ß-N-Methylamino-L-Alanine Toxicity in PC12: Excitotoxicity vs. Misincorporation.

The implication of ß-N-methylamino-L-alanine (BMAA) in the development of neurodegenerative diseases worldwide has led to several investigations of the mechanism, or mechanisms, of toxicity of this cyanobacterially produced amino acid. The primary mechanism of toxicity that was identified is excitotoxicity, with a second possible mechanism, the misincorporation of BMAA into the primary protein structure and consequent cell damage, having been more recently reported. However, studies on excitotoxicity and misincorporation have been conducted independently and there are therefore no data available on the relative contribution of each of these mechanisms to the total toxicity of BMAA. The rat pheochromocytoma cell line PC12 is an ideal model for a study of this type, as glutamate receptor expression is modified by cell differentiation, which can be affected by exposure to nerve growth factor. In this study, the PC12 cell line was evaluated as a model to study BMAA toxicity via the two proposed mechanisms: excitotoxicity and protein misincorporation. BMAA and canavanine treatment of cultures of PC12 were evaluated for depolarization of the mitochondrial membrane. In canavanine-treated cultures, this was evident after 9 days of treatment and was attributed to the primary mechanism of canavanine toxicity, protein misincorporation. However, no membrane depolarization was observed for BMAA-treated cultures even after 21 days of continuous treatment at 500 µM. Short-term exposure to both BMAA and canavanine resulted in a slight increase in necrosis in undifferentiated cells that was prevented in canavanine-treated cultures by co-incubation with arginine, but not in BMAA-treated cultures by co-incubation with serine. A slight increase in apoptosis was observed in undifferentiated cells treated with either BMAA or glutamate, and ROS production increased in glutamate-treated cells. However, the excitotoxicity was less pronounced than reported in previous studies with neuronal cells. In contrast, apoptosis was greatly increased in both BMAA- and glutamate-treated cells after differentiation and resulting mGluR1 increase, indicating that excitotoxicity is the main, if not only, mechanism of toxicity in PC12.

JLK1486, a N,N-[(8-hydroxyquinoline)methyl]-substituted benzylamine analogue, inhibits melanoma proliferation and induces autophagy.

OBJECTIVES: To investigate anti-proliferatory activity of a selected N,N-[(8-hydroxyquinoline)methyl]-substituted benzylamine (JLK1486) on melanoma cells and to characterize its mechanism of cell population growth inhibition. MATERIALS AND METHODS: In vitro cultures of B16F10 (mouse melanoma) cells were used as a model to characterize anti-proliferatory activity of JLK1486 using MTT growth assay, trypan blue viability assessment, cell cycle analysis, melanin production, ß-galactosidase and acridine orange staining. RESULTS: Proliferating B16F10 and also MeWo (human melanoma) cells were strongly growth inhibited by JLK1486, displaying IC50 values of 196 nm and 110 nm respectively. Anti-proliferatory effects were independent of cell death and were characterized by a distinct accumulation of cells in G0 /G1 phase. Tyrosinase activity and relative melanin content remained unchanged indicating that the anti-proliferatory activity was not due to phenotype differentiation. Although treated B16F10 cells stained strongly positive for senescence marker ß-galactosidase, cells regained near normal proliferatory activity after removal of JLK1486. Increased acridine orange staining and presence of perinuclear vacuoles suggested induction of autophagy in B16F10 cells. Furthermore, JLK1486 pre-treatment completely abolished melphalan and antimycin A-induced apoptosis. CONCLUSION: JLK1486 provides a promising chemical scaffold to develop new anti-melanoma drugs or combination therapies, due to its potent inhibition of cell proliferation and induction of autophagy, at pharmacologically relevant concentrations.

Sutherlandia frutescens prevents changes in diabetes-related gene expression in a fructose-induced insulin resistant cell model.

