants in both conditions had to choose 8 out of 16 pieces of information. The results show that once again a stronger preference for supporting information arises when the information is presented sequentially compared to simultaneously. The alternative explanation mentioned above could thus be ruled out.

Insulin receptor in Aplysia neurons: characterization, molecular cloning, and modulation of ion currents.

We have isolated the cDNA for a tyrosine kinase receptor that is expressed in the nervous system of Aplysia californica and that is similar to the vertebrate insulin receptor. Binding studies and immunocytochemical staining show that the receptor is abundant in the bag cell neurons. Application of vertebrate insulin to clusters of bag cell neurons stimulates the phosphorylation of the receptor on tyrosine residues, and exposure of isolated bag cell neurons to insulin produces an increase in height and a decrease in duration of the action potentials that can be detected within 15-30 min. These effects were not seen with insulin-like growth factor-1. In voltage-clamped neurons, insulin produces an increase in the amplitude of the voltage-dependent Ca2+ current that can be blocked by preincubation with herbimycin A, an inhibitor of tyrosine kinases. Insulin also enhances a delayed K+ current. We suggest that insulin-like peptides regulate the excitability of the bag cell neurons.

The effect of parenteral lipid emulsion-induced hyperlipidemia on prostaglandin E1 modulation of platelet function.

The purpose of this study is to better understand how hyperlipidemia alters the modulating action of prostaglandin E1 (PGE1) on platelet function. Using our previously characterized rat model of atherogenesis, we demonstrate that the parenteral lipid emulsions, Lipofundin-S and Liposyn, significantly (p < or = 0.05) enhance baseline platelet aggregation. In addition, dose response curves show that in all animals, PGE1 substantially inhibits platelet aggregation at 10(-7) to 10(-6) M, while significantly stimulating platelet function at lower doses. However, at all PGE1 concentrations, aggregation values are higher in platelets from lipid-treated vs. control rats, showing that hyperlipidemia significantly reduces the ability of high concentrations of PGE1 to inhibit platelet activity, based on the absolute values of the controls. Also, dose response curves for PGE1 on platelet aggregation show a marked similarity in shape for control ratsvs. normal humans. Thus, this study demonstrates that hyperlipidemia significantly alters the platelet modulating action of prostaglandin E1, and it shows that PGE1 can either inhibit or stimulate platelet activity in both rat and human platelets.

The effect of atherogenic infusions of the triglyceride-rich, lipid emulsion, Lipofundin-S, on the in vitro growth characteristics of rat aortic smooth muscle cells.

This study shows that arterial smooth muscle cells (SMC) isolated from rats receiving atherogenic doses of the lipid emulsion, Lipofundin-S, alter their in vitro growth properties. Compared to cells from control animals, SMC isolated from Lipofundin-S-infused rats show a reduction in both saturation density and response to increasing serum concentrations, without a change in the baseline proliferation. Also, SMC isolated from lipid-treated animals and grown for five days in the presence of 30, 150, or 300 pg/ml estradiol show a 30% increase in growth vs. cells from controls. Epinephrine at 1 microM stimulates growth in SMC from control rats, while causing no growth enhancement over five days in cells from lipid-infused animals. Thus, atherogenic infusions of Lipofundin-S into rats cause phenotypic changes in arterial SMC which can be passed to successive cell generations in vitro.

Parenteral lipid emulsion-induced atherosclerosis in the obese Zucker rat and its lean littermate.

