Biospeciation of Oxidovanadium(IV) Imidazolyl-Carboxylate Complexes and Their Action on Glucose-Stimulated Insulin Secretion in Pancreatic Cells.

The reaction of the vanadyl ion (VO2+) with imidazole-4-carboxylic acid (Im4COOH), imidazole-2-carboxylic acid (Im2COOH) and methylimidazole-2-carboxylic acid (MeIm2COOH), respectively, in the presence of small bioligands (bL) [oxalate (Ox), lactate (Lact), citrate (Cit) and phosphate (Phos)] and high-molecular-weight (HMW) human serum proteins [albumin (HSA) and transferrin (hTf)] were studied in aqueous solution using potentiometric acid-base titrations. The species distribution diagrams for the high-molecular-mass (HMM) proteins with oxidovanadium(IV) under physiological pH were dominated by VO(HMM)2, VOL(HMM) for unsubstituted ligands (L- = Im4COO- and Im2COO-). However, for the N-substituted MeIm2COOH, the species distribution diagrams under physiological pH were dominated by VOL2, VO(HMM)2 and VO2L2(HMM). These species were further confirmed by LC-MS, MALDI-TOF-MS and EPR studies. The glucose-stimulated insulin secretion (GSIS) action of the complexes was investigated using INS-1E cells at a 1 µM concentration, which was established through cytotoxicity studies via the MTT assay. The neutral complexes, especially VO(MeIm2COO)2, showed promising results in the stimulation of insulin secretion than the cationic [VO(MeIm2CH2OH)2]2+ complex and the vanadium salt. Oxidovanadium(IV) complexes reduced insulin stimulation significantly under normoglycaemic levels but showed positive effects on insulin secretion under hyperglycaemic conditions (33.3 mM glucose media). The islets exposed to oxidovanadium(IV) complexes under hyperglycaemic conditions displayed a significant increase in the stimulatory index with 1.19, 1.75, 1.53, 1.85, 2.20 and 1.29 observed for the positive control (sulfonylurea:gliclazide), VOSO4, VO(Im4COO)2, VO(Im2COO)2, VO(MeIm2COO)2 and VO(MeIm2CH2OH)22+, respectively. This observation showed a potential further effect of vanadium complexes towards type 2 diabetes and has been demonstrated for the first time in this study.

Inhibitory effects of selected cannabinoids against dipeptidyl peptidase IV, an enzyme linked to type 2 diabetes.

Ethnopharmacological relevance: In recent times the decriminalisation of cannabis globally has increased its use as an alternative medication. Where it has been used in modern medicinal practises since the 1800s, there is limited scientific investigation to understand the biological activities of this plant. Aim of the study: Dipeptidyl peptidase IV (DPP-IV) plays a key role in regulating glucose homeostasis, and inhibition of this enzyme has been used as a therapeutic approach to treat type 2 diabetes. However, some of the synthetic inhibitors for this enzyme available on the market may cause undesirable side effects. Therefore, it is important to identify new inhibitors of DPP-IV and to understand their interaction with this enzyme. Methods: In this study, four cannabinoids (cannabidiol, cannabigerol, cannabinol and Δ9-tetrahydrocannabinol) were evaluated for their inhibitory effects against recombinant human DPP-IV and their potential inhibition mechanism was explored using both in vitro and in silico approaches. Results: All four cannabinoids resulted in a dose-dependent response with IC50 values of between 4.0 and 6.9 µg/mL. Kinetic analysis revealed a mixed mode of inhibition. CD spectra indicated that binding of cannabinoids results in structural and conformational changes in the secondary structure of the enzyme. These findings were supported by molecular docking studies which revealed best docking scores at both active and allosteric sites for all tested inhibitors. Furthermore, molecular dynamics simulations showed that cannabinoids formed a stable complex with DPP-IV protein via hydrogen bonds at an allosteric site, suggesting that cannabinoids act by either inducing conformational changes or blocking the active site of the enzyme. Conclusion: These results demonstrated that cannabinoids may modulate DPP-IV activity and thereby potentially assist in improving glycaemic regulation in type 2 diabetes.

The development of folate-functionalised palladium nanoparticles for folate receptor targeting in breast cancer cells.