ETHNOPHARMACOLOGICAL RELEVANCE: The African medicinal plant Sutherlandia frutescens (L.) R.Br. (Fabaceae) is traditionally used to treat diabetes and has been shown to have anti-diabetic properties in animal models. The present study investigated the capacity of an aqueous extract of Sutherlandia frutescens to prevent insulin resistance (a precursor of type 2 diabetes) in a human liver cell culture and to identify genes regulated by Sutherlandia frutescens treatment. MATERIALS AND METHODS: A combination of insulin and fructose was used to generate an in vitro model of insulin resistance in human liver cells to compare untreated control, insulin resistant and Sutherlandia frutescens treated insulin resistant cultures. Insulin resistance and its prevention by Sutherlandia frutescens were measured by glucose uptake, gluconeogenesis and lipid accumulation in the cell cultures. Changes in gene expression were quantified using the RT(2)Profiler(TM) PCR Array of 84 diabetes-related genes. RESULTS: The insulin resistant Chang liver cells took up significantly less 2-[(3)H]-deoxyglucose (p<0.05) than controls, released more glucose into the culture medium (p<0.05) and accumulated more intracellular lipid (p<0.05). Simultaneous treatment with Sutherlandia frutescens prevented development of these insulin resistance parameters (p<0.05). A total of 27 potential gene targets of Sutherlandia frutescens were significantly up or down regulated in the Sutherlandia frutescens treated insulin resistant cells. The gene VAMP3, which plays a role in vesicle transport, was down-regulated by insulin resistance, and up-regulated by Sutherlandia frutescens. Twenty six other genes encoding vesicle transporters, receptors, signalling molecules, transcription factors, and metabolic enzymes were significantly regulated by Sutherlandia frutescens. CONCLUSION: These results confirm that Sutherlandia frutescens can prevent insulin resistance in hepatocytes. The identified changes in gene expression indicate several potential mechanisms of anti-diabetic action for Sutherlandia frutescens, reflecting the multiple bioactive compounds previously identified in aqueous extracts of Sutherlandia frutescens.

Production of polysaccharide and surfactin by Bacillus subtilis ATCC 6633 using rehydrated whey powder as the fermentation medium.

The aim of this research was to assess the amounts of polysaccharide and surfactin produced by Bacillus subtilis ATCC 6633 in rehydrated whey powder (RWP) as the growth medium. One-day-old cultures of B. subtilis (∼4.6 log cfu/mL) were inoculated into 100mL of 10, 15, or 20% (wt/vol) RWP and incubated at 30°C for 72 h. To analyze the effects of lactose and protein on polysaccharide and surfactin production, 6 RWP solutions containing different levels of lactose and protein were also used as media. The number of vegetative cells and spores, pH, viscosity, and the concentration of lactose were determined at 0, 24, 48, or 72 h of fermentation. The levels of polysaccharide and surfactin produced after 72 h of fermentation were measured using HPLC and the phenol-sulfuric acid method, respectively. During 72 h of fermentation, B. subtilis populations increased from 4.6 to 10.54, 9.82, and 9.67 log(10) cfu/mL in 10, 15, and 20% RWP, respectively. The number of B. subtilis spores in 10% RWP increased from 3.91 to 4.72 log(10) cfu/mL after 48 and 72 h of fermentation, respectively. The increased level of lactose or protein in RWP did not significantly change the vegetative growth. After 72h of fermentation, the pH of RWP decreased from 5.70 to 4.99 with a slight increase in viscosity. Polysaccharide levels in 10, 15, and 20% RWP after fermentation were 513.6, 613.5, and 768.3mg/L, respectively, with B. subtilis producing 0.18 to 0.29 g/L of surfactin after 72 h of fermentation. The polysaccharide or surfactin production was not changed significantly by addition of protein or lactose to RWP. These results indicate that RWP is a good fermentation substrate for surfactin and polysaccharide production.

Hypoglycemic evaluation of a new triterpene and other compounds isolated from Euclea undulata Thunb. var. myrtina (Ebenaceae) root bark.

