Maternal undernutrition around the time of conception and embryo number each impact on the abundance of key regulators of cardiac growth and metabolism in the fetal sheep heart.

Poor maternal nutrition before and during pregnancy is associated with an increased risk of cardiovascular disease in later life. To determine the impact of maternal undernutrition during the periconceptional (PCUN: -45 days to 6 days) and preimplantation (PIUN: 0-6 days) periods on cardiac growth and metabolism, we have quantified the mRNA and protein abundance of key regulators of cardiac growth and metabolism in the left ventricle of the sheep fetus in late gestation. The cardiac protein abundance of AMP-activated protein kinase (AMPK), phospho-acetyl CoA carboxykinase (ACC) and pyruvate dehydrogenase kinase-4 (PDK-4) were decreased, whereas ACC was increased in singletons in the PCUN and PIUN groups. In twins, however, cardiac ACC was decreased in the PCUN and PIUN groups, and carnitine palmitoyltransferase-1 (CPT-1) was increased in the PIUN group. In singletons, the cardiac abundance of insulin receptor ß (IRß) was decreased in the PCUN group, and phosphoinositide-dependent protein kinase-1 (PDPK-1) was decreased in the PCUN and PIUN groups. In twins, however, the cardiac abundance of IRß and phospho-Akt substrate 160kDa (pAS160) were increased in the PIUN group. The cardiac abundance of insulin-like growth factor-2 receptor (IGF-2R), protein kinase C alpha (PKCα) and mammalian target of rapamycin (mTOR) were decreased in PCUN and PIUN singletons and extracellular-signal-regulated kinase (ERK) was also decreased in the PIUN singletons. In contrast, in twins, cardiac abundance of IGF-2R and PKCα were increased in the PCUN and PIUN groups, phospho-ribosomal protein S6 (pRPS6) was increased in the PCUN group, and ERK and eukaryotic initiation factor 4E (eIF4E) were also increased in the PIUN fetuses. In conclusion, maternal undernutrition limited to around the time of conception is sufficient to alter the abundance of key factors regulating cardiac growth and metabolism and this may increase the propensity for cardiovascular diseases in later life.

Effect of Cymbopogon citratus and Citral on Vascular Smooth Muscle of the Isolated Thoracic Rat Aorta.

Cymbopogon citratus has been shown to have antioxidant, antimicrobial, antispasmodic and chemo-protective properties. Citral, is the major constituent of C. citratus. This study investigated the effects of methanolic extracts of leaves (LE), stems (SE), and roots (RE) of C. citratus and citral on vascular smooth muscle and explored their possible mechanisms of action. The experiment was conducted using isolated tissue preparations, where citral, LE, SE, and RE were added separately into a tissue bath that contained aortic rings, which were pre-contracted with phenylephrine (PE). Citral, LE, and RE exhibited a dose-dependent relaxant effect on the PE-induced contractions. Citral appeared to partially act via NO as its vasorelaxant effect was attenuated by L-NAME. However, the effect of LE may involve prostacyclin as indomethacin reversed the relaxant effect of LE on the PE-induced contraction. Furthermore, citral, LE, and RE abolished the restoration of PE-induced contraction caused by the addition of increasing doses of calcium in both endothelium intact and denuded rings. These findings suggest that the relaxation effect of citral, LE, and RE is endothelium-independent and may be mainly by affecting the intracellular concentration of calcium. Citral may partially act through the NO pathway while a vasodilator prostaglandin may mediate the effect of LE.

Enzymatic and toxinological activities of Hypnale hypnale (hump-nosed pit viper) venom and its fractionation by ion exchange high performance liquid chromatography

