Carminita L. Frost

Comparative blood coagulation studies in the ostrich.

Blood coagulation of the ostrich was compared to that of mammalian (man and sheep), avian (chicken) and reptilian (puff adder) systems. The international normalised ratio (INR), partial thromboplastin time (PTT), thrombin time and fibrin degradation were determined, as well as the various coagulation factors in venous ostrich plasma, using human physiological substrates. Thromboplastin was isolated from fresh brain tissue with the exception of the reptile for which lung tissue was used. The levels of markers of the coagulation [antithrombin III (AT), factor X (FX) and prothrombin], the fibrinolytic (alpha2-antiplasmin) and the kallikrein system were determined using chromogenic substrates. Elevated values for INR, PTT and thrombin time were obtained as compared to known human standards. It was found that factors VII, IX, X, XI and XII were absent from ostrich plasma. A study of the homologous and heterologous thromboplastin activities indicated that ostrich plasma exhibited a lower thromboplastic activity when compared to human standards, but was comparable to avian and reptilian values. Ostrich plasma revealed 42.2% FX, 72.9% AT, 35.3% prothrombin, 115.6% alpha2-antiplasmin and 19.8% plasma kallikrein, relative to human plasma. All the results suggest that the ostrich coagulatory system has not evolved to include all the complex myriad of reactions found in the human system.

Marrubiin, a constituent of Leonotis leonurus, alleviates diabetic symptoms

AIMS Marrubiin and an organic extract of Leonotis leonurus were tested in vitro and in vivo for their antidiabetic and anti-inflammatory activities. MATERIALS AND METHODS INS-1 cells were cultured under normo- and hyperglycemic conditions conditions. An in vivo animal model confirmed the biological activities of marrubiin and the organic extract observed in the studies in vitro. RESULTS The stimulatory index of INS-1 cells cultured under hyperglycemic conditions was significantly increased in cells exposed to the organic extract and marrubiin, relative to the hyperglycaemic conditions. Insulin and glucose transporter-2 gene expressions were significantly increased by the organic extract and marrubiin. Similarly, the extract and marrubiin resulted in an increase in respiratory rate and mitochondrial membrane potential under hyperglycaemic conditions. Marrubiin increased insulin secretion, HDL-cholesterol, while it normalized total cholesterol, LDL-cholesterol, atherogenic index, IL-1β and IL-6 levels in an obese rat model. CONCLUSION The results provide evidence that marrubiin, a constituent of Leonotis leonurus, alleviates diabetic symptoms.

Real-time monitoring of 3T3-L1 preadipocyte differentiation using a commercially available electric cell-substrate impedance sensor system

Real-time analysis offers multiple benefits over traditional end point assays. Here, we present a method of monitoring the optimisation of the growth and differentiation of murine 3T3-L1 preadipocytes to adipocytes using the commercially available ACEA xCELLigence Real-Time Cell Analyser Single Plate (RTCA SP) system. Our findings indicate that the ACEA xCELLigence RTCA SP can reproducibly monitor the primary morphological changes in pre- and post-confluent 3T3-L1 fibroblasts induced to differentiate using insulin, dexamethasone, 3-isobutyl-1-methylxanthine and rosiglitazone; and may be a viable primary method of screening compounds for adipogenic factors.

Biological effects of THC and a lipophilic cannabis extract on normal and insulin resistant 3T3-L1 adipocytes

Type 2 diabetes, a chronic disease, affects about 150 million people world wide. It is characterized by insulin resistance of peripheral tissues such as liver, skeletal muscle, and fat. Insulin resistance is associated with elevated levels of tumor necrosis factor alpha (TNF-alpha), which in turn inhibits insulin receptor tyrosine kinase autophosphorylation. It has been reported that cannabis is used in the treatment of diabetes. A few reports indicate that smoking cannabis can lower blood glucose in diabetics. Delta(9)-tetrahydrocannabinol (THC) is the primary psychoactive component of cannabis. This study aimed to determine the effect of a lipophilic cannabis extract on adipogenesis, using 3T3-L1 cells, and to measure its effect on insulin sensitivity in insulin resistant adipocytes. Cells were cultured in Dulbeccos modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and differentiated over a 3 day period for all studies. In the adipogenesis studies, differentiated cells were exposed to the extract in the presence and absence of insulin. Lipid content and glucose uptake was subsequently measured. Insulin-induced glucose uptake increased, while the rate of adipogenesis decreased with increasing THC concentration. Insulin-resistance was induced using TNF-alpha, exposed to the extract and insulin-induced glucose uptake measured. Insulin-induced glucose was increased in these cells after exposure to the extract. Semiquantitative real time polymerase chain reaction (RT-PCR) was performed after ribonucleic acid (RNA) extraction to evaluate the effects of the extract on glucose transporter isotype 4 (GLUT-4), insulin receptor substrate-1 (IRS-1) and IRS-2 gene expression.

Cannabis exposure associated with weight reduction and β-cell protection in an obese rat model.

