I. A. Tavares

Cytosolic phospholipase A2, cyclo-oxygenases and arachidonate in human stomach tumours.

Human stomach tumours usually form more prostaglandins (PGs) than their associated normal mucosa/submucosa, but the mechanisms are not fully understood. The key enzymes are cytosolic phospholipase A2 (cPLA2, Mr 85,000) and the cyclo-oxygenases (COXs) which exist in constitutive (COX-1) and inducible forms (COX-2). In human stomach tumours and associated macroscopically normal tissues, we determined the fatty acid composition by gas chromatography, amounts of cPLA2, COX-1 and COX-2 by immunoblotting with specific antibodies and cPLA2 enzyme activity using a tritiated substrate. Although compared to normal mucosa there was less arachidonate in tumours (P < 0.05), the arachidonate/total fatty acid ratio was higher. Mean amounts of cPLA2 and COX-1 and cPLA2 activity were similar in tumours and normal mucosa. However, substantial amounts of COX-2 were found in the tumours but not in the mucosa, which may explain why many gastric tumours form increased amounts of PGs.

Smoking, eicosanoids and ulcerative colitis.

Abstract— In this study, which is the first of its kind using normal tissue samples that are very difficult to obtain, we have investigated the hypothesis that smoking protects against ulcerative colitis by altering the colonic mucosal formation of prostaglandins and related substances. Colonic mucosa biopsied from healthy young men produced prostaglandin E, 6‐keto‐PGF1α (formed from PGI2), leukotriene B4 and leukotriene C4/D4/E4 as determined by radioimmunoassay. With each substance, the median yield was lower in the group of smokers who smoked 3 cigarettes in the 2 h before biopsy, than in the non‐smokers. However, with each eicosanoid the statistical probability approached only the 10% level, but the fact that the trend was the same for all eicosanoids somewhat strengthens the possibility of a real difference between the groups.

Prostate-derived Sterile 20-like Kinase 2 (PSK2) Regulates Apoptotic Morphology via C-Jun N-terminal Kinase and Rho Kinase-1

We have reported previously that human prostate-derived sterile 20-like kinase (PSK) 1 alters actin cytoskeletal organization and binds to microtubules, regulating their organization and stability. We have shown a structurally related protein kinase PSK2, which lacks a microtubule-binding site, activated c-Jun N-terminal kinase (JNK), and induced apoptotic morphological changes that include cell contraction, membrane blebbing, and apoptotic body formation. Apoptotic stimuli increased the catalytic activity of endogenous PSK2 and JNK, and dominant negative JNK or a physiological inhibitor of JNK blocked these apoptotic morphological responses to PSK2, demonstrating a requirement for JNK. PSK2 also stimulated the cleavage of Rho kinase-1 (ROCK-I), and the activity of ROCK-I was required for PSK2 to induce cell contraction and membrane blebbing. The activation of caspases was also needed for the induction of membrane blebbing by PSK2, which was itself a substrate for caspase 3. PSK2 therefore regulates apoptotic morphology associated with the execution phase of apoptosis, which involves dynamic reorganization of the actin cytoskeleton, via downstream targets that include JNK and ROCK-I. Our findings suggest that PSKs form a subgroup of sterile 20 (STE20)-like kinases that regulate different cytoskeletal processes.

High molecular weight phospholipase A2 and fatty acids in human colon tumours and associated normal tissue

Human colon tumours usually form more prostaglandins (PGs) than associated normal tissues, but the mechanism(s) are not fully understood. We analysed fatty acid compositions, in particular arachidonate, and measured the amount and the activity of high molecular weight cytoplasmic phospholipase A2 (cPLA2) of these tissues. Total lipids extracted from homogenised surgical specimens were transesterified and fatty acids were analysed by gas chromatography. cPLA2 was separated by SDS-PAGE, Western-blotted, immunoblotted using a specific antibody to cPLA2 and semiquantified following enhanced chemiluminescence using a scanning densitometer. cPLA2 biological activity was also assayed using 1-stearoyl, 2-[1-14C]-arachidonyl, L-3-phosphatidylcholine. Compared with normal mucosa/submucosa, there was more total arachidonate in tumours (P < 0.01), and increased levels of cPLA2 occurred in 6 of 17 tumours. In conclusion, the higher amounts of tumour total arachidonate and the sometimes higher levels of cPLA2, might help to explain why some human colon tumours form increased amounts of PGs.

