Biologically active bisbenzylisoquinoline alkaloids from the root bark of Epinetrum villosum.

Methanol and water extracts of the root of Epinetrum villosum (Exell) Troupin (Menispermaceae) were found to exhibit antimicrobial and antiplasmodial activities. Investigation of the active methanol fraction led to the isolation of four bisbenzylisoquinoline alkaloids, i.e., cycleanine, cycleanine N-oxide, isochondodendrine and cocsoline. Structures were established by spectroscopic methods. Cocsoline displayed antibacterial and antifungal activities (MIC values of 1000-15.62 and 31.25 microg/ml, respectively). Isochondodendrine was found to have the most potent antiplasmodial activity (IC50 = 0.10 microg/ml), whereas the IC50 on HCT-116 human colon carcinoma cells was 17.5 microg/ml (selectivity index 175). Cycleanine acted against HIV-2 (EC50=1.83 microg/ml) but was at least 10-fold less active against HIV-1. Cycleanine N-oxide showed no activity towards all tested microorganisms.

Pyruvate-induced long-term maintenance of glutathione s-transferase in rat hepatocyte cultures.

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, mu and alpha class GST activities were well maintained, whereas GST pi activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: mu,class proteins remained relatively constant, whereas a decrease in the alpha class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, pi class GST activity was increased, but total, mu, and alpha class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.

Antidiarrhoeal activity of root extracts from Roureopsis obliquifoliolata and Epinetrum villosum.

The methanol extracts of Roureopsis obliquifoliolata and Epinetrum villosum root significantly reduced castor oil-induced diarrhoea in mice. This effect supports the use of these plants in Congolese folk medicine as antidiarrhoeal remedies.

Hormonal regulation of glutathione S-transferase expression in co-cultured adult rat hepatocytes.

Glutathione S-transferases (GSTs) are subject to regulation by thyroid and sex hormones and by GH. We have used an in vitro experimental system comprising adult rat hepatocytes co-cultured with rat liver epithelial cells of primitive biliary origin, to distinguish between direct and indirect effects of various hormones on GSTs; to identify the GST subunits affected by individual hormones; and to investigate the level at which the hormones act. Tri-iodothyronine (T3), thyroxine (T4) and 17beta-oestradiol (OE2) reduced GST activities, whereas testosterone, dihydrotestosterone, and human growth hormone (hGH) had little effect on total GST activity. HPLC separation of the various GST subunits revealed that T3 and T4 reduced total GST content, in particular the abundance of subunits M1 and M2. The amount of the Pi-class subunit P1 was reduced by OE2. Treatment of the co-cultured cells with this hormone altered the GST subunit profile to one that is more similar to that observed in freshly isolated hepatocytes. Analysis of mRNAs demonstrated that some of the hormones act at a pre-translational level, whereas others act at a translational or post-translational level to regulate the expression of various GST subunits.

Contribution to the ethnobotanical, phytochemical and pharmacological studies of traditionally used medicinal plants in the treatment of dysentery and diarrhoea in Lomela area, Democratic Republic of Congo (DRC).

In order to collect ethnobotanical information about antidiarrhoeal plants, we performed inquiries among traditional healers, community leaders, and native people of Lomela villages in Congo. Six medicinal plants widely used in this region were designated as having antidysenteric and antidiarrhoeal properties. These six medicinal plants were screened for groups of phytochemical compounds with antibacterial and antiamoebic activities. They were found to contain tannins, alkaloids, saponins, flavonoids, sterols and/or triterpenes and reducing sugars. Of the six tested plants, three showed prominent antibacterial activity whereas two acted against Entamoeba histolytica. The usefulness of the phytochemical bases and biological activities of these plants as potential source of antidiarrhoeal remedies is discussed.

In vitro antimicrobial activity of six medicinal plants traditionally used for the treatment of dysentery and diarrhoea in Democratic Republic of Congo (DRC).

Twenty-four crude extracts derived from six medicinal plants highly valued as antidiarrhoeal agents in Congolese folk medicine were screened for antimicrobial activity against several enteric pathogens. The results of this study indicated that the methanolic and aqueous extracts derived from three of them (Roureopsis obliquifoliolata, Epinetrum villosum and Cissus rubiginosa) possessed prominent antibacterial activity, therefore supporting the ethnomedical uses of these species. In addition, phytochemical analysis of these medicinal plants showed that 1/6 plant sample contained alkaloids, 6/6 triterpenes and/or sterols, 4/6 flavonoids, 3/6 tannins and 5/6 saponins. Anthraquinones were not detected in any of these plants.

