Reversal of mitochondrial permeability transition pore and pancreas degeneration by chloroform fraction of Ocimum gratissimum (L.) leaf extract in type 2 diabetic rat model.

Introduction: Unmanaged Diabetes Mellitus (DM) usually results to tissue wastage because of mitochondrial dysfunction. Adverse effects of some drugs used in the management of DM necessitates the search for alternative therapy from plant origin with less or no side effects. Ocimum gratissimum (L.) (OG) has been folklorically used in the management of DM. However, the mechanism used by this plant is not fully understood. This study was designed to investigate the effects of chloroform fraction of OG leaf (CFOG) in the reversal of tissue wastage in DM via inhibition of mitochondrial-mediated cell death in streptozotocin (STZ)-induced diabetic male Wistar rats. Methods: Air-dried OG leaves were extracted with methanol and partitioned successively between n-hexane, chloroform, ethylacetate and methanol to obtain their fractions while CFOG was further used because of its activity. Diabetes was induced in fifteen male Wistar rats, previously fed with high fat diet (28 days), via a single intraperitoneal administration of STZ (35 mg/kg). Diabetes was confirmed after 72 h. Another five fed rats were used as the normal control, treated with corn oil (group 1). The diabetic animals were grouped (n = 5) and treated for 28 days as follows: group 2 (diabetic control: DC) received corn oil (10 mL/kg), groups 3 and 4 were administered 400 mg/kg CFOG and 5 mg/kg glibenclamide, respectively. Body weight and Fasting Blood Glucose (FBG) were determined while Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) and beta cell (HOMA-ß), and pancreatic tissue regenerating potential by CFOG were assessed. Activity-guided purification and characterization of the most active principle in CFOG was done using chromatographic and NMR techniques. The animals were sacrificed after 28 days, blood samples were collected and serum was obtained. Liver mitochondria were isolated and mitochondrial permeability transition (mPT) was investigated by spectrophotometry. Results: CFOG reversed diabetic-induced mPT pore opening, inhibited ATPase activity and lipid peroxidation. CFOG reduced HOMA-IR but enhanced HOMA-ß and caused regeneration of pancreatic cells relative to DC. Lupanol was a major metabolite of CFOG. Discussion: Normoglycemic effect of CFOG, coupled with reversal of mPT, reduced HOMA-IR and improved HOMA-ß showed the probable antidiabetic mechanism and tissue regenerating potentials of OG.

Effects of different fractions of Calliandra portoricensis root bark on isolated rat liver mitochondrial membrane permeability transition pore.

BACKGROUND: Mitochondrial membrane permeability transition (MMPT) pore ha s emerged as a promisingtarget for various pharmacological interventions because of the consequent release of cytochrome c upon the opening of the pore which is the point of no return for apoptosis, a form of programed cell death that is down regulated in cancercells. AIM: To evaluate the modulatory effects of fractions (Chloroform fraction of calliandra portoricensis( CFCP), Aqueous fraction of calliandra portoricensis (AFCP), and Ethylacetate fraction of Calliandra portoricensis (EFCP) of methanol extracts of the root bark of Calliandra portoricensis (MECP), a medicinal plant used in the traditional treatment of prostate tumour, on mitochondrial membrane permeability transition (MMPT) pore. METHODOLOGY: Opening of the pore was assessed as mitochondrial swelling and was monitored spectrophotometrically as changes in absorbance at 540nm. RESULTS: Varying concentrations of MECP (10microg/ml, 20microg/ml, 40microg/ml, and 60microg/ml) induced opening of the pore, in the absence of calcium, by 1.1, 2.8, 4.5, 13.8 folds, respectively while spermine reversed this inductive effect. Interestingly, unlike MECP, EFCP and AFCP did not have any effect at lower concentrations (<40microg/ml) but induced pore opening at 60microg/ml, 80microg/ ml, 100microg/ml and 120microg/ml by 1.6, 3.1, 12.7, 16.7folds, respectively for EFCP and 1.4, 5.4, 7 and 10 folds respectively, for AFCP. In the presence ofcalcium, the pore was slightly further opened by MECP, EFCP andAFCP. The CFCP however did not have any significant effect on the pore either in the presence or absence of calcium. CONCLUSION: These findings suggest that the bioactive agents that induced the opening of the pore are present in the most potent ethylacetate fraction of the root bark of C. portoricensis. This fraction will therefore be useful for the structural elucidation of the bioactive principle in the plant and for further studies in diseases that require increased apoptosis such as cancer.