Hypnale hypnale (hump-nosed pit viper) has been recently identified as one of the medically important venomous snakes in Sri Lanka and on the southwestern coast of India. The characterization of its venom is essential for understanding the pathophysiology of envenomation and for optimizing its management. In the present study, the biological properties of Hypnale hypnale venom and venom fractions obtained using Resource Q ion exchange chromatography were determined. The venom exhibited toxic activities typical of pit viper venom, comparable to that of its sister taxon, the Malayan pit viper (Calloselasma rhodostoma). Particularly noteworthy were its high activities of thrombin-like enzyme, proteases, phospholipase A2, L-amino acid oxidase and hyaluronidase. The thrombin-like enzyme was mainly acidic and distributed over several chromatography fractions, indicating its existence in multiple isoforms. The hemorrhagic and necrotic activities of the venom were likely associated with the proteolytic enzyme found mainly in the basic fraction. Phospholipase A2 and phosphomonoesterase exist in both acidic and basic isoforms, while L-amino acid oxidase and hyaluronidase are highly acidic. The venom clotting activity on fibrinogens showed distinct species specificity in the following increasing order for clotting time: bovine < rabbit < goat < human < horse < < dog, and was comparable to that of C. rhodostoma venom. Its clot formation on human fibrinogen is gradual and prolonged, a phenomenon suggestive of consumptive coagulopathy as a complication observed clinically. At an intramuscular sublethal dose, the venom did not cause acute kidney injury in a rodent model, contrary to the positive control group treated with Daboia russelii venom. Nephrotoxicity may result from higher venom doses in the context of coagulopathy, as a complication provoked by venom hematoxicity.(AU)

Protective effects of Mucuna pruriens seed extract pretreatment against cardiovascular and respiratory depressant effects of Calloselasma rhodostoma (Malayan pit viper) venom in rats.

The protective effects of Mucuna pruriens seed extract (MPE) against the cardio-respiratory depressant and neuromuscular paralytic effects induced by injection of Calloselasma rhodostoma (Malayan pit viper) venom in anaesthetized rats were investigated. While MPE pretreatment did not reverse the inhibitory effect of the venom on the gastrocnemius muscle excitability, it significantly attenuated the venom-induced cardio-respiratory depressant effects (p < 0.05). The protection effects may have an immunological mechanism, as indicated by the presence of several proteins in the venom that are immunoreactive against anti-MPE. However, we cannot rule out the possibility that the pretreatment may exert a direct, non-immunological protective action against the venom.

The protective effects of Mucuna pruriens seed extract against histopathological changes induced by Malayan cobra (Naja sputatrix) venom in rats.

Seed of Mucuna pruriens (Velvet beans) has been prescribed by traditional medicine practitioners in Nigeria as a prophylactic oral antisnake remedy. In the present studies, we investigated the protective effects of M. pruriens seed extract (MPE) against histopathological changes induced by intravenous injection of Naja sputatrix (Malayan cobra) venom in rats pretreated with the seed extract. Examination by light microscope revealed that the venom induced histopathological changes in heart and blood vessels in liver, but no effect on brain, lung, kidney and spleen. The induced changes were prevented by pretreatment of the rats with MPE. Our results suggest that MPE pretreatment protects rat heart and liver blood vessels against cobra venom-induced damages.

Many roads lead to Rome--is that true of the teaching and learning of pharmacology? Lessons from three medical schools in Malaysia.

In Malaysia many new medical schools (both public and private) have been set up in the last 12 years. As a result of global changes and local adjustments made in medical training, cross-breeds of different medical curricula have produced a wide spectrum of teaching-learning methods in these medical schools. In this paper, we have selected three medical schools--two public (Universiti Malaya and Universiti Putra Malaysia) and one private (International Medical University) to illustrate different approaches in the teaching-learning of pharmacology that exist in Malaysia. How do these different teaching-learning approaches affect the students' interest and ability to "master" pharmacology and in turn to develop a good prescribing practice?

Encouraging learning how to fish: an uphill but worthwhile battle.