We have shown previously that parenterally-administered lipid emulsions can be utilized to induce early atherosclerosis in the aortas of Sprague-Dawley rats. In order to evaluate the effect of obesity on lipid-induced atherogenesis, we have utilized this same approach in the present study to demonstrate that i.v. infusions of the parenteral lipid emulsion, Lipofundin-S, will induce in the genetically obese Zucker rat and its lean littermate aortic endothelial and myofibroelastic changes indicative of early atherogenesis. Four groups of rats were used: 1) obese controls, 2) obese lipid-infused, 3) lean littermate controls, and 4) lean littermate lipid-infused. Observations were made with light and transmission electron microscopy (TEM), using qualitative morphological criteria to evaluate the results. Based on the fact that both untreated control and Lipofundin-S-induced atherosclerosis was more frequent and generally more advanced in the obese animals than in their respective lean counterparts, it appears that the obese Zucker rat is more susceptible to both spontaneous and hyperlipidemia-induced atherosclerosis than its respective lean littermate. Thus, obesity in these animals, as might be the case in humans, could potentiate an atherogenic process already enhanced by hyperlipidemia.

Platelet aggregability in rats with early atherosclerotic changes induced by parenterally-administered lipid emulsions.

The present study is the first work to evaluate thrombin-, ADP-, and collagen-induced platelet aggregation in laboratory rats receiving alimentation with the parenterally-administered lipid emulsion, Lipofundin-S, in doses sufficient to induce early atherosclerotic changes in the aorta. The aggregometry parameters of percent maximum aggregation, slope, and b2 or b20 almost uniformly indicate that such lipid treatments result in a statistically significant increased sensitivity of the platelets to ADP and collagen, while no change is noted with thrombin as the aggregating agent. By varying the amounts of ADP and collagen during aggregometry, we also demonstrate that the concentrations of these reagents necessary for equivalent platelet aggregation is substantially lower in lipid-infused rats than in controls. We conclude from this study that such lipid infusions can cause increased platelet aggregation, and that these lipids probably act in a synergistic fashion by affecting a variety of components which comprise the atherogenic process and its clinical endpoint. In addition, we believe that this experimental approach is of interest in that infusions of clinically-useful lipid emulsions are easily controlled, while alterations in platelet physiology and aortic structure occur concurrently and rapidly.

Induction of early atherosclerosis in rats using parenterally-administered lipid emulsions.

It has been demonstrated by us and other workers that rats receiving I.V. infusions of Lipofundin-S will develop aortic changes indicative of early atherosclerosis. However, different lipid emulsions which are used in the clinical setting for parenteral nutrition vary substantially in chylomicron size and fatty acid composition. Therefore, in an attempt to better understand the mechanism by which a lipid emulsion might induce vessel lesions, we compared the nature of potential aortic changes resulting from infusions of Liposyn, Intralipid, or Lipofundin-S into the tail veins of Sprague-Dawley rats. Three groups of animals received either Liposyn (N = 10), Intralipid (N = 5), or Lipofundin-S (N = 9) at the rate of 6 g fat/kg body wt/day for 10 consecutive days. A fourth group (N = 5) received saline in equivalent dose to evaluate the effect of injection volume on vessel lesion formation. The other controls (N = 6) received no injections. Rats were sacrificed 24 hrs after the last infusion, and 1 mm rings from the top of the aortic arch and proximal third of the thoracic aorta were prepared for transmission electron microscopy (TEM). Examination by TEM allows two main conclusions to be drawn for both segments of the aorta. First, all three emulsions are capable of inducing early vessel changes which include endothelial damage, platelet adherence to damaged endothelium or subendothelial collagen, intimal phagocytic cells, and intimal smooth muscle cells surrounded by collagen bundles and elastin plates. Saline-infused rats only show occasional subendothelial swelling. None of the above-described changes are seen in any of the uninjected controls. Second, Lipofundin-S induces smooth muscle penetration of the intima in 7 of 9 rats, while Liposyn causes such changes in 2 of 10 animals. This difference in the efficiency with which the two emulsions induce the most advanced changes is statistically significantly by Chi Square (p less than 0.05). Intralipid produces smooth muscle penetration of the intima in 2 of 5 rats. The composition of the three emulsions suggests that the lower percent of linoleic acid and larger chylomicron size in Lipofundin-S may account for these differences, at least in part.