Therapeutic effects of various solvent fractions of Alstonia boonei (apocynaceae) stem bark on Plasmodium berghei-induced malaria.

Malaria, the most important parasitic disease afflicting man is the leading cause of mortality and morbidity in the world. Chemotherapy remains the mainstay for the treatment and prevention of the disease in the absence of an effective vaccine. The incidence of resistance of malaria parasites to chemotherapy is increasing and complicated. This study was therefore undertaken in order to evaluate the therapeutic effects of fractions of the stem bark of A. boonei on P. berghei-induced malaria using chloroquine as control. Different doses (200 mg/kg and 400 mg/kg body weight) of methanolic extract (ME), n-hexane (HF), chloroform (CF), ethylacetate(EF) and aqueous (AF) fractions of the stem bark of A. boonei were administered orally to albino mice. Five milligrammes chloroquine base per kilogramme body weight (5 mg/kg bw) was used as positive control while the negative control mice received only the vehicle (5% v/v tween 80). The results obtained showed that the 400 mg/kg bw dose was more effective with respect to the parasite clearance than the 200 mg/kg bw dose. The 400 mg/kg bw dose of ME gave 68.1% percent parasite clearance. The CF gave the highest clearance of 98.4% at 400 mg/kg bw after 7 days treatment while chloroquine at 5 mg/kg bw gave 100% parasite clearance. The order of increasing potency of the fractions (parasite clearance) was (EF 50.0% < AF 60.3% < HF 63.1%, < CF 98.4%) indicating that the active principle in the stem bark was highest in the CF. Percentage parasitemia following exposure to these fractions also decreased in all groups in the same order and was only significant (p < 0.05) in CF (0.11%) compared to the untreated control group. The ME of A. boonei also caused increase in PCV by 15.5%. Purification enhanced PCV value as the HF and CF fractions gave 19.0% and 24.5% increases, respectively. However, 31.5% increase in PCV was obtained in the albino mice treated with chloroquine. The EF and AF gave increase of 10.0% and 11.0% increase relative to the negative control treated mice. The high bioactivity of CF and HF indicate that the putative compound(s) in A. boonei are lipophillic and further purification could enhance greater activity. Further work is required to isolate the bioactive compound for a promising antimalarial drug from the chloroform fraction.

Human erythrocyte membrane Ca(2+)-ATPase during occupational exposure to lead.

OBJECTIVE: Erythrocyte membrane Ca(2+)-ATPase activity was determined in workers occupationally exposed to lead because of the prevalence of elevated blood lead in auto-mobile workers in some urban areas in Nigeria. MATERIALS AND METHODS: Blood lead levels, biochemical profiles, lipid peroxidation, basal and calmodulin-stimulated Ca(2+)-ATPase activities were determined in erythrocytes of different categories of workers occupationally exposed to lead. These subjects were mainly battery chargers (BC), spray painters (SP) and auto mechanics (MC). RESULTS: Estimation of erythrocyte Ca(2+)-ATPase activity in the absence of calmodulin (basal activity) in test groups indicated that there were significant reductions in the pump function and this correlated very well with the levels of lead in their blood. Specifically, blood lead levels were of the order: BC (5.5 folds) > SP (4 folds) > MC, although there was no significant difference between the blood lead levels in MC (10.60 +/- 2.55 microgPb2+/dl) and CT (8.51 +/- 4.55 microgPb2+/dl). Similarly, the order of reduction in Ca(2+)-ATPase activity was BC (69.8%) > SP (52.8%) > MC (32.6%). There was significant difference in the values obtained for MC and CT, ATPase activity being lower in MC compared to CT or healthy individuals. In the presence of calmodulin, basal ATPase activity was increased by at least four fold in erythrocytes from healthy subjects (CT) while the basal activity of the enzymes in membranes of BC, SP and MC was enhanced by about one and half times the activity of the pump in membranes from CT. The levels of total serum protein and albumin increased significantly in BC, SP and MC when compared to healthy subjects (CT). C-reactive proteins (C-RP) levels were higher in BC, SP and MC in comparison to CT. The levels of MDA were high in all lead-exposed workers, BC > SP > MC relative to CT. Although, there were significant decreases in the PCV values of all the groups occupationally exposed to lead compared to values obtained for CT, cholesterol level increased significantly only in BC when compared to the other groups. CONCLUSION: These observations are probably due to the integrity of the plasma membrane of these workers and the ability of the heavy metal to compete with Ca2+ in the catalytic cycle and Ca2+ transport mechanism of the pump protein.