AIM OF THE STUDY: Investigate the hypoglycaemic activity of the four isolated compounds from a crude acetone extract of the root bark of Euclea undulata var. myrtina, which is used by traditional healers in the Venda area, Limpopo Province in the treatment of diabetes. MATERIAL AND METHODS: The hypoglycaemic activity of the four compounds isolated from Euclea undulata was determined by in vitro screening of glucose utilization by C2C12 myocytes at a concentration of 25 µg/ml or 50 µg/ml. The inhibition of α-glucosidase was also tested at concentrations ranging from 0.02 to 200.00 µg/ml. RESULTS: Assay-guided isolation of the crude acetone extract of the root bark of Euclea undulata var. myrtina afforded a new triterpene, α-amyrin-3O-ß-(5-hydroxy) ferulic acid (1), in addition to three known compounds; betulin (2), lupeol (3) and epicatechin (4). The in vitro results on C2C12 myocytes suggest that compound 4 may have some effect to lowers blood glucose levels, whereas compound 1 has the ability to inhibit α-glucosidase at a concentration of 200.0 µg/ml with an IC50 value of 4.79 that correlates with that of the positive control acarbose IC50 value 4.75. CONCLUSION: The results suggest that 4 may have some ability to lower blood glucose levels, whereas 1 has the ability to inhibit α-glucosidase. ETHNOPHARMACOLOGICAL RELEVANCE: These findings corroborate the ethnomedicinal use of Euclea undulata by traditional healers for the treatment of diabetes as two substances was isolated from the acetone plant extract that exhibit hypoglycaemic activity.

Combination treatment with oxaliplatin and mangiferin causes increased apoptosis and downregulation of NFκB in cancer cell lines.

Mangiferin-mediated down-regulation of NFκB showed potential for chemotherapeutic agent-mediated cell death, suggesting a role in combination therapy for cancer. In this study the combined mechanism of the anticancer action of oxaliplatin and mangiferin was investigated. MTT dose response curves, trypan blue staining, caspase 3 assays as well as DNA cell cycle analyses were performed on HeLa, HT29 and MCF7 cancer cell lines, with and without the addition of 10 µg/ml mangiferin. Mitochondrial membrane potential, DNA fragmentation, resistance induction studies and NFκB assays were performed on HT29 cells only. Addition of 10 µg/ml mangiferin reduced oxaliplatin IC(50) values in HT29 (3.4 fold) and HeLa (1.7 fold) cells in the MTT assay while reducing trypan blue staining. This was accompanied by increased caspase 3 activation and DNA fragmentation and a delay in the S-phase of the cell cycle. Mitochondrial membrane permeabilization was not enhanced in the combination treatment. Mangiferin was shown to cause a reduction of NF-κB activation in HT29 cells rendered resistant to oxaliplatin. The present study indicates that mangiferin in combination with oxaliplatin favours apoptotic cell death and thereby improves the efficacy of oxaliplatin in vitro. In addition, combination therapy with mangiferin may also counteract the development of resistance in cancer cell lines.

Chemical composition and the antioxidative properties of Nigerian Okra Seed (Abelmoschus esculentus Moench) Flour.

Studies on the chemical composition and the antioxidative properties of Nigerian Okra Seed (Abelmoschus esculentus Moench) Flour were carried out. This is done to establish the nutritional composition and the antioxidative potentials of the seeds, both of which are highly implicated in health. Okra seeds were roasted at 160 degreeC for 10-60 mins. The roasted seeds were subjected to proximate, yield and antioxidative activity determination. Pre-treatment by roasting was found to increase the yield, but was found to be time dependent. The range means obtained for protein, fat, ash, fiber and sugar contents were 42.14-38.10, 31.04-17.22, 4.06-3.42, 3.45-3.60 and 8.82-8.65, respectively. The antioxidant activity was significantly increased by roasting, while in vitro digestibility showed that most antioxidative activities were available in the intestinal phase of gastrointestinal tracts.

Hypoglycaemic activity of four plant extracts traditionally used in South Africa for diabetes.

AIM: To validate plant species for hypoglycaemic activity. MATERIALS AND METHODS: Four plants were investigated for hypoglycaemic activity by evaluating inhibiting effects on carbohydrate-hydrolising enzymes: alpha-glucosidase and alpha-amylase. Acetone plant extracts were screened against C2C12 myocytes, 3T3-L1 preadipocytes and Chang liver cells by measuring glucose uptake. Cytotoxicity was done in preadipocytes and hepatocytes. RESULTS: Extract of Euclea undulata rootbark exhibited highest activity, displaying a glucose uptake of 162.2% by Chang liver cells at 50 microg/ml. An inhibition concentration of 50% for Euclea undulata was found to be 49.95 microg/ml for alpha-glucosidase and 2.8 microg/ml for alpha-amylase. No cytotoxicity was recorded for Euclea undulata, while Schkuhria pinnata and Elaeodendron transvaalense exhibited cytotoxicity at 12.5 microg/ml. Alpha-glucosidase and alpha-amylase assays showed inhibitory activity on enzymes for three plant extracts. CONCLUSION: Euclea undulata, Schkuhria pinnata and Elaeodendron transvaalense showed in vitro hypoglycaemic activity. Schkuhria pinnata and Elaeodendron transvaalense indicated cytotoxicity on 3T3-L1 preadipocytes and Chang liver cells. Euclea undulata, Pteronia divaricata and Elaeodendron transvaalense inhibited alpha-glucosidase and alpha-amylase enzymes. ETHNOPHARMACOLOGICAL RELEVANCE: Screening of plant extracts scientifically validated traditional use of Euclea undulata for treatment of diabetes. Cytotoxicity results revealed that acetone extracts of Schkuhria pinnata and Elaeodendron transvaalense are toxic and raise concern for chronic use.