INTRODUCTION: Encouraging teaching practices such as problem-based learning (PBL) amongst undergraduate students within a lecture-based, system-based integrated curriculum is a challenge. Students are apprehensive about developing an organised framework for acquiring knowledge while lecturers are required to reframe their views on the educational process and their role as educators. MATERIALS AND METHODS: Lecturers and students in the Phase (Year) II programme were asked to fill questionnaires following the second and fourth PBL cases. The two sets of survey responses were compared to see whether the students' and teachers' perceptions had changed over the 5-month period. RESULTS: Students' responses from both surveys (1 and 2) were similar in that a majority agreed that the PBL tutorials had encouraged the seeking of information (66% and 67%, respectively), had improved understanding (57% and 56%), integration (65% and 70%) and application (50% and 64%) of knowledge. However, the views given in the form of written comments, following their positive responses, were somewhat contradictory. A large number of students (38% and 40%) faced difficulties in getting involved in discussions during the PBL tutorial and a majority (73% and 82%) preferred the normal subject-based tutorials. The reasons given by approximately 20% of the students were that the subject-based tutorials were more efficient for obtaining information and/or that the information had been pre-selected by the lecturers. More than 80% of the lecturers (in both surveys) perceived that the students had identified the appropriate learning objectives and covered the subject matter. The percentage of lecturers who agreed that PBL tutorials encouraged rapport and teamwork amongst students had increased in the second survey, from 70% to 92% and 55% to 83% respectively. CONCLUSION: Implementing PBL is not simply a matter of developing new teaching materials and new effective ways of presenting them. It requires a paradigm shift, a change in the roles of students and teachers, and time.

Effect of ribavirin on zidovudine efficacy and toxicity in vitro: a concentration-dependent interaction.

Zidovudine (ZDV) is converted to its active triphosphate (ZDVTP) by intracellular kinases. The intermediate ZDV monophosphate (ZDVMP) is believed to play a major role in ZDV toxicity. Manipulation of ZDV phosphorylation is a possible therapeutic strategy for altering the risk-benefit ratio. Here we investigate whether combining RBV with ZDV is able to modulate efficacy and toxicity of ZDV. We have measured the intracellular activation of ZDV (0.3 microM) in the absence and presence of ribavirin (RBV; 2 and 20 microM) in Molt 4 and U937 cells. MTT cytotoxicity of ZDV (10-1000 microM) was also measured with and without RBV (2 microM) in Molt 4 and U937 cells. Measurement of endogenous deoxythymidine triphosphate (dTTP) allowed investigation of the dTTP/ZDVTP ratio. The antiviral efficacy of ZDV in combination with RBV (2 microM) was assessed by HIV p24 antigen measurements. In the presence of RBV (2 and 20 microM) a decrease in total ZDV phosphates was observed, owing mainly to an effect primarily on ZDVMP rather than the active ZDVTP. RBV also increased endogenous dTTP pools in both cell types, resulting in an increase in the dTTP/ZDVTP ratio. ZDV alone significantly reduced p24 antigen production, with an IC50 of 0.34 microM. Addition of RBV increased the IC50 approximately fivefold (1.52 microM). However, at higher concentrations of ZDV (10 and 100 microM) the antagonistic effect of RBV (2 microM) on ZDV was lost. The RBV-mediated decrease in ZDVMP may explain the reduction in ZDV toxicity when combined with RBV (2 microM). Cytotoxicity of ZDV was reduced in the presence of RBV (2 microM) at all concentrations in both cell lines, probably owing to saturation of ZDVTP formation. The interaction of ZDV and RBV is concentration dependent.

Allergens of Bipolaris species.

Skin prick tests done previously revealed a significantly higher percentage of sensitization to an extract of Bipolaris sp. among atopic individuals (34/147, 23.1%) compared to non-atopic individuals. Bipolaris-specific IgE levels were quantified in sera from a representative group of 38 individuals using the Fluorescence Allergosorbent Test (FAST). Result obtained by FAST were found to be comparable to the skin prick test results (r2 = 0.60, p < 0.001 for IgE levels vs wheal sizes; r2 = 0.44, p < 0.001 for IgE levels vs erythema sizes). Characterisation of the extract's allergenic component by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed 28 protein bands with molecular weights (MW) ranging from 11 kDa to above 100 kDa. Immunoblotting with sera of 10 Bipolaris-sensitive (skin prick test, 3 +) individuals showed that Bipolaris spore extract contained at least 4 IgE binding proteins (MW 11-13 kDa, 16-17 kDa, 20-22 kDa and 36 kDa). All 10 sera reacted to the protein at MW 20-22 kDa, 2 sera with MW 11-13 kDa, 3 sera with 16-17 kDa and 6 sera with 36 kDa. This study has thus demonstrated that spores of Bipolaris sp. contain allergenic components which may elicit IgE-mediated reactions.

