Induction of foetal haemoglobin synthesis in erythroid progenitor stem cells: mediated by water-soluble components of Terminalia catappa.

Current novel therapeutic agents for the treatment of sickle cell anaemia (SCA) focus on increasing foetal haemoglobin (HbF) levels in SCA patients. Unfortunately, the only approved HbF-inducing agent, hydroxyurea, has long-term unpredictable side effects. Studies have shown the potential of plant compounds to modulate HbF synthesis in primary erythroid progenitor stem cells. We isolated a novel HbF-inducing Terminalia catappa distilled water active fraction (TCDWF) from Terminalia catappa leaves that induced the commitment of erythroid progenitor stem cells to the erythroid lineage and relatively higher HbF synthesis of 9.2- and 6.8-fold increases in both erythropoietin (EPO)-independent and EPO-dependent progenitor stem cells respectively. TCDWF was differentially cytotoxic to EPO-dependent and EPO-independent erythroid progenitor stem cell cultures as