Effect of lignocaine on eicosanoid synthesis by pieces of human gastric mucosa

Abstract— Lignocaine can affect prostaglandin synthesis in various tissues, and it has anti‐inflammatory activity. No studies have been made previously on human isolated gut tissues. When concentrations of 5, 50 and 250 μg mL−1 lignocaine were incubated with human gastric mucosa/submucosa at 37°C for 30 min, only the highest concentration reduced the levels of prostaglandin E, thromboxane B2 and 6‐keto‐PGF1α in the incubates, and leukotriene C4/D4 was unaffected. Therapeutically relevant amounts of lignocaine given parenterally would therefore seem unlikely to alter gastric mucosal prostanoids, but high doses can be given orally because of extensive first‐pass metabolism in the liver.

Prostate-derived sterile 20-like kinase 1-alpha induces apoptosis - JNK- and caspase-dependent nuclear localization is a requirement for membrane blebbing

We have demonstrated previously that full-length prostate-derived sterile 20-like kinase 1-α (PSK1-α) binds to microtubules via its C terminus and regulates their organization and stability independently of its catalytic activity. Here we have shown that apoptotic and microtubule-disrupting agents promote catalytic activation, C-terminal cleavage, and nuclear translocation of endogenous phosphoserine 181 PSK1-α and activated N-terminal PSK1-α-induced apoptosis. PSK1-α, unlike its novel isoform PSK1-β, stimulated the c-Jun N-terminal kinase (JNK) pathway, and the nuclear localization of PSK1-α and its induction of cell contraction, membrane blebbing, and apoptotic body formation were dependent on JNK activity. PSK1-α was also a caspase substrate, and the broad spectrum caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or mutation of a putative caspase recognition motif (916DPGD919) blocked nuclear localization of PSK1-α and its induction of membrane blebs. Additional inhibition of caspase 9 was needed to prevent cell contraction. PSK1-α is therefore a bifunctional kinase that associates with microtubules, and JNK- and caspase-mediated removal of its C-terminal microtubule-binding domain permits nuclear translocation of the N-terminal region of PSK1-α and its induction of apoptosis.

The effect of garlic extracts on contractions of rat gastric fundus and human platelet aggregation

Garlic has been extracted and separated chromatographically into various fractions which show different degrees of activity as inhibitors of platelet aggregation and smooth muscle. The most potent smooth muscle inhibitor fraction had little activity on platelet aggregation, but μg ml−1 concentrations greatly reduced the contractions of rat gastric fundus to prostaglandin E2 and acetylcholine. Material in this fraction may contribute to some of the claimed therapeutic effects of garlic involving smooth muscle. Its identity is not known, but is different from allyl sulphide, dimethyl sulphide and diallyl disulphide. These compounds eluted earlier on liquid chromatography than the most active fraction, and they showed only modest inhibitory activity against prostaglandin E2 and acetylcholine on rat fundus.

Effects of amide local anaesthetics on eicosanoid formation in burned skin

Bacground: Previous studies have demonstrated potent inhibition of burn oedema and progressive ischaemia by local anaesthetics. Since eicosanoids have been suggested to play an important role in the pathophysiology of burns, we compared in the present ex vivo study the effects of topical lidocaine/prilocaine cream (EMLA®, ASTRA, Sweden) and intravenous lidocaine with that of saline on eicosanoid formation by normal and burned rat skin.

Effect of the flavonoid galangin on urinary bladder rat contractility in-vitro

Galangin is a flavanol with several biological activities. We have evaluated the effect of galangin on the contractile response elicited by electrical field stimulation (EFS) in the rat isolated urinary bladder. Galangin (10−8‐ 10−4m) produced a concentration‐dependent inhibition of the EFS contractile response without modifying the contractions produced by exogenous acetylcholine (10−6 m). Blockade of adrenergic and cholinergic nerves with a combination of atropine (10−6 m), phentolamine (10−6 m) and propranolol (10−6 m) or blockade of tachykinin NK1 and NK2 receptors with SR140333 (10−7 m) and SR48968 (10−6 m) did not modify the inhibitory effect of galangin. However, verapamil (10−7 m) significantly reduced the inhibitory effect of galangin. It is concluded that the galangin inhibits EFS‐induced contractions of the rat urinary bladder by acting on L‐type calcium channels on presynaptic nerves.

Stimulation of Gastric and Colonic Mucosal Eicosanoid Synthesis by Plantain Banana

Abstract— Extracts of plantain banana (Musa sapientum Linn var. paradisiaca) were studied on the accumulation of eicosanoids in incubates of human gastric and colonic mucosa. The ethanolic extract caused a concentration‐dependent increase in the eicosanoid accumulation but the water extract was ineffective. Since all the eicosanoids studied tended to increase, banana may act by increasing the availability of arachidonate. In control tissues the accumulation of PGE and TXB2 in the incubates decreased with time while that of 6‐keto‐PGF1α increased (colon only, studied).