Protective Effect of Plumbago zeylanica Against Cyclophosphamide-Induced Genotoxicity and Oxidative Stress in Swiss Albino Mice

Plumbago zeylanica, commonly known as white leadwort, found abundantly in the plains of Bengal and southern India, was tested for its possible in vivo protective effect against cyclophosphamide-induced genotoxicity and oxidative stress in Swiss albino mice. Pretreatment with the alcoholic root extract of Plumbago zeylanica (250 and 500 mg/kg body weight orally for 5 days) significantly reduced the frequency of micronucleated polychromatic erythrocytes (MnPCEs), increased the PCE/NCE (normochromatic erythrocyte) ratio in the bone marrow, and decreased the levels of lipid peroxidation products with concomitant changes in the status of antioxidants. Both doses of Plumbago zeylanica were effective in exerting a protective effect against cyclophosphamide-induced genotoxicity and oxidative stress.     

Factor Xa-like and fibrin(ogen)olytic activities of a serine protease from Hippasa agelenoides spider venom gland extract

In the recent past, a low molecular mass serine protease, the Hag-protease that caused pro-coagulant activity and as well as local toxicity was isolated and characterized from the Hippasa agelenoides spider venom gland extract (Devaraja et al., Toxicon 52:130–138, 2008). In the current study, the pro-coagulant property has been investigated further and the results are presented. The Hag-protease reduced the re-calcification time of citrated human plasma. It reduced the activated partial thromboplastin time (APTT), and prothrombin time (PT) suggesting its participation in common pathway of blood coagulation. Interestingly, it coagulated the citrated human plasma in the absence of CaCl₂ but, it was lacking thrombin-like activity as it did not clot the purified fibrinogen. Strikingly, the enzyme coagulated the factor X deficient congenital human plasma, suggesting the factor Xa-like activity. However, the cumulative augmented activity was observed in presence of CaCl₂ and phospholipids. Further, the Hag-protease preferentially hydrolyzed the Aα chain and then the Bβ-chain, but not the γ-chain. As a result, truncated fibrinogen generated was lacking in the polymerization property. It hydrolyzed all the subunits of partially cross-liked fibrin clot (α-polymer, α-chain, β-chain, and γ–γ dimers). Further, at low concentrations, the Hag-protease stimulated the aggregation of human platelets in platelet rich plasma, but at high concentrations caused spontaneous clumping. In contrast, it inhibited the collagen induced aggregation of washed human platelets. In summary, the present study for the first time reporting the factor Xa-like activity of a serine protease especially from the spider venom that exhibited opposing effects on hemostasis, the pro-coagulant activity and the anti-coagulant activity including fibrin(ogen)olytic and platelet aggregation inhibition activities.     

Effects of CRL 41034 on the adrenergic neuroeffector interaction in the canine saphenous vein

Experiments were designed to determine the effect of CRL 41034, a buflomedil analogue, on the adrenergic responsiveness of canine veins. Rings of saphenous vein (without endothelium) were suspended for isometric tension recording in modified Krebs-Ringer bicarbonate solution at 37 degrees C. CRL 41034 produced a concentration-dependent inhibition of the contractions evoked by the alpha adrenergic agonists norepinephrine, phenylephrine and UK 14304 which was insensitive to the blockade of neuronal uptake by cocaine. CRL 41034 was more potent in inhibiting the concentration-dependent contractions evoked by UK 14304 than those by phenylephrine and the antagonism it caused against the response to UK 14304 fulfilled the criteria for competitivity. CRL 41034, at 10(-5) M significantly depressed, and at 10(-4) M abolished the contractions induced by electrical stimulation of the adrenergic nerves and those evoked by the indirect sympathomimetic amine tyramine. Strips of canine saphenous vein were superfused after incubation with [3H] norepinephrine. During sympathetic nerve activation, CRL 41304 increased the stimulation-evoked overflow of [3H] norepinephrine and 3-methoxy-4-dihydroxyphenylglycol; in the presence of rauwolscine the compound only increased the stimulation-evoked overflow of 3,4-dihydroxyphenylglycol. These experiments suggest that the major vascular effects of CRL 41034 in canine veins are blockade of alpha 2-adrenoceptors on vascular smooth muscle, and inhibition of prejunctional alpha 2-adrenoceptors on adrenergic nerve endings.     