Effects of extracts of the leaves of Brysocarpus coccineus on rat liver mitochondrial membrane permeability transition (MMPT) pore.

OBJECTIVE: To examine the influence or the effect of the extracts of Brysocarpus coccineus leaves on the mitochondrial membrane permeability transition (MMPT) pore opening in rats with a view to establishing if any bioactive constituent of the plant could become useful in the chemotherapy of cancer. MATERIALS AND METHODS: The effects of extracts of the leaves of Brysocarpus coccineus, a medicinal plant with anti-tumour, anti-inflammatory and analgesic properties, were assessed on rat liver mitochondrial membrane permeability transition (MMPT) pore in the presence and absence of calcium in vitro and in vivo. RESULTS: The results obtained show that calcium ions induced the opening of MMPT pore significantly (P < 0.05) in rat liver mitochondria, while spermine inhibited calcium-induced opening of pore, indicating that the mitochondria were intact ab initio. The results further revealed the inhibitory effects of different concentrations (200, 600, 1000, 1400, and 1800 microg/ml) of the various extracts of the leaves compared with spermine. Specifically, the data revealed that chloroform and ethylacetate extracts reversed calcium-induced opening of MMPT pore in a concentration-dependent manner (74%, 79%, 85%, 86%, 87%) for the chloroform extract and (36%, 37%, 59%, 71% and 83%) for the ethylacetate extract, respectively. On the contrary, pre-incubation of normal healthy mitochondria with the extracts in the absence of calcium resulted in the induction of the MMPT pore opening to varying degrees by these concentrations of the extracts. The chloroform extract induced pore opening in a concentration-dependent manner in the order 2.4, 2.4, 2.5, 2.6 and 3.0 folds while the ethylacetate extracts induced the opening of the pore by 1.1, 1.2, 1.3, 1.3 and 1.4 folds between 200-1800 microg/ml, respectively. The results obtained using rats orally exposed to various doses of methanol extract of the leaves of B. coccineus for fourteen days showed that there was significant (p < 0.05) induction of mitochondrial membrane permeability transition pore opening in the absence of calcium in a dose-dependent manner. Maximum induction of 26-fold was obtained at 200 mg/kgbwt while the least dose (50 mg/kgbwt) gave 17 fold induction. CONCLUSION: The ability of the extracts of B. coccineus to induce MMPT pore opening in the absence of calcium in vitro and in vivo suggest that the leaves of the plant contain certain bioactive substances capable of inducing MMPT opening either in the original form or as formed biotrans derivative with eventual release of apoptotic proteins which may lead to apoptosis. The property of the extracts could be exploited for cancer chemotherapy when increased rate of apoptosis is required.

Modulation of opening of rat liver mitochondrial membrane permeability transition pore by different fractions of the leaves of Cnestis ferruginea. D.C.