The aim of this study was to investigate the effect of an organic cannabis extract on β-cell secretory function in an in vivo diet-induced obese rat model and determine the associated molecular changes within pancreatic tissue. Diet-induced obese Wistar rats and rats fed on standard pellets were subcutaneously injected with an organic cannabis extract or the vehicle over a 28-day period. The effect of diet and treatment was evaluated using the intraperitoneal glucose tolerance tests (IPGTTs) and qPCR analysis on rat pancreata harvested upon termination of the experiment. The cafeteria diet induced an average weight difference of 32g and an overall increase in body weight in the experimental groups occurred at a significantly slower rate than the control groups, irrespective of diet. Area under the curve for glucose (AUC(g)) in the obese group was significantly lower compared to the lean group (p<0.001), with cannabis treatment significantly reducing the AUC(g) in the lean group (p<0.05), and remained unchanged in the obese group, relative to the obese control group. qPCR analysis showed that the cafeteria diet induced down-regulation of the following genes in the obese control group, relative to lean controls: UCP2, c-MYC and FLIP. Cannabis treatment in the obese group resulted in up-regulation of CB1, GLUT2, UCP2 and PKB, relative to the obese control group, while c-MYC levels were down-regulated, relative to the lean control group. Treatment did not significantly change gene expression in the lean group. These results suggest that the cannabis extract protects pancreatic islets against the negative effects of obesity.

Purification and characterization of ostrich prothrombin.

The work focused on the penultimate enzyme, prothrombin, in the coagulation cascade. Prothrombin was purified and characterized from ostrich plasma. The results obtained contribute to a better understanding of blood coagulation in the ostrich and the evolution of prothrombin and the coagulation cascade. Prothrombin was purified from ostrich plasma by barium chloride precipitation, ammonium sulfate fractionation, and DEAE-cellulose and Cu(2+)-chelate Sepharose chromatography. Ostrich prothrombin exhibited a M(r) of 72,800 and a pI of 6.9 using SDS-PAGE and PAG-isoelectrofocusing, respectively. The N-terminal sequence of ostrich prothrombin showed 78 and 87% identity with human and bovine, respectively. The cDNA was isolated from ostrich liver and the predicted amino acid sequence compared with those from other species. Ostrich prothrombin shares sequence identity with chicken (84%), human (60%), bovine (59%), rat (60%), mouse (59%) and hagfish (50%) prothrombin, suggesting a common function of prothrombin in these vertebrates. Amino acid sequence identities indicate that the thrombin beta-chain (62%) and the propeptide-Gla (75%) domains are the regions most constrained for the common functions of vertebrate prothrombins. Ostrich prothrombin, therefore, shows similarity in structure to other vertebrate prothrombins.

Ostrich antithrombin III: kinetics and mechanism of inhibition of ostrich thrombin.

A kinetic investigation of ostrich thrombin specificity, its regulation and evolutionary development in comparison to those of other well-characterised species may contribute to the understanding of the structure-function relationships of thrombin. Antithrombin III (ATIII) was purified from ostrich plasma by heparin-Sepharose and Super Q-650S chromatography. It exhibited a M(r) of 59.2K and a pI in the range of 5.2-6.0. The ostrich N-terminal sequence was compared to those of other known species and showed the highest identity with rabbit ATIII (31%). Inhibition studies included the interaction of ostrich and human ATIII with bovine, human and ostrich thrombin. At a 2:1 molar ratio of ostrich ATIII to enzyme, 20 and 40% remaining activity was found for bovine and ostrich thrombin, respectively. Ostrich thrombin exhibited a pH and temperature optimum of 9.0 and 60 degrees C, respectively. Hydrolysis of seven peptide p-nitroanilide substrates by ostrich thrombin revealed D-Phe-Pip-Arg-pNA (k(cat)/K(m)=9.65 microM(-1)s(-1)) as the substrate with the highest catalytic efficiency. The effect of monovalent cations on ostrich thrombin catalysis revealed enhanced activity with Na(+). The calculated K(i) values for the complex formation between ostrich thrombin and ostrich (9.29 x 10(-11)M) and human (9.66 x 10(-11)M) ATIII are comparable to reported results. The results obtained from the present study confirmed that ostrich thrombin and ATIII are closely related to the corresponding molecules of other species in terms of physicochemical and kinetic properties.

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor

Abstract One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

pH-metric chemical speciation modeling and studies of in vitro antidiabetic effects of bis[(imidazolyl)carboxylato]oxidovanadium(IV) complexes.

A range of bidentate N,O-donor ligands of the imidazolyl-carboxylate moiety, which partially mimic naturally occurring bioligands, were prepared and reacted with the oxidovanadium(IV) ion to form the corresponding bis-coordinated oxidovanadium(IV) complexes. The aqueous pH-metric chemical speciation was investigated using glass electrode potentiometry, which allowed for the determination of protonation and stability constants of the ligands and complexes, respectively. The species distribution diagrams generated from this information gave evidence that the bis[(imidazolyl)carboxylato]oxovanadium(IV) complexes possess a broad pH-metric stability. The complexes improved glucose uptake in cell cultures using 3T3-L1 adipocytes, C2C12 muscle cells and Chang liver cells. The PTP inhibition studies indicated that the mechanism underlying insulin-stimulated glucose uptake was possibly via the protein tyrosine phosphorylation through the inhibition of the protein tyrosine phosphatase 1B (PTP 1B). The vanadium compounds also demonstrated the inhibition of D-dimer formation, suggesting that these compounds could potentially relieve a hypercoagulative state in diabetic patients.

Selected terpenoids from medicinal plants modulate endoplasmic reticulum stress in metabolic disorders.

The majority of research performed on cellular stress and apoptosis focuses on mitochondrial dysfunction; however, the importance of the endoplasmic reticulum dysfunction and the link to metabolic diseases has gained a substantial interest. This review focuses on the potential of terpenoids to influence endoplasmic reticulum stress and the possible role terpenoids play as the treatment of metabolic diseases.