Folate receptor-targeted therapy has excellent prospects for the treatment of breast cancer. A non-toxic concentration of folate-conjugated palladium-based nanoparticles was used to target the overexpressed folate receptor on breast cancer cells. The folate-conjugated nanoparticles were tailored to accumulate selectively in cancer cells relative to normal cells via the folate receptor. The MDA-MB-231, MDA-MB-468, MCF-7 breast cancer cell lines, and MCF-10A normal cell lines were used in the study. Qualitative and quantitative analysis of nanoparticle cellular uptake and accumulation was conducted using transmission electron microscopy and inductively coupled plasma-optical emission spectroscopy. The findings proved that folate-conjugated palladium nanoparticles successfully and preferentially accumulated in breast cancer cells. We conclude that folate-conjugated palladium nanoparticles can be potentially used to target breast cancer cells for radiopharmaceutical applications.

Effect of oral cannabis administration on the fat depots of obese and streptozotocin-induced diabetic rats.

The prevalence of obesity and insulin-resistance is on the rise, globally. Cannabis have been shown to have anti-diabetic/obesity properties, however, the effect mediated at various fat depots remains to be clarified. The aim of this study was to (1) investigate the anti-diabetic property of an oral cannabis administration in an obese and streptozotocin-induced diabetic rat model and (2) to determine and compare the effect mediated at the peritoneal and intramuscular fat level. Cannabis concentration of 1.25 mg/kg body weight (relative to THC content) was effective in reversing insulin-resistance in the rat model, unlike the other higher cannabinoid concentrations. At the peritoneal fat level, gene expression of fat beigeing markers, namely Cidea and UCP1, were significantly increased compared to the untreated control. At the intramuscular fat level, on the other hand, CE1.25 treatment did not promote fat beigeing but instead significantly increased mitochondrial activity, relative to the untreated control. Therefore, these findings indicate that the mechanism of action of oral cannabis administration, where glucose and lipid homeostasis is restored, is not only dependent on the dosage but also on the type of fat depot investigated.

Cannabis and metformin on diabetic male Wistar rat sperm and reproductive organ parameters.

Purpose: Cannabis use has reportedly increased in type 2 diabetic users as a possible co-treatment for associated pain and inflammation. Both cannabis and metformin (an anti-diabetic drug) have a limited number of studies completed on their effect on male reproductive parameters in a diabetic model. This study determined if cannabis and metformin administration alter various reproductive parameters in diabetic male rats. Methods: Male Wistar rats (n = 35) were fed on a high fat diet and injected with streptozotocin (30 mg/kg rat) to induce a type-2 diabetic model. Treatment groups received cannabis based on Delta-9-Tetrahydrocannabinol (THC) concentrations of 1.25, 2.5 and 5 mg/kg per rat and metformin (50 mg/kg) every alternate day for 10 weeks. Organ weight; serum testosterone levels and sperm count, motility, lipid peroxidation, citrate synthase and lactate dehydrogenase activities were measured. Results: Cannabis treatment induced a significant concentration dependent decrease in sperm motility at 5 mg/kg rat THC (P = 0.009) administration. Metformin significantly (P = 0.035) increased sperm counts and lactate dehydrogenase activity (P = 0.002). Both cannabis and metformin negatively affected testosterone concentrations. Conclusions: Cannabis needs to be used cautiously as an alternative treatment in diabetic males based on the negative effects observed for the various reproductive parameters in this diabetic rat model.

Cannabis with breast cancer treatment: propitious or pernicious?

Cannabis has been used for various medicinal applications including, but not limited to, cancer: most commonly to treat chemotherapy-associated side effects. Cannabis is often used for its palliative effects in the form of purified cannabinoids, or as extracts. This study was conducted using two breast cancer cell lines and aimed to evaluate potential anti-proliferative "intra-entourage effects" between purified phytocannabinoids resembling the THC and CBD ratios of medicinal and recreational cannabis strains, as well as to investigate potential "inter-entourage effects" between the different ratios and the phytochemicals found in a Cannabis sativa extract. This study also aimed to evaluate the potential interaction between cannabinoids and chemotherapeutic agents. The data identified an intra-entourage effect present in the MCF-7 cells when treated with a recreational, but not a medicinal, cannabis formulation. This effect may be due to THC partially exerting its anti-proliferative effects through the estrogen receptor (ER), present in the MCF-7 cell line. Little to no intra-entourage effects were observed in the MDA-MB-231 cell line and no inter-entourage effects were observed in either cell line. The simultaneous treatment of the MCF-7 cell line with various cannabinoid formulations and the common breast cancer treatment, tamoxifen, resulted in the diminished anti-proliferative activity of tamoxifen, an effect that was more evident when combined with recreational cannabis formulations. Since cannabis is commonly used in palliative care to treat chemotherapy-associated side effects, further research is required to investigate the potential interference of various cannabis formulations to ensure that the efficacy of chemotherapeutic agents is not compromised. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03102-1.