In vitro anti-HIV activity of five selected South African medicinal plant extracts.

AIM OF THE STUDY: Five South African medicinal plants, Bulbine alooides (L.) Willd. (Asphodelaceae), Crinummacowani Baker (Amaryllidaceae), Hypoxis sobolifera var. sobolifera (Jacq.) Nel (Hypoxidaceae), Leonotisleonurus (L.) R.Br. (Lamiaceae) and Tulbaghiaviolacea Harv (Liliaceae) used for the treatment of various ailments, including infectious diseases, were screened for activity against human immunodeficiency virus (HIV). MATERIALS AND METHODS: Aqueous and ethanol extracts were tested for inhibitory activity in HIV-1 infected CEM.NK(R)-CCR5 cells, and against HIV-1 reverse transcriptase (RT) and HIV-1 protease (PR). RESULTS: In CEM.NK(R)-CCR5 cells, ethanol extracts of Leonotisleonurus inhibited HIV-1 significantly (33% reduction in HIV-1 p24, P<0.05). HIV-1 RT inhibition (> or =50%) was shown for extracts of Bulbine alooides (aqueous and ethanol), Hypoxis sobolifera (aqueous and ethanol) and Leonotisleonurus (aqueous), but inhibitory activity was lost upon dereplication for removal of non-specific tannins/polysaccharides. HIV-1 PR inhibition was observed for extracts of Hypoxis sobolifera (aqueous), Bulbine alooides (aqueous and ethanol) and Leonotisleonurus (ethanol). Only ethanolic extracts of Bulbine alooides and Leonotisleonurus retained HIV-1 PR inhibition after dereplication with IC50 of 94 microg/ml and 120 microg/ml, respectively. CONCLUSION: The dereplicated ethanolic extracts of Leonotisleonurus and Bulbine alooides showed the greatest anti-HIV potential in this study through inhibition of HIV-1 PR.

Influence of pre-treatment on yield chemical and antioxidant properties of a Nigerian okra seed (Abelmoschus esculentus moench) flour.

Okra seeds are reported to be limited to re-generational purpose in Nigeria while majority are discarded as unfit for this purpose. Studies were carried out to evaluate the effect of soaking and blanching on the yield, proximate composition and antioxidant activity of okra seed flour. Pre-treatment by soaking and blanching were found to increase yield which was time dependent. The range mean obtained for protein, fat, ash and fiber contents were 46.10-38.99, 28.08-25.08, 3.95-3.15 and 3.76-3.10, respectively. Slight but significant DPPH radical scavenging activity increase was observed in soaked samples at 18th-h while blanching resulted into progressive decrease.

Cytotoxic activity of selected Nigerian plants.

Cancer is one of the most prominent human diseases which has stimulated scientific and commercial interest in the discovery of new anticancer agents from natural sources. The current study investigates the cytotoxic activity of ethanolic extracts of sixteen Nigerian plants used locally for the treatment of cancer using the MTT assay on the HeLa cell line. Sapium ellipticum leaves showed activity comparable to the reference compound Cisplatin and greater cytotoxic activity than Combretum paniculatum, Celosia trigyna, Drymaria cordata, Cyathula achyranthoides and Cyathula prostata. Justica extensa, Pupalia lappacea, Hedranthera barteri leaves, Alternanthera sessilis, Ethulia conyzoides leaves, Combretum zenkeri root, Sapium ellipticum stembark and Lannea nigritana stembark showed very low activity while Combretum molle, Adenanthera parvoniana and Lannea acida showed no activity. The results justify the use of Sapium, Combretum, Celosia, Drymaria and Cyathula in traditional treatment of cancer.