BACKGROUND: Increased attention is now directed towards the search for novel naturally occurring anticancer agents that can induce mitochondrial membrane permeability transition (MMPT) pore opening and cell death as a chemotherapeutic mechanism to combat cancer incidence. AIM: The inductive effects of partially purified fractions of leaves of Cnestis ferruginea- on rat liver MMPT pore opening was investigated. METHOD: De-fatted methanol extract of leaves of Cnestis ferruginea was partitioned between water, chloroform, ethylacetate, or butanol separately in succession. The extract solutions were concentrated at 40 degrees C to obtain water (WF), chloroform (CF), ethylacetate (EF) and butanol (BF) fractions. The effects of these fractions (0.2- 1.4 mg/ml) on MMPT pore opening or mitochondrial swelling in the presence and absence of calcium were evaluated The effects of these fractions on the rat liver mitochondrial F0F1-ATPase activity were also assessed. RESULTS: Ca(2+)-induced MMPT pore opening was inhibited by 1 mg/ml each of MECF, CF, BF, WF and EF by 75.0%, 83.0%, 88.0%, 68.0%, and 71.0%, respectively and compared with the effect of spermine, a standard inhibitor. However, in the absence of Ca2+, the fractions significantly induced MMPT pore opening in intact mitochondria by 7.0, 5.7, 0.7, 4.8, 10.9 folds, respectively. In normal rat liver mitochondria, F1F0-ATPase activity was stimulated maximally by MECF, CF, EF, BF and WF by 4.7, 12.7, 1.6, 3.6 and 1.5 folds, respectively, thus indicating that the chloroform fraction is the most potent and therefore contains the active principle in the plant. CONCLUSION: The present study revealed that the leaves of Cnestis ferruginea contain bioactive substances that induced mitochondrial membrane permeability transition and activated the specific activity of F0F1 ATPase. Thus, suggesting strongly that these bioactive agents may serve as a useful chemotherapeutic strategy in cancer therapy.

Sulphadoxine-pyrimethamine alters the antioxidant defense system in blood of rabbit.

Sulphadoxine-pyrimethamine (SP) despite reported resistance remains an important drug of choice for the treatment and control of malaria in most endemic areas. Exacerbation of intra-erythrocytic oxidative stress might contribute to the process of elimination of malaria parasites in the body. The effect of treatment with SP on the antioxidant defense system was investigated using rabbit as a model. Ten male rabbits were divided into two groups of five animals each. The first group was administered with normal saline and served as control. The second group received a single dose of SP (26.25mg/kg body weight). Blood samples were collected before and at 6, 12 and 24 h after drug administration. Activity of cellular enzymatic antioxidants, superoxide dismutase (SOD) and catalase (CAT), and level of reduced glutathione (GSH) were assayed using standard spectrophotometric methods. Serum lipid peroxidation was assessed by the formation of thiobarbituric acid reactive species (TBARS) while protein content was assayed by the method of Lowry et al., 1951. SOD activity was observed to increase progressively by 4.9, 63.4 and 120.8% at 6, 12 and 24 h respectively, after drug administration. Similarly, CAT activity increased by 44.5, 82.6 and 116.3% at 6, 12 and 24 h, respectively. TBARS level also increased significantly by 45.5, 118.2 and 186.4%, respectively. However, the level of GSH decreased by 41.9% at 6 h and remained so up till the 12 h, but by 24 h after drug administration, the level of the thiol substance has increased considerably up to 48.4% above the baseline level. SP treatment altered the antioxidant defense system in blood and may therefore induce oxidative stress by generating reactive oxygen species. This might play significant role in the therapeutic and adverse effects associated with the drug.

Effects of the leaf decoction of Momordica charantia (bitter melon) on Mitochondrial Membrane Permeability Transition Pore (MMPTP) and fertility in normal male albino rats.

Momordica charantia (M. charantia), a medicinal plant of the family, Cucurbitaceae, is used in treating an array of ailments including diabetes, heamorrhoids, fevers and various cancers. Programmed cell death may be modulated by an intrinsic pathway involving the release of cytochrome C when the mitochondrial membrane permeability transition (MMPTP) pore is opened. Opening of MMPT pore was assayed using the method of Lapidus and Sokolove. The results obtained revealed that there was a dose-dependent and significant increase in the opening of the MMPT pore in rats orally administered the decoction with maximum induction (11-fold increase) at 55mg/100g body weight (bw), although the extent of opening of the pore was reduced at 65mg/100g bw (9-fold increase). An assessment of the blood parameters of animals orally exposed to the decoction showed significant decrease (p<0.05) in lymphocytes and a significant increase (p<0.05) in neutrophils at 55mg/ 100g bw. Moreover, significant increases (p<0.05) in RBC levels at 45 and 65mg/100g bw, were observed. Similarly, PCV and Heamoglobin values were also elevated at 65mg/100g bw while there was a significant reduction (p<0.05) in MCV and MCH values at 45, 55 and 65mg/100g bw. MCHC values were reduced only in animals that received 65mg/ 100g when compared to control animals. Analysis of the spermiogram of the experimental rats showed significant reductions (p<0.05) in sperm motility and sperm cell concentrations for all animals that were orally exposed to the decoction. There was a significant reduction (p<0.05) in percentage viability in animals that received 45mg/100g bw and above. Morphological abnormalities of sperm cells above the proposed percentage range (10%) were also observed in animals that received 45mg/100g bw and above. However, decoction did not show any significant effect on ALT and AST levels but there were significant increases (p<0.05) in a somewhat dose-dependent pattern in ALP and ãGT levels for all groups in comparison to the control group. These findings thus suggest dose-related negative or toxic effects of sub acute (30-day) oral administration of leaf decoction of M. charantia in albino rats and may therefore pose some danger to humans especially in regard to male fertility in individuals who rely on oral administration of the decoction in treating various ailments.

Effects of methanolic and chloroform extracts of leaves of Alstonei boonei on rat liver mitochondrial membrane permeability transition pore.

The effects of methanolic and chloroform extracts of the leaves of Alstonei boonei, a medicinal plant with anti-malarial, anti-inflammatory and analgesic properties, were assessed on liver mitochondrial membrane permeability transition (MMPT) pore were assessed in experimental animals in vitro and in vivo. The results obtained showed that calcium ions induced the opening of MMPT pore significantly (P< 0.05) in rat liver mitochondria, while spermine (0.1mM) inhibited calcium-induced opening of MMPT pore of these mitochondria thus, indicating that the mitochondria were intact, ab initio. The results further revealed the inhibitory effects of different concentrations of the various extracts of leaves of Alstonei boonei (200 microg/ml, 600 microg/ml, 1000 microg/ml, 1400 microg/ml) compared with spermine. Specifically, the data revealed that methanolic and chloroform extracts of leaves of Alstonei boonei reversed calcium-induced opening of MMPT pore in a concentration-dependent manner (26.5%, 27.4%, 56.4%, 69.3%) for methanolic extract of Alstonei boonei and (9.6%, 34.9%, 51.5% and 82.1%) for chloroform extract of Alstonei boonei, respectively. Although, the MMPT pore was not affected by low concentrations of the methanolic extract of Alstonei boonei (200 microg/ml and 600 microg/ml) in the absence of calcium, the extract at higher concentrations (1000 microg/ ml and 1400 microg/ml) induced the opening of the pore in a concentration-dependent manner. Mitochondria isolated from Wistar strain albino rats orally exposed to various doses of the methanolic extract of Alstonei boonei exhibited pore opening in the absence of calcium. In this respect, maximum (112%) induction of pore opening was obtained at 250mg/kg body weight, while minimum (31%) induction of pore opening was obtained at 200mg/kg body weight. Calcium further increased the extent of opening of the MMPT pore in animals previously exposed to methanolic extract of Alstonei boonei. These findings suggest that certain bioactive components of Alstonei boonei may be involved in inhibiting the opening of the MMPT pore in-vitro and in the induction of the opening of the pore at high doses of the extract with the eventual release of cytochrome C which is a prelude to the progression of programmed cell death.

Induction of membrane permeability transition (MPT) pore opening by Buccholzia coriacea (Engl.) seeds in vitro in rat liver mitochondria.

Apoptosis involves a phenomenon termed mitochondrial permeability transition (MPT) which induces permeability of a voltage-dependent pore to solutes of smaller than 1500Da. Induction of MPT pore is beneficial in case of tumour cells while inhibition of the pore is relevant in conditions such as tissue wastage. The effects of methanol extracts of Buccholzia coriacea (MEBC) commonly known as 'wonder kola' on MPT wa s assessed in vitro in normal rats inthe presence and absence of exogenous calcium- the triggering agent. MPT was estimated by the extent of mitochondrial swelling monitored spectrophotometrically as decreases in absorbance at 540nm. The results revealed that in the absence ofexogenous calcium, MPT pore opening was induced by MEBC at 200 microg/ml, 600 microg/ml, 1000 microg/ml and 1400 microg/ml in a concentration-dependent manner by 21.0, 7.6, 4.2, 3.5 folds, although higher concentrations of MEBC reduced pore opening. Pre-incubation of mitochondria with similar concentrations of MEBC for 5 minutes in the absence of calcium induced pore opening by 1.47, 10, 8.7 and 10.1 folds, respectively. Furthermore, mitochondrial membrane treated with MEBC (200 microg/ml, 600 microg/ml, 1000 microg/ml and 1400 microg/ml) in the presence of exogenous calcium induced pore opening by 63.9%, 44.0%, 23.4% and 64.4%, respectively. Oral administration of MEBC at varying doses of 50 - 200mg/kg b.w to rats for 30 days had no significant effects (p>0.05) on MPT pore opening in the absence of calcium when compared to untreated animals. The liver function tests revealed that the activities of alanine and aspartate amino transferases, alkaline phosphatase, and ã-glutammyl transferase were significantly (p>0.05) increased in serum of animals exposed to MEBC compared to control animals. Overall, Buccholzia coriacea induced MPT pore opening in vitro thus suggesting that certain bioactive components in the extract may prove useful in chemotherapy of tumor cells however, these bioactive agents seem to have been completely metabolized in vivo.

Chloroquine Resistant Plasmodium falciparum in Nigeria: Relationship between pfcrt and pfmdr1 Polymorphisms, In-Vitro Resistance and Treatment Outcome.

This study was designed to evaluate the association between polymorphisms in pfcrt and pfmdr1 genes and in-vitro chloroquine (CQ) sensitivity in fresh isolates of P. falciparum and patients' treatment outcome. The modified schizont inhibition assay was used to determine in-vitro sensitivity of P. falciparum. Polymorphisms in pfcrt and pfmdr1 genes were detected using nested PCR and RFLP techniques in 84 P. falciparum isolates obtained from patients with acute uncomplicated malaria.Eighty five percent (71/84) and 15% (13/84) of the parasites were resistant and sensitive in-vitro to CQ respectively. Molecular analysis showed presence of mutant pfcrtT76, pfmdr1Y86 and pfmdr1F184 alleles in 60%, 33% and 14% of the isolates respectively. There was a significant association between in-vitro and in-vivo CQ resistance (p=0.029) and also between the presence of mutant pfcrtT76+pfmdr1 Y86-Y184 haplotype and in-vitro (p=0.013) or in-vivo CQ resistance (p=0.024).Overall results from this study demonstrates that the presence of pfcrtT76+ pfmdr1 Y86-Y184 haplotype in Nigerian isolates of Plasmodium falciparum is predictive of in-vitro and in-vivo CQ resistance and therefore may be useful for monitoring resistance to this drug.

Building bioinformatics capacity in West Africa.

African scientists need more bioinformatics training in order to make innovative contributions to global biotechnology. To address the bioinformatics skills gap in West Africa, various training initiatives have been established in the sub-region. We present the activities of the West African Biotechnology Workshops (http://www.wabw.org/) in the past three years, and report on a symposium on bioinformatics and applied genomics in West Africa. To establish and sustain regional and national networks, stronger and increased government commitment by way of financial and infrastructural support for bioinformatics capacity building in West Africa is required.

Toxic effects of chromatographic fractions of Phyllanthus amarus on the serum biochemistry of rats.

Chromatographic fractions obtained from Phyllanthus amarus were tested for toxicity on the serum biochemistry of rats. The results revealed that some fractions of P. amarus had potentially deleterious effects on the blood and therefore caution should be exercised in the use of P. amarus as a medicinal plant.

Morphometric and histopathological studies on the effects of some chromatographic fractions of Phyllanthus amarus and Euphorbia hirta on the male reproductive organs of rats.

The aqueous crude extracts of P. amarus and E. hirta were administered to thirty eight-week old sexually mature male albino to determine the effects of these extracts on the male reproductive organs of these animals. The results from this study revealed that the aqueous crude extracts of P. amarus and E. hirta caused varying degrees of testicular degeneration as well as reduction in the mean seminiferous tubular diameter (STD) in the treated rats. It thus shows that the aqueous crude extracts of P. amarus and E. hirta have potentially deleterious effects on the testes and accessory organs of rats. Great caution should therefore be exercised in the use of these plants for medicinal purpose.

Erythrocyte membrane protein alteration in diabetics.

OBJECTIVES: To study the protein components of the erythrocyte membranes of diabetic Nigerians and to compare the results with the erythrocyte membrane protein components of normal healthy Nigerians. DESIGN: Laboratory based cross-sectional study. SETTING: Department of Medicine, University College Hospital (UCH), Ibadan and Biomembrane Research Laboratory, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria. SUBJECTS AND METHODS: The study was carried out in patients with insulin-dependent diabetes mellitus--IDDM (Type 1 diabetes), non-insulin dependent diabetes mellitus--NIDDM (Type 2 diabetes) and healthy human volunteers (HHm), which served as controls. The subjects were aged 30-65 years. There were 12 subjects in each of the IDDM and NIDDM) and 18 subjects in the HHm group. Blood samples (20 ml per subject) were obtained from each subject and erythrocyte ghost membranes were isolated separately from each sample. Total erythrocyte membrane protein concentration of each sample was determined using bovine serum albumin (BSA) as standard. The protein components of the erythrocyte ghost membranes were determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis. STATISTICAL METHOD: All values given are the mean +/- standard deviation (+/- SD) of the parameters measured. Significance was assessed using Student's t-test and P values of 0.05 or less were taken as statistically significant. RESULTS: The total protein concentration of HHm was 5.5 +/- 0.01 micrograms/ml, total protein concentration of IDDM was 4.5 +/- 0.01 micrograms/ml while NIDDM was 5.1 +/- 0.02 micrograms/ml. The spectrin alpha and beta-chain bands are heavily present in the healthy human erythrocyte membranes and are absent in the insulin dependent diabetic membranes. The ankyrin band, band six and below are more pronounced in IDDM and NIDDM but are relatively absent in the healthy humans. CONCLUSIONS: The results obtained provide evidence of profound quantitative and qualitative alteration of the erythrocyte membrane proteins in diabetic Nigerians. This may likely have serious functional implications of the diabetic patients.

Ca++, Mg++-ATPase activity in insulin-dependent and non-insulin dependent diabetic Nigerians.

A study of Ca++, Mg++-ATPase activity was carried out in normal (HHm) and diabetic Nigerians of both sexes with insulin-dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM). The results showed that protein concentration of erythrocyte ghost membranes of healthy humans (HHm) was the highest when compared with protein concentrations of IDDM and NIDDM patients. The protein concentration was lowest in IDDM, while the value in NIDDM was between those of HHm and IDDM. The basal activities of erythrocyte Ca++-ATPase from IDDM and NIDDM were determined and were found to be significantly lower than that of HHm. The addition of calmodulin (CaM) 2 microg/ml stimulated the activity of the calcium pump in all the groups (IDDM, NIDDM and HHm). The effects of calcium (Ca++) and adenosine triphosphate (ATP) on the activity of the pump from each group were determined. Enzyme kinetics (Km and Vmax) revealed that the activity of Ca++, Mg++-ATPase was initiated by ATP in the presence of Ca++ in a dose-dependent manner. Calmodulin also enhanced the activity of the enzyme in the presence of Ca++ in all the groups, though activities in IDDM and NIDDM were significantly lower than in HHm. There was no significant difference in the activities between IDDM and NIDDM. These results suggest a defective calcium translocating mechanism in diabetic Nigerians.

Reversal of sodium arsenite inhibition of rat liver microsomal Ca2+ pumping ATPase by vitamin C.

Sodium arsenite (NaAsO2), at 10% of its median lethal dose, was administered to rats with and without vitamin C pretreatment. Liver microsomal fraction was isolated and the activity of Ca2+-ATPase was assayed. Sodium arsenite was found to inhibit the activity of the liver microsomal Ca2+-ATPase to 50% to that of control rats. The specific activity of the enzyme in rats administered sodium arsenite with vitamin C pretreatment was not significantly different from that of control rats.

Iron-induced oxidative stress in erythrocyte membranes of non-insulin-dependent diabetic Nigerians.

The presence of higher level of endogenous free radical reaction products in the erythrocyte ghost membrane (EGM) of Non-insulin-dependent diabetes mellitus (NIDDM) subjects compared with that of normal healthy controls has been demonstrated. The EGMs of NIDDM subjects were also shown to be more susceptible to exogenously generated oxidative stress than those of normal healthy individuals. The decreased level of reactive thiol groups in the EGM of NIDDM individuals supported this observation. We propose that the presence of significant levels of non-heme iron in the EGM of NIDDM subjects is an indication of the potential for iron-catalysed production of hydroxy and other toxic radicals which could cause continuous oxidative stress and tissue damage. Oxygen free radicals could therefore be responsible for most of the erythrocyte abnormalities associated with non-insulin-dependent diabetes and could indeed be intimately involved in the mechanism of tissue damage in diabetic complications.

The anticalmodulin effect of aflatoxin B1 on purified erythrocyte Ca(2+)-ATPase.

The genotoxic carcinogen aflatoxin B1 (AFB1) inhibited the calmodulin-stimulated membrane-bound (Ca2+Mg2+)-ATPase. Using the purified enzyme, 12 nmoles per ml of AFB1 caused maximum inhibition of 28% and 50%, of the acidic phospholipid-stimulated and calmodulin-activated Ca(2+)-ATPase activity respectively. Treatment of red cell ghosts with increasing concentrations of Triton X-100, a non-ionic detergent caused a progressive loss of both the basal and calmodulin-stimulated Ca(2+)-ATPase activity. The activity of the phospholipid-free, detergent-solubilized enzyme was almost fully restored by phosphatidyl serine (PS) and its sensitivity to calmodulin was restored in the presence of phosphatidyl choline (PC). Analysis of the results obtained using varying concentrations of ATP shows that AFB1 did not affect the Km and Vmax of the unstimulated enzyme whereas these parameters were reduced by about 75% and 50%, respectively, in the presence of calmodulin. Using the product of limited proteolysis by trypsin i.e. the 90 kDa fragment which still retains its calmodulin binding-domain and the 76 kDa fragment which has lost this domain, kinetic studies on the enzyme activity revealed that AFB1 inhibited the calmodulin-activated 90 kDa fragment by about 50% while the 76 kDa was not affected at all by the toxin and calmodulin. The toxin had no significant affect on the basal activity of the 90 kDa limited proteolysis fragment of the enzyme. These observations suggest that AFB1 inhibits the activated Ca(2+)-ATPase by binding to an important site in the calmodulin-binding domain of the enzyme. It seems likely that the toxin binds to tryptophan in the calmodulin-binding domain, thus causing a reduction in the rate at which this domain can interact with Ca(2+)-calmodulin or acidic phospholipids. The implication of these observations is that Ca(2+)-extrusion and other calmodulin-activated enzymes and processes may be slowed down during prolonged exposure to AFB1 because of its anticalmodulin effect.