CBD activation of TRPV1 induces oxidative signaling and subsequent ER stress in breast cancer cell lines.

Endoplasmic reticulum (ER) stress is an imbalance between the protein-folding load and capacity of ER. It can be induced by various physiological conditions, activating the unfolded protein response (UPR) to re-establish homeostasis, promoting cell survival. Under severe or chronic stress, apoptosis is induced. Normal cells generally do not experience continuous ER stress induction. The stressful conditions experienced in the tumor microenvironment facilitate chronic ER stress and UPR activation, which plays a pivotal role in tumour survival. Exacerbation of pre-existing ER stress can trigger cancer cell death, with a minimal effect on normal cells. Current literature suggests that cannabinoid treatment may induce cancer cell death via ER stress; however, little is known about the mechanisms of induction. This study proposed that cannabidiol (CBD) mechanism occurred through the influx of Ca2+ via the TRPV1 receptor, and increasing reactive oxygen species (ROS) production affects protein folding and induces ER stress. ER stress was induced, and detection and quantification were completed using Thioflavin T staining and GRP78 by western blot analysis. The effect of cannabinoid treatment on ROS production and Ca2+ influx was measured. CBD was the most potent ER stress inducer, significantly increasing Ca2+ and ROS accumulation. Concomitant treatment with CBD and an antioxidant significantly increased cell viability and decreased ER stress induction in the MCF7 cell line. Concomitant treatment with a TRPV1 antagonist increased viability in this cell line. In conclusion, the data suggested that CBD induced ER stress via Ca2+ influx through the TRPV1 receptor, thereby elevating intracellular ROS levels and disrupting protein folding.

Tulbaghia violacea (Harv) Exerts its Antifungal Activity by Reducing Ergosterol Production in Aspergillus flavus.

Opportunistic infections in immunosuppressed patients have led to an increase in fungal infections, with Aspergillus being one of the main causative agents. Medicinal plants exhibiting antifungal activity have the potential to be used as chemotherapeutic agents. However, often their mechanisms of action are not fully researched. Tulbaghia violacea exhibits antifungal activity towards Candida, Aspergillus flavus and Aspergillus parasiticus but its mode of action has only recently begun to be investigated. This study aimed to ascertain the effect of T. violacea rhizome extracts on ergosterol production in A. flavus and the mechanism of inhibition. The MIC of a T. violacea rhizome extract against A. flavus was first determined, using a broth dilution assay, to be 15 mg/ml. Thereafter, the culture was subjected to sub-inhibitory concentrations of the extract before sterol intermediates of the ergosterol biosynthetic pathway were isolated and analysed for dose-dependent accumulation. Analysis by reverse-phase HPLC displayed a decline in ergosterol production in a dose-dependent manner when exposed to increasing concentrations of T. violacea extract. Quantification of the sterol intermediates of the ergosterol pathway indicated a definite accumulation of 2,3-oxidosqualene. The results prove that the plant extract affected ergosterol synthesis by inhibiting oxidosqualene cyclase. This prevented the formation of downstream intermediates of the ergosterol pathway ultimately resulting in inhibition of ergosterol production.

Cannabinoid Combination Induces Cytoplasmic Vacuolation in MCF-7 Breast Cancer Cells.

This study evaluated the synergistic anti-cancer potential of cannabinoid combinations across the MDA-MB-231 and MCF-7 human breast cancer cell lines. Cannabinoids were combined and their synergistic interactions were evaluated using median effect analysis. The most promising cannabinoid combination (C6) consisted of tetrahydrocannabinol, cannabigerol (CBG), cannabinol (CBN), and cannabidiol (CBD), and displayed favorable dose reduction indices and limited cytotoxicity against the non-cancerous breast cell line, MCF-10A. C6 exerted its effects in the MCF-7 cell line by inducing cell cycle arrest in the G2 phase, followed by the induction of apoptosis. Morphological observations indicated the induction of cytoplasmic vacuolation, with further investigation suggesting that the vacuole membrane was derived from the endoplasmic reticulum. In addition, lipid accumulation, increased lysosome size, and significant increases in the endoplasmic reticulum chaperone protein glucose-regulated protein 78 (GRP78) expression were also observed. The selectivity and ability of cannabinoids to halt cancer cell proliferation via pathways resembling apoptosis, autophagy, and paraptosis shows promise for cannabinoid use in standardized breast cancer treatment.