Oxidized low-density lipoprotein and atherosclerosis

Atherosclerosis is the leading cause of morbidity and mortality in western society. The most important risk factors for atherosclerosis include smoking, hypertension, dyslipidemia, diabetes and a family history of premature atherosclerosis. Several studies indicate that an increased plasma low density lipoprotein (LDL) cholesterol constitutes a major risk factor for atherosclerosis. Many data support a proatherogenic role for oxidized LDL and its in vivo existence. The oxidative susceptibility of LDL is increased with established cardiovascular risk factors, such as diabetes, smoking and dyslipidemia. Supplementation with antioxidants such as ascorbate and alpha-tocopherol can decrease LDL oxidation as well as cardiovascular mortality and thus shows promise in the prevention of atherosclerosis.     

A new combined in-vitro test model for the identification of substances affecting essential sperm functions [published erratum appears in Hum Reprod 1997 Nov;12(11):2580]

Binding of mammalian spermatozoa to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. It has been postulated that xenobiotics that are released in the environment as well as exposure to pharmaceutical medications may be associated with reproductive problems in men and wildlife. Examining physiological and non-physiological effects of particular compounds on sperm functions requires high quality in-vitro test systems. We established a reliable combined in-vitro test system with bovine gametes and evaluated if aliquots of pooled post-thaw spermatozoa are suitable for examining essential sperm functions. Using cryopreserved semen, the PSA-FITC/Hoechst 33258 staining procedure was applicable to evaluate the acrosomal status and cell viability. In the bovine hemizona assay, hemizona indices revealed no differences between cryopreserved and fresh semen. Treatment of post-thaw bovine spermatozoa with progesterone (1 microM or bovine follicular fluid (20%) induced the acrosome reaction from 12% (untreated spermatozoa) to 25% (P < 0.001) and to 22% [corrected] (P < 0.01), respectively. Incubation of both compounds (1 microM progesterone and 20% follicular fluid) raised the percentage of acrosome-reacted spermatozoa to 30% (P < 0001). Our results demonstrate that cryopreserved semen can be integrated into an in-vitro screening model for reproductive toxicology testing. Pooled, cryopreserved bovine spermatozoa will thus permit reproducible experiments for clinical and basic science purposes and may also be applicable for the human system.      

Cloning, functional activities and in vivo tissue distribution of rat NKR-P1+ TCR alpha beta + cells

We have successfully cloned nine NKR-P1+ TCR alpha beta + cells from PVG rat spleens, utilizing murine macrophage inflammatory protein-1 alpha (MIP-1 alpha) and IL-2. These clones are either double negative (DN, CD4-CD8-), which included clones 3.31, 3.71, 4.19, 4.59 and 4.65, or single positive (SP, CD4+CD8-), which included clones 1.64, 3.8, 3.76 and 3.78. No CD8+ clone was recovered. All nine clones are restricted in terms of their expression of the V beta antigens, since they express V beta 8.2 but not V beta 8.5, V beta 10 or V beta 16. These clones are agranular and they fall to generate NK or LAK activity upon incubation with IL-2, IL-12 or their combination. On the basis of their production of intracellular cytokines they can be divided into three categories: (I) SP clones (1.64, 3.8, 3.76 and 3.78) do not produce IL-2 or IL-4, but produce IFN-gamma and IL-12, and they vary in their production of IL-1, RANTES or tumor necrosis factor (TNF)-alpha; (II) DN clones 4.59 and 4.65 produce IL-1 alpha and IFN-gamma only, and fall to produce other cytokines; and (III) DN clones 3.31, 3.71 and 4.19 produce IL-1 alpha, IL-1 beta, IL-2, IL-12, IFN-gamma, RANTES and TNF-alpha. From all the clones examined only DN clones 3.31 and to a lesser degree 4.19 produce IL-4. In vivo tissue localization of clones 3.8, 3.31 and 4.59 shows that these cells distribute into the liver and bone marrow 24 h post i.v. administration. Their accumulation in the liver and bone marrow along with their ability to secrete various cytokines suggest that these cells may influence the generation, differentiation or apoptosis of immune or hematopoietic cells.