In vitro anti-HIV-1 properties of ethnobotanically selected South African plants used in the treatment of sexually transmitted diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected The plants selected in this study are used traditionally in the treatment of sexually transmitted diseases and traditional healers interviewed claimed these plants can also help AIDS patients. AIM: To evaluating the in vitro anti-HIV properties of selected plants in various bioassays. MATERIALS AND METHODS: The extracts were evaluated for their inhibition against alpha-glycohydrolase, reverse transcriptase and viral proteins (NF-kappaB and Tat) which play a significant role in the HIV life cycle. RESULTS: Terminalia sericea extract (IC(50)=92mg/ml) exhibited a considerable alpha-glucosidase inhibitory activity which was better than acarbose (IC(50)=131mg/ml) under our assay conditions. In the reverse transcriptase assay, T. sericea also showed good inhibitory activity (IC(50)=43mg/ml), which was higher than that of the reference drug, Adriamycin (IC(50)=100mg/ml). The ethyl acetate extract of Elaeodendron transvaalense exhibited the most potent inhibitory activity in both the NF-kappaB and Tat assays with inhibitory activity of 76% and 75% respectively at a concentration of 15mg/ml. The acetone and chloroform extracts of E. transvaalense and Zanthoxylum davyi also showed good activity in the NF-kappaB and Tat assays.

The synthesis and anticancer activity of selected diketopiperazines.

Six selected diketopiperazines, cyclo(Gly-Val), cyclo(Gly-D-Val), cyclo(Gly-Leu), cyclo(Gly-Ile), cyclo(Phe-Cys) and cyclo(Tyr-Cys), were synthesized via various synthetic routes. Their potential to inhibit cancer cell growth in HT-29, HeLa and MCF-7 cells was determined. Cyclo(Tyr-Cys) caused the greatest inhibition in cervical carcinoma cells with near equivalent activity against HT-29 and MCF-7 cells. The other cyclic dipeptides tested were effective in the inhibition of colon, cervical and breast carcinoma cells, respectively, but the percentage inhibition was lower than for cyclo(Tyr-Cys).

Shifts in metabolic parameters surrounding glucose homoeostasis resulting from tricyclic antidepressant therapy: implications of insulin resistance?

This study displayed the physiological effects the tricyclic antidepressants amitriptyline or trimipramine have on glucose homoeostasis in male Wistar rats. An insulin secreting cell line (INS-1) was also used to determine effects tricyclic antidepressants have on insulin secretion and insulin displacement. Thirty rats each received a 1 mg kg-1 dose of amitriptyline or trimipramine for a period of 14 weeks; another 14 rats served as the control group. Blood glucose, serum insulin and muscle and liver glycogen levels were determined. Kidney, liver and muscle insulin degradation was measured and compared with insulin degrading enzyme concentrations in the latter two tissues. INS-1 cells were used to determine the effect 1 microM amitriptyline has on insulin secretion. Displacement studies for [3H]glibenclamide by amitriptyline or trimipramine were undertaken on INS-1 cells. A significant increase in blood glucose (P<0.01) was found for both test groups after 6 and 14 weeks of receiving the medication, which may be related to a significant decrease in liver and muscle glycogen levels (P<0.001). Serum insulin levels remained unchanged, although a significant increase in insulin degradation was observed in the muscle, liver and kidney, which may be related to a significant increase in insulin degrading enzyme (P<0.001) that was found. A significant increase in insulin secretion was observed for the INS-1 cells treated with amitriptyline, although no significant displacement for the [3H]glibenclamide was evident for amitriptyline or trimipramine. The significant alterations in glucose homoeostasis observed, as well as the significant changes associated with insulin secretion and degradation associated with amitriptyline or trimipramine treatment, imply that prolonged use of these medicines may lead to insulin resistance and full blown diabetes.

Anticoagulant effects of a Cannabis extract in an obese rat model.