Synthesis, Characterization and Biological Activity of Some Dithiourea Derivatives.

Novel dithiourea derivatives have been designed as HIV-1 protease inhibitors using Autodock 4.2, synthesized and characterized by spectroscopic methods and microanalysis. 1-(3-Bromobenzoyl)-3-[2-([(3-bromophenyl)formami-do]methanethioylamino)phenyl]thiourea (10) and 3-benzoyl-1[(phenylformamido)methanethioyl]aminothiourea (12) gave a percentage viability of 17.9 ± 5.6% and 11.2 ± 0.9% against Trypanosoma brucei. Single crystal X-ray dif-fraction analysis of 1-benzoyl-3-(5-methyl-2-[(phenylformamido)methanethioyl]aminophenyl)thiourea (1), 3-ben-zoyl-1-(2-[(phenylformamido)methanethioyl]aminoethyl)thiourea (11), 3-benzoyl-1-[(phenylformamido)methan-ethioyl]aminothiourea (12) and 3-benzoyl-1-(4-[(phenylformamido)methanethioyl]aminobutyl)thiourea (14) have been presented. 1-(3-Bromobenzoyl)-3-[2-([(3-bromophenyl)formamido]methanethioylamino)phenyl]thiourea (10) gave a percentage inhibition of 97.03 ± 0.37% against HIV-1 protease enzyme at a concentration of 100 ?M.

Purification and biochemical characterisation of a putative sodium channel agonist secreted from the South African Knobbly sea anemone Bunodosoma capense.

Voltage gated ion channels have become a subject of investigation as possible pharmaceutical targets. Research has linked the activity of ion channels directly to anti-inflammatory pathways, energy homeostasis, cancer proliferation and painful diabetic neuropathy. Sea anemones secrete a diverse array of bioactive compounds including potassium and sodium channel toxins. A putative novel sodium channel agonist (molecular mass of 4619.7 Da) with a predicted sequence: CLCNSDGPSV RGNTLSGILW LAGCPSGWHN CKKHKPTIGW CCK was isolated from Bunodosoma capense using a modified stimulation technique to induce the secretion of the neurotoxin rich mucus confirmed by an Artemia nauplii bio-assay. The peptide purification combined size-exclusion and reverse-phase high performance liquid chromatography. A thallium-based ion flux assay confirmed the presence of a sodium channel agonist/inhibitor and purity was determined using a modified tricine SDS-PAGE system. The peptide isolated indicated the presence of multiple disulfide bonds in a tight ß-defensin cystine conformation. An IC50 value of 26 nM was determined for total channel inhibition on MCF-7 cells. The unique putative sodium channel agonist initiating with a cystine bond indicates a divergent evolution to those previously isolated from Bunodosoma species.

Comparative detection method of early onset cytokine-induced stress in ß-cells (INS-1E).

ß-Cells contain a prominent endoplasmic reticulum (ER), disrupting ER homeostasis and function, activating the unfolded protein response (UPR). Currently, no direct protocols measure the UPR initiation. Current methods to measure ER stress include the quantification of nitric oxide (NO) (indirect method), Western blotting, and qRT-PCR of downstream components. However, these methods do not account for the overlap with mitochondrial dysfunction. In this study, INS-1E cells were exposed to proinflammatory cytokines to induce ER stress, as determined using NO, thioflavin T (ThT) binding, and ß-cell functionality (insulin production). ER stress was confirmed through the upregulation of CHOP. Cell viability was monitored using MTT, sulforhodamine B, and the xCELLigence system. Morphological changes were monitored using electron microscopy. IL-1ß exposure-induced ß-cell stress after 4 H, decreased insulin levels, and increased thioflavin binding were noted. Increased NO production was only detected after 10 H, highlighting its lack of sensitivity, and the need for a continuous, selective, rapid, convenient, and economical detection method for early onset of ER stress. Standard methods (MTT and NO) failed to detect early ER stress. The xCELLigence coupled with a functional assay such as the detection of insulin levels or ThT are better predictors of ER stress in INS-1E cells.

Improving the understanding of plasma kallikrein contribution to arterial thrombus formation using two plant protease inhibitors.