The effect of various drugs on the glucuronidation of zidovudine (azidothymidine; AZT) by human liver microsomes.

1. Zidovudine (3'-azido-3'-deoxythymidine; AZT) is the drug of proven efficacy available for the treatment of patients with AIDS or ARC. It is eliminated mainly by hepatic glucuronidation. Therefore, interference with this metabolic pathway may lead to enhancement of AZT effect or to increased toxicity of the drug. We have examined the effect of a number of drugs which themselves undergo glucuronidation on AZT conjugation by human liver microsomes in vitro. 2. AZT glucuronidation followed Michaelis-Menten kinetics. The apparent Km and Vmax values (mean +/- s.d., n = 5), were 2.60 +/- 0.52 mM and 68.0 +/- 23.4 nmol h-1 mg-1, respectively, as determined from Eadie-Hofstee plots. 3. Dideoxyinosine, sulphanilamide and paracetamol were essentially non-inhibitory at concentrations up to 10 mM (4 times the concentration of AZT in the incubation). The most marked inhibitory effects were seen with indomethacin, naproxen, chloramphenicol, probenecid and ethinyloestradiol, with enzyme activity decreased by 97.7, 94.9, 88.7, 83.4% and 79.0%, respectively, at a concentration of 10 mM. Other compounds producing some inhibition of AZT conjugation were oxazepam, salicylic acid and acetylsalicylic acid. 4. Further studies are necessary to characterise the inhibition observed but the method described enables a screen of potentially important drug interactions to be carried out.

Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ--II. Studies with the Doluisio technique.

The absorption of oestrone (E1), oestrone glucuronide (E1G) and oestrone sulphate (E1S) from the small intestine of anaesthetized rats has been evaluated using the Doluisio in situ technique. Luminal disappearance of E1 was biphasic, which is consistent with a 3-compartment model; t1/2 alpha (first phase) was less than 5 min and t1/2 beta (second phase) approx 27 min for each concentration of steroid studied (trace identical to 10 nM, 1 microM and 10 microM). In contrast, luminal disappearance of E1G and E1S was monoexponential; t1/2 for E1G was 159, 229 and 299 min (trace identical to 200 nM, 10 microM and 100 microM respectively) and for E1S, 215, 174 and 192 min (trace identical to 10 nM, 10 microM and 100 microM respectively). There was a good correlation between the luminal disappearance data and recovery of steroid in bile. Adsorption of E1S was estimated from the initial rapid fall in luminal content within the first 5 min after drug administration. The study provides further evidence that E1S can be absorbed intact. Since saccharolactone only caused a reduction in E1G absorption of 32% we also conclude that part of the administered E1G was absorbed intact.

Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ--I. Importance of hydrolytic enzymes on conjugate absorption.

The biliary excretion of steroid after administration of [3H]oestrone ([3H]E1), [3H]oestrone glucuronide ([3H]E1G) and [3H]oestrone sulphate ([3H]E1S) into the hepatic portal vein of anaesthetized rats was very rapid with more than 70% of E1S and greater than 80% of E1 and E1G excreted in the first 30 min. There was a lag period in the biliary excretion of E1S, this was less apparent with E1 and absent with E1G. Biliary excretion accurately reflects the amount of steroid in the portal circulation and was therefore used as an assessment of absorption from the gastrointestinal (GI) tract. Absorption (as judged by excretion in bile) was least after administration of each steroid into the stomach. The extent of absorption correlated well with the lipophilicity of the steroids as shown by their relative partition coefficients between n-octanol and pH 6.5 phosphate-buffered saline (E1 greater than or equal to E1S greater than or equal to E1G). There was no significant difference in excretion profile when the steroids were given into the caecum (at 5 h, E1, 46.3 +/- 9.1%; E1G, 42.2 +/- 14.5%; E1S, 39.9 +/- 7.1%). The similarity, despite marked differences in physicochemical properties, suggested conjugate hydrolysis to the parent steroid. In contrast, after administration into the small intestine, excretion of E1 was very rapid and was maximal at 1 h (72.5 +/- 8.0%); E1G showed a near-linear excretion rate (1 h, 14.4 +/- 3.0%; 5 h, 80.0 +/- 11.7%), whereas in comparison E1S excretion was low (1 h, 12.1 +/- 2.4%; 5 h, 36.9 +/- 2.7%). The involvement of hydrolytic enzymes in conjugate absorption was assessed. Ampicillin pretreatment (200 mg/kg/day for 2 days) reduced the absorption of E1G from both the proximal and distal small intestine (by approximately 50%) but had no effect on the absorption of E1S. There was, therefore, evidence that quantitative absorption of E1G requires prior hydrolysis (by mammalian and/or microbial enzymes) but intact absorption of E1S from this region of the tract was implicated. Ampicillin pretreatment reduced the absorption of both conjugates (greater with E1S) from the caecum; hydrolysis clearly precedes absorption from the caecum. The above findings were supported by an in vitro study which showed that ampicillin pretreatment abolished the hydrolysis of E1S by caecal contents but only partially reduced the hydrolysis of E1G. The presence of mammalian glucuronidase enzyme may account for this difference.