Blood coagulation studies were conducted to determine the possible anti-/prothrombotic effect of an organic cannabis extract and the three major cannabinoids, THC, CBD and CBN. The in vitro effect of the cannabis extract on thrombin activity produced an IC50 value of 9.89 mg/ml, compared to THC at 1.79 mg/ml. It was also found that the extract, THC and CBN showed considerable inhibition of thrombin-induced clot formation in vitro with IC50 values of 600, 87 and 83 microg/ml for the extract, THC and CBN respectively. In an in vivo model used to determine clotting times of lean and obese rats treated with a cannabis extract, 50% clotting times were found to be 1.5 and 2 fold greater than their respective control groups, supporting the results obtained in the in vitro model. The study thus shows that Cannabis sativa and the cannabinoids, THC and CBN, display anticoagulant activity and may be useful in the treatment of diseases such as type 2 diabetes in which a hypercoagulable state exists.

The biological activity of the histidine-containing diketopiperazines cyclo(His-Ala) and cyclo(His-Gly).

Two cyclic dipeptides, cyclo(His-Ala) and cyclo(His-Gly,) were synthesized from their linear counterparts and their structures elucidated using standard elucidation techniques. Molecular modeling and predictive NMR results indicated that the majority of energetically favourable conformers adopted a boat conformation with respect to the diketopiperazine ring. Cyclo(His-Ala), at concentrations of 100 microM inhibited the growth, in vitro, of various cancer cell lines, including HT-29, MCF-7 and HeLa carcinoma cells while cyclo(His-Gly) inhibited the growth of MCF-7 cells. While the antibacterial potential of these two compounds was limited, both cyclic dipeptides significantly inhibited the growth of C. albicans. Both compounds at a concentration of 100 microM resulted in a decrease in heart rate, coronary flow rate and left ventricular systolic pressure in the isolated rat heart. Inhibition of thrombin, amounting to a 63.3% and 36.7% reduction in the rate of fibrin formation, was noted for cyclo(His-Ala) and cyclo(His-Gly), respectively. While cyclo(His-Ala) showed no notable effects on platelet aggregation, cyclo(His-Gly) significantly inhibited both pathways tested with greatest effects on thrombin-induced platelet aggregation, yielding an IC(50) of 0.0662 mM (R(2)=0.989). The results of the anticancer and hematological studies indicate that histidine-containing diketopiperazines have potential as a novel group of cytotoxic agents with antithrombotic effects.

Microcystin content of Microcystis aeruginosa is modulated by nitrogen uptake rate relative to specific growth rate or carbon fixation rate.

Modulation of microcystin production has been extensively studied in both batch and continuous cultures. Positive correlations with medium nitrogen, medium phosphorous, light intensity, inorganic carbon availability, and growth rate have been reported. Negative correlations have been reported between microcystin content and medium phosphorous. The only reported quantitative relationship between any variable and microcystin production was that of growth rate. Microcystis aeruginosa PCC7806 was therefore cultured under continuous culture conditions in a bubble-lift reactor at a growth rate of 0.01 h(-1) in modified BG11 (constant phosphate concentration of 0.195 mM and varying nitrate from 0.125 to 18 mM) and sampled at steady states for analysis of cell number, microcystin content, cellular N and P, residual medium nutrient concentration, and carbon fixation rate. Cellular microcystin quotas showed significant positive correlation with both nitrate uptake and cellular nitrogen content and were negatively correlated with carbon fixation rate, phosphate uptake, and cellular phosphorous. Thus, the ratio of nitrate uptake to phosphate uptake, cellular N to cellular P, and nitrate uptake to carbon fixation were positively correlated to cellular microcystin. Microcystin quotas increased 10-fold from the lowest to the highest steady-state values. Cellular microcystin content therefore is controlled to a significant extent by variables other than growth rate, as was previously reported, with nitrogen the most significant modulator. Batch culture in BG11 under identical conditions yielded increased microcystin when nitrogen uptake exceeded relative growth rate, confirming the importance of nitrogen uptake in the modulation of microcystin content for a specific growth rate.

Anti-HIV activities of organic and aqueous extracts of Sutherlandia frutescens and Lobostemon trigonus.

A screening process was applied to extracts made from Sutherlandia frutescens (L.) R. Br (Fabaceae) and Lobostemon trigonus (Boraginaceae) as identified by the Botany Department, University of Port Elizabeth to detect if any of the extracts inhibited the human immunodeficiency virus (HIV). For purposes of dereplication, sulphated polysaccharides were removed and bovine serum albumin (BSA) was included in the assays to adsorb non-specific tannins potentially present. In the reverse transcriptase (RT) assay, an aqueous extract of the Lobostemon leaves inhibited HIV-1 RT with an IC50 value of 49 microg/ml, while in the protease assay no inhibition was seen. In the alpha- and beta-glucosidase assays, no significant inhibition was seen with the inclusion of BSA, indicating tannin-based inhibitory effects on these two enzymes. The beta-glucuronidase inhibitory activity, however, was retained in the presence of BSA. The study shows that Sutherlandia extracts contain inhibitory compounds active against HIV target enzymes, while aqueous Lobostemon leaf extracts contain a potent HIV-1 RT inhibitor, thus showing a potential mechanistic action of these plants in aiding HIV-positive patients.

An investigation into the biological activity of the selected histidine-containing diketopiperazines cyclo(His-Phe) and cyclo(His-Tyr).

Although cyclic diketopiperazines have been known since the beginning of the century, only now have they attracted considerable interest with respect to their biological activity. The aim of this study was to determine if the diketopiperazines cyclo(L-histidyl-L-phenylalanyl) (cyclo(His-Phe)) and cyclo(L-histidyl-L-tyrosyl) (cyclo(His-Tyr)) have significant biological activity relevant to the treatment of cardiovascular-related disease states, cancer and infectious diseases. Haematological studies were performed, including thrombin substrate binding, blood clotting time, platelet adhesion, platelet aggregation and fibrinolysis assays. A cytotoxicity screening utilizing a tetrazolium-based assay on the cell lines HeLa, WHCO3, and MCF-7 was performed. The whole-cell patch-clamp technique was used to investigate ion-channel activity in ventricular myocytes of rats, and isolated rat heart studies were performed to investigate the cardiac effects involving heart rate and coronary flow rate. Cyclo(His-Tyr) produced a significant prolongation of blood clotting time, slowing of clot lysis and inhibition of ADP-induced platelet adhesion and aggregation (P < 0.05). Cyclo(His-Phe) showed significant (P < 0.05) anti-tumour activity, causing greatest reduction of cell viability in cervical carcinoma cells. Preliminary results from patch-clamp studies indicate that both diketopiperazines caused blocking of sodium and calcium ion channels, but opening of inward rectifying potassium ion channels. In the rat isolated heart studies, cyclo(His-Phe) caused a gradual reduction in heart rate (P = 0.0027) and a decrease in coronary flow rate (P = 0.0017). Cyclo(His-Tyr) significantly increased the heart rate (P = 0.0016) but did not cause any significant change of coronary flow rate (P > 0.05). Cyclo(His-Tyr) showed notable (P < 0.05) antibacterial activity and both diketopiperazines showed excellent antifungal activity (P < 0.05). These observations reveal diketopiperazines to be ideal lead compounds for the rational design of an agent capable of preventing metastasis, inhibiting tumour growth, and as potential chemotherapeutic, antiarrhythmic and antihypertensive agents, as well as potential antibacterial and antifungal agents.

Interaction of plasma lipoprotein subfractions with differentiating 3T3-L1 and human mammary preadipocytes in culture.

Differentiating 3T3-L1 preadipocytes (murine fatty fibroblasts) and human preadipocytes interact with human lipoprotein subfractions (HDL2 and LDLII/III) at all stages of the differentiation program, displaying saturable binding behavior. Both cell types interact similarly with LDLII/III as differentiation proceeds, showing increased binding affinities and capacities and maximal rates of uptake in the mature cells, as compared with the preadipocyte stage. These changes coincide with the intracellular appearance of lipid droplets. However, with regard to HDL2, a markedly different pattern of interaction is evident in both cell types. For 3T3-L1 cells, lowered binding and uptake affinities and capacities are apparent in the fully differentiated state for HDL2, as compared with LDLII/III. Human preadipocytes displayed two distinct affinity binding sites for HDL2 during the early stages of differentiation (days 2 and 3), as compared with a single affinity site for LDLII/III at all stages. However, in the fully differentiated human cells, only a single affinity site, indistinguishable from the high-affinity site present on day 2, is evident, and probably represents the only binding site of physiological significance in these cells. All the cellular developments appear to be largely unaffected by exposure of both preadipocyte types to added lipoproteins (HDL + LDL) in the medium during the early stages of the conversion process.