Prolonged exposure of pancreatic beta cells to raised glucose concentrations results in increased cellular content of islet amyloid polypeptide precursors.

Most non-insulin dependent diabetic patients have amyloid deposits in their pancreatic islets. It is not known whether chronic hyperglycaemia contributes to the formation of amyloid fibrils from the islet amyloid polypeptide that is produced by the pancreatic beta cells. Since islet amyloid exhibits islet amyloid polypeptide precursors immunoreactivity, we examined whether sustained in vitro exposure to raised glucose increases the abundance of these precursors in human beta cells. After 6 days stimulation with 20 mmol/l glucose the cellular content of insulin but not islet amyloid polypeptide was decreased leading to an increase in the ratio of the latter over insulin (3.0 +/- 0.6 vs 1.8 +/- 0.3 after 6 mmol/l glucose culture, p < 0.05). Similar changes occurred in rat beta cells cultured for 3 days in the presence of 20 mmol/l glucose plus 3-isobutyl-1-methylxanthine. Western blot analysis of cellular islet amyloid polypeptide after prolonged exposure to high glucose indicated the presence of higher proportions of its precursor- and intermediate forms. In human beta cells cultured in 20 mmol/l glucose, the major form corresponds to an intermediate species which exhibits an immunoreactivity for the N-flanking peptide, as is also the case in islet amyloid. We concluded that prolonged in vitro exposure of beta cells to raised glucose concentrations increases the relative proportion of islet amyloid polypeptide over insulin, as well as of its precursors over the mature form of islet amyloid polypeptide.

Effect of glucose on production and release of proinsulin conversion products by cultured human islets.

Isolated human islets were examined for the rates of conversion and release of newly formed (pro)insulin-like peptides. The rate of proinsulin (PI) conversion was 2-fold slower in human beta-cells (t(1/2) = 50 min) than in rat beta-cells (t(1/2) = 25 min). During the first hour following labeling of newly synthesized proteins, PI represented the main newly formed hormonal peptide in the medium; its release was stimulated 2-fold over the basal level by 20 mmol/L glucose. During the second hour, newly synthesized hormone was mainly released as insulin, with 10- to 20-fold higher rates at 20 mmol/L glucose. Prolonged preculture of the islets at 20 mmol/L glucose did not delay PI conversion, but markedly increased the release of newly formed PI, des(31,32)-PI, and insulin at both low and high glucose levels. Our data demonstrate that 1) the release of PI provides an extracellular index for the hormone biosynthetic activity of human beta-cells; 2) an acute rise in glucose exerts a stronger amplification of the release of converted hormone than in that of nonconverted hormone; and 3) prolonged exposure to high glucose levels results in an elevated basal release of converted and nonconverted PI; this elevation is not associated with a delay in PI conversion, but is attributed to the hyperactivated state of the human beta-cell population, which was recently found to be responsible for an elevation in basal rates of hormone synthesis. These in vitro observations on human beta-cells provide a possible explanation for the altered circulating (pro)insulin levels measured in nondiabetic and noninsulin-dependent diabetic subjects.

Differential expression of rat insulin I and II messenger ribonucleic acid after prolonged exposure of islet beta-cells to elevated glucose levels.

Prolonged exposure of rat islet beta-cells to 10 mmol/liter glucose has been previously shown to activate more cells into a glucose-responsive state (>90%) than has exposure to 6 mmol/liter glucose (50%). The present study demonstrates that this recruitment of more activated cells results in 4- to 6-fold higher levels of proinsulin I and proinsulin II messenger RNA (mRNA). However, only the rate of proinsulin I synthesis is increased. Failure to increase the rate of proinsulin II synthesis in the glucose-activated cells results in cellular depletion of the insulin II isoform, which can be responsible for degranulation of beta-cells cultured at 10 mmol/liter glucose. Higher glucose levels (20 mmol/liter) during culture did not correct this dissociation between the stimulated insulin I formation and the nonstimulated insulin II formation. On the contrary, the rise from 10 to 20 mmol/liter glucose resulted in a 2-fold reduction in the levels of proinsulin II mRNA, but not of proinsulin I mRNA; this process further increased the ratio of insulin I over insulin II to 5-fold higher values than those in freshly isolated beta-cells. The present data suggest that an elevated insulin I over insulin II ratio in pancreatic tissue is a marker for a prolonged exposure to elevated glucose levels. The increased ratio in this condition results from a transcriptional and/or a posttranscriptional failure in elevating insulin II formation while insulin I production is stimulated in the glucose-activated beta-cells.

Separation of a triterpenoid saponin mixture from Maesa lanceolata: semipreparative reversed-phase wide pore high performance liquid chromatography with temperature control.

A mixture of triterpenoid saponins derived from the dried leaves of Maesa lanceolata was separated, without structure deterioration, in its components. Seven fractions (I-VII) of high molecular weight (1234-1358) saponins were obtained on a semipreparative scale using wide pore reversed-phase high performance liquid chromatography with an acetonitrile trifluoroacetic acid (500:0.3 w/w)-water-trifluoroacetic acid (391:0.3, w/w) gradient from 35 to 56% in 30 min. The mobile phase was cooled in an ice bath (0 degrees C) during chromatography in order to prevent bubble formation and to improve the quality of the separation. Freeze-dried fractions IV, V, VI and VII were further separated using solvent systems developed for each of the fractions. Fourteen pure triterpenoid saponins were isolated in this way and their molar weight determined.

Proinsulin and its conversion intermediates in human pancreas and isolated islet tissue: kinetics and steady-state analysis.

In non-insulin-dependent diabetes, circulating insulin-related immunoreactivity (IRI) is often composed of a higher fraction of the incompletely converted forms proinsulin and des-31,32 proinsulin. The present study describes an immunoadsorption method for measuring the proportions of proinsulin, its two split products, and insulin in human pancreatic tissue and for determining their rates of formation in human isolated islets. The method uses two junction-specific monoclonal proinsulin antibodies in a protein G fractionation; it is validated by > or = 90% specificity and recovery. The peptide contents measured in tissue extracts were comparable to those determined in a previously developed immunoradiometric assay. In the nine tissue extracts from nondiabetic donor organs, 97% of IRI corresponded to insulin, 1% to proinsulin, 2% to the des-31,32 proinsulin conversion product, and 0.1% to des-64,65 proinsulin. Two samples from non-insulin-dependent diabetics under sulfonylurea treatment contained a fourfold lower content of IRI but the peptide distribution was comparable except for a low percentage (0.3) of proinsulin in one case. In pulse-chase experiments on three-preparations of human islets isolated from nondiabetic donors, proinsulin represented the major (> 90%) IRI that was synthesized at the end of the 30-min pulse; a subsequent 90-min chase at either 2.5 or 10 mM glucose resulted in conversion of 75% of proinsulin to des-31,32 (20%) and des-64,65 (2%) intermediates and to insulin (50%); after a 180-min chase, 88% of proinsulin was converted to insulin, but 10% remained present as proinsulin. In a pulse-chase experiment on islets isolated from tissue with a high proportion of des-31,32 intermediate (5% instead of 2%), the conversion process was slower (45% after 90 min and 70% after 180 min) and resulted in a higher fraction of des-31,32 intermediate, suggesting that the elevated tissue content in this intermediate is caused by a reduced PC2 converting activity. These data confirm that des-31,32 proinsulin represents the major conversion intermediate in normal human islets and indicate the existence of slow converters, possibly as a result of decreased enzymatic processing of the prohormone's AC junction.

Abnormal circulating pancreatic enzyme activities in more than twenty-five percent of recent-onset insulin-dependent diabetic patients: association of hyperlipasemia with high-titer islet cell antibodies. Belgian Diabetes Registry.

Pancreatic amylase and lipase activities were measured in sera of 307 Caucasian insulin-dependent diabetes mellitus patients (IDDM) at clinical onset, 303 nondiabetic siblings of registered patients, and 207 control subjects under age 40 years. In all subject groups lipasemia and pancreatic (but not salivary) amylasemia increased with age and were significantly correlated. Using age-dependent reference ranges, reduced pancreatic enzyme levels were measured in 18% of patients, 6% of siblings, and only 2% of control subjects (p < 0.001). Increased lipase levels were noted in 10% of patients and in only 3% of siblings and 2% of control subjects (p < 0.001). Using both univariate and multivariate statistical analysis, elevated lipase activities at clinical onset were associated with higher titers of autoantibodies against islet cell cytoplasmic antigens and glucagon, but not against insulin or the 65-kDa isoform of glutamic acid decarboxylase (GAD65-Ab), or with markers of genetic predisposition or metabolic dysregulation. These findings indicate the presence of modest, but statistically significant, variations in circulating pancreatic enzyme levels in 28% of IDDM patients at clinical onset (p < 0.001 vs. 5% in control subjects). Increased lipase levels may express a form or a stage of the disease with exocrine cell damage; their association with higher titers of islet cell and glucagon autoantibodies is not yet explained. Lower lipase and isoamylase levels are thought to result from the reduced acinar cell function in the vicinity of insulin-depleted islets. It must be tested whether pancreatic enzyme activities in serum can also be altered during the preclinical stage and can thus be considered as an additional marker for the disease process in the pancreas.

Effect of ethanol on glutathione S-transferase expression in co-cultured rat hepatocytes.

To examine the effects of ethanol (EtOH) on rat liver glutathione S-transferase (GST, E.C. 2.5.1.18), a key phase 2 biotransformation enzyme, an in vitro system consisting of rat hepatocytes co-cultured with rat epithelial cells derived from primitive biliary duct cells was used. Cells were either untreated or treated for 10 days with 0.1 or 0.4% EtOH. Cytosolic GST activities were assayed spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-dichloro-4-nitrobenzene (DCNB) as substrates. When cultures were exposed to 0.4% EtOH the total GST activity towards the substrate CDNB increased by 35%. However, the activity of GSTs towards DCNB did not increase significantly in the cells treated with this concentration of EtOH. Affinity chromatography followed by reversed phase HPLC was used to separate the various GST subunits. EtOH was found to affect the GST subunit pattern. Total GST proteins increased after exposure to 0.4% EtOH; this was largely due to a three-fold and 2.5-fold increase in the GST mu-class subunits 3 and 4 respectively. Since EtOH induces GST expression in liver parenchymal cells, particularly the mu-class GSTs, its addition as a solvent to cultured hepatocytes or its use in vivo may result in important changes in the metabolism and toxicity of the xenobiotics under study.

Formation of poliomyelitis subviral particles in the yeast Saccharomyces cerevisiae.

The sequence of the poliovirus genome encoding 3CD (a protease) was transferred to the yeast Saccharomyces cerevisiae on expression vectors with either a constitutive or an inducible promoter. Transformants could only be obtained with vectors carrying the inducible transcription unit. Extracts of induced cells were able to cleave cell-free synthesized P1, the precursor of the poliovirus capsid proteins, into VP0, VP3 and VP1. In yeast cells constitutively expressing P1, induction of 3CD expression resulted in only trace amounts of processed products. Processing could be improved considerably by simultaneous induction of both P1 and 3CD expression. Analysis of extracts of such induced cells revealed the presence of particles that resembled authentic subviral particles.

Comparison of capillary zone electrophoresis with high-performance liquid chromatography for the determination of additives in foodstuffs.

A capillary zone electrophoretic (CZE) method was developed to determine caffeine, aspartame and benzoic acid in diet cola soft drinks and in artificial sweetening powders. The effects of pH, ionic strength, organic solvents and different buffers were investigated to select the optimum conditions. These consisted of a sodium phosphate buffer at pH 11 and ionic strength 0.025. The running voltage was set at 15 kV and the injection was performed hydrostatically for 30 s. The CZE method was then compared with a previously developed high-performance liquid chromatographic (HPLC) method in terms of repeatability, reproducibility, accuracy, linearity, sensitivity and separation efficiency. Both methods gave good repeatability. The relative standard deviations for reproducibility were significantly higher in CZE than in HPLC. The main reason for this is probably the condition of the wall of the capillary, which was difficult to keep constant between the days of analysis. The separation efficiency of CZE was 65-110 times higher than that of HPLC; on the other hand, 10-20 times lower detection limits were obtained in HPLC. Both methods were linear, but the linear ranges were different owing to the lower detection limit of HPLC. In CZE, the effect of the matrix was higher.

Detection of autoantibodies against islet amyloid polypeptide in human serum. Lack of association with type 1 (insulin-dependent) diabetes mellitus, or with conditions favouring amyloid deposition in islets. The Belgian Diabetes Registry.

A radiobinding assay for the detection of autoantibodies against islet amyloid polypeptide was developed, analytically validated, and--in parallel with a similar assay for the detection of autoantibodies against insulin--applied to sera from recent-onset Type 1 (insulin-dependent) diabetic patients and from age- and sex-matched control subjects. There was no difference in islet amyloid polypeptide autoantibody titres between patient groups and matched control subjects, nor within subject groups according to age. At onset of Type 1 diabetes, elevated islet amyloid polypeptide-autoantibody levels (> 97th percentile of control subjects) were only detected in 1 of 30 patients aged 0-19 years and in 2 of 35 patients aged 20-39 years. By contrast, insulin autoantibodies were frequently demonstrated, in particular at onset of diabetes under age 20 (0-19 years: 18 of 30 patients; 20-39 years: 10 of 35 patients; p < 0.01 vs matched control subjects). Islet amyloid polypeptide autoantibodies were not detectable in 3 insulinoma patients nor in 37 patients (aged 33-70 years) with Type 2 diabetes (vs 1 of 40 in matched control subjects). In positive serum, adsorption onto protein A-Sepharose removed islet amyloid polypeptide binding activity, hereby confirming its antibody nature. In conclusion, Type 1 diabetes is associated with an age-dependent autoantibody reaction against insulin but not against islet amyloid polypeptide. Conditions associated with amyloid deposition in islets (Type 2 diabetes, insulinoma and ageing) do not favour the formation of autoantibodies against islet amyloid polypeptide.

Presence of islet amyloid polypeptide in rat islet B and D cells determines parallelism and dissociation between rat pancreatic islet amyloid polypeptide and insulin content.

The islet amyloid polypeptide (IAPP) immunoreactivity of the adult rat pancreas is located in insulin-containing B cells as well as in somatostatin-containing D cells. In both cell types, the IAPP immunoreactivity is identical to rat synthetic IAPP in terms of its elution position after reversed phase HPLC and its binding to IAPP antibodies. The IAPP content per 10(6) B-cells is more than 100 fold lower than the corresponding insulin content, but comparable to the IAPP content of D cells. After induction of diabetes by streptozotocin, pancreatic IAPP seems predominantly located in somatostatin-containing cells. In normal rats, pancreatic insulin and IAPP content increase 20 fold from birth to 12 weeks of age; beyond week 12, the further rise in pancreatic insulin was not paralleled by an increase in IAPP content.

In vitro assembly of poliovirus 14 S subunits: identification of the assembly promoting activity of infected cell extracts.

Purified poliovirus 14 S subunits are assembled into empty capsids in vitro only if their concentration exceeds a 1.6 nM threshold. This also holds true for the 14 S subunits in unpurified extracts of infected cells. Such an extract may promote the assembly of extraneous 14 S subunits, but only if it contributes enough 14 S subunits to raise their total concentration over the threshold. As a result of assembly, the concentration of 14 S subunits in an infected cell extract decreases exponentially, with a half life of 15 min at 37 degrees. When purified 14 S subunits of serotype 1 are mixed with extracts of cells infected with type 2 or 3, chimeric empty capsids are formed, thus showing the pooling of endogenous and extraneous 14 S subunits. In conclusion, the assembly promoting activity of infected cell extracts amounts to nothing more than the supply of endogenous 14 S subunits.

Purification of poliovirus 14 S subunits by sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography.

Purification of 14 S subunits from extracts of poliovirus-infected HeLa cells was achieved by a combination of sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography. The particles were free of admixtures of other subviral particles, of nonstructural viral proteins, and of host cell proteins. The purified material retained the physical and antigenic properties of native 14 S subunits fully, as well as their ability to assemble to empty capsids in vitro.

Changes in expression and "de novo" synthesis of glutathione S-transferase subunits in cultured adult rat hepatocytes.

Glutathione S-transferase (GST) isoenzymes of conventionally and co-cultured adult rat hepatocytes were purified and the GST subunits were separated by reversed phase HPLC in order to study the development of the GST subunit composition as a function of culture time and culture conditions. Several media conditions were tested, namely medium with and without fetal calf serum and with nicotinamide or dimethyl sulphoxide. Compared to the GST subunit composition of freshly isolated hepatocytes, changes in culture and media conditions result in a modification of the subunit profile. General observations are a decrease of subunits 1 and 2, an increase of subunit 3, a stabilization of subunit 4 and "de novo" expression of subunit 7. When [35S] methionine was added to the various culture media, and the thus labelled subunits were purified and separated, it was shown that cultured adult rat hepatocytes are able to synthesize the different GST proteins. Furthermore, the GST subunit composition, measured during various culture conditions, is probably a reflection of the "de novo" synthesis in vitro.