Distribution of estrone sulfatase activity in the intestine of germfree and conventional rats.

The intestinal content, the mucosa and the rest of the intestinal wall of germfree (GF) and conventional ( CVL ) rats were tested for in vitro hydrolysis of [3H]estrone sulfate. In homogenates from GF rat intestine some estrone sulfate hydrolysis was detected in those from the proximal small intestine (PSI) (4.2 +/- 0.1% hydrolyzed after 4 h), but not in those from the distal small intestine (DSI) and the caecum. Estrone sulfate was also hydrolyzed by the homogenates of the mucosa and the rest of the intestinal wall from each of the segments tested (PSI: 12.8 +/- 0.4% (mucosa) and 21.5 +/- 2.1 (wall); DSI: 8.2 +/- 0.9% (mucosa) and 17.3 +/- 1.7% (wall); caecum: 8.8 +/- 1.6% (mucosa) and 17.3 +/- 0.5% (wall) ). In the homogenates of CVL rat intestine, the estrone sulfatase activity in the rest of the intestinal wall did not differ considerably from the values for GF rats, when expressed per mg protein of the homogenate. The mucosa of the CVL rats, however, showed higher rates of hydrolysis than the mucosa of the GF rats. The microbial estrone sulfatase activity in the intestinal content of CVL rats, tested by anaerobic incubation, was high in the caecum (91.7 +/- 6.6% after 4 h), but very low in the PSI (2.2 +/- 0.7%) and DSI (1.3 +/- 0.5%). Serial dilutions of the caecal content also showed higher viable numbers of estrone sulfate hydrolyzing bacteria. These results add further weight to the suggestion that estrone sulfate may be absorbed from the small intestine, but has to be hydrolyzed in the caecum by the gut microflora prior to absorption.

Gastrointestinal absorption of estrone sulfate in germfree and conventional rats.

Steroids are extensively excreted in the bile of rats. There was no significant difference in biliary excretion of steroid following administration of [3H]-estrone sulfate into the proximal small intestine (PSI) of conventional (CVL; 17.8 +/- 62%; mean +/- SD) or germfree (GF; 28.2 +/- 5.3) rats. A similar finding resulted from administration into the distal small intestine (DSI)-CVL, 22.3 +/- 11.8%; GF, 11.4 +/- 3.7%. However, when the drug was given into the caecum, excretion in the bile of CVL rats after 5 h was 59.1% whereas in GF rats it was only 1.7%. When estrone was injected into the PSI and DSI of CVL and GF rats, absorption (as judged by excretion in bile) was more rapid than that seen with estrone sulfate. Five hours after injection into the PSI, biliary excretion was, in CVL 88.2% and in GF 81.7% and after injection into the DSI excretion was, in CVL 84.7% and in GF 83.6%. Absorption of estrone from the caeca of GF rats was apparently reduced (49.0% and 25.3% excreted in the bile of CVL and GF rats respectively). There was no significant difference in bile flow rate between CVL and GF rats. These results give unequivocal evidence of intact absorption of estrone sulfate from the small intestine of the rat. The rate of absorption is however very much reduced compared to the non-sulphated steroid. Estrone sulfate is not absorbed intact in the caecum but is hydrolysed by the gut microflora prior to absorption.