The purpose of antithrombotic therapy is the prevention of thrombus formation and/or its extension with a minimum risk of bleeding. The inhibition of a variety of proteolytic processes, particularly those of the coagulation cascade, has been reported as a property of plant protease inhibitors. The role of trypsin inhibitors (TIs) from Delonix regia (Dr) and Acacia schweinfurthii (As), members of the Kunitz family of protease inhibitors, was investigated on blood coagulation, platelet aggregation, and thrombus formation. Different from Acacia schweinfurthii trypsin inhibitor (AsTI), Delonix regia trypsin inhibitor (DrTI) is a potent inhibitor of FXIa with a Kiapp of 1.3 × 10-9 M. In vitro, both inhibitors at 100 µg corresponding to the concentrations of 21 µM and 15.4 µM of DrTI and AsTI, respectively, increased approximately 2.0 times the activated partial thromboplastin time (aPTT) in human plasma compared to the control, likely due to the inhibition of human plasma kallikrein (huPK) or activated factor XI (FXIa), in the case of DrTI. Investigating in vivo models of arterial thrombus formation and bleeding time, DrTI and AsTI, 1.3 µM and 0.96 µM, respectively, prolonged approximately 50% the time for total carotid artery occlusion in mice compared to the control. In contrast to heparin, the bleeding time in mice treated with the two inhibitors did not differ from that of the control group. DrTI and AsTI inhibited 49.3% and 63.8%, respectively, ex vivo murine platelet aggregation induced by adenosine diphosphate (ADP), indicating that these protein inhibitors prevent arterial thrombus formation possibly by interfering with the plasma kallikrein (PK) proteolytic action on the intrinsic coagulation pathway and its ability to enhance the platelet aggregation activity on the intravascular compartment leading to the improvement of a thrombus.

In Vitro Screening of Synthetic Fluorogenic Substrates for Detection of Cancer Procoagulant Activity.

Cancer procoagulant (CP), a direct activator of coagulation factor X, is among one of the tumour cell products or activities which may promote fibrin formation and has been suggested to be selectively associated with the malignant phenotype. At present, the most reliable assay for the quantification of CP activity is the three-stage chromogenic assay which utilises the ability of CP to activate factor X. In this assay, the activation of factor X leads to the formation of activated thrombin from prothrombin and the eventual hydrolyses of a thrombin chromogenic substrate which contains a p-nitroaniline leaving group. The complexity of the three-stage chromogenic assay suggests a need for a direct method of assaying CP activity. This study focuses on the design of a fluorogenic substrate that would enable the direct quantification of CP activity. The results of the study show two promising substrates for the determination of CP activity: Boc-PQVR-AMC and PQVR-AMC. Further analysis showed that Boc-PQVR-AMC could be excluded as a potential substrate for CP since it was also cleaved by collagenase.

Nanoformulation of Leonotis leonurus to improve its bioavailability as a potential antidiabetic drug.

Nanostructured lipid carriers (NLCs) of Leonotis leonurus were successfully produced using high-pressure homogenisation (HPH) on a LAB 40 homogeniser. The particle size was determined for the formulation as well as short-term stability study. The formulation was exposed to Chang liver cells for a glucose uptake study and to INS-1 cells for a chronic insulin release study under normoglycaemic and hyperglycaemic conditions. The particle size of the extract NLC was 220 nm with a PdI of 0.08 after homogenisation at 800 bar. The formulation was stable at the tested temperatures. The extract NLC formulation at 1 µg/ml improved glucose uptake, relative to the control liver cells. Insulin release in INS-1 cells was also elevated under hyperglycaemic conditions when exposed to the NLCs, in comparison with the control untreated cells and the non-formulated extract. The plant extract encapsulated in NLC improved the uptake of glucose and enhanced the insulin sensitivity in vitro, compared to the extract.

The proteolytic profile of human cancer procoagulant suggests that it promotes cancer metastasis at the level of activation rather than degradation.

Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.

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.

OBJECTIVES: 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. KEY FINDINGS: Metabolic diseases develop as a result of a cascade of cellular pathways. In most cases, cells are able to compensate for the disruption of the cellular homeostasis although the initiation of response pathways; however, chronic stress initiates apoptotic pathways. This reviewed (1) showed the importance of phytoterpenoids to influence endoplasmic reticulum (ER) stress and homeostasis, (2) showed how regulating ER stress affect the cell survival and death, and (3) highlighted some examples of how the progression of metabolic diseases can be influenced by ER. SUMMARY: Due to the substantial number of terpenoids that have been identified in literature, this review gave examples of 21 terpenoids that have been documented to have an effect on the different proteins associated with ER stress, how these plant terpenoids influence ER dysfunction and metabolic diseases such as diabetes, cancer, liver, and neurological diseases and parasitic infections.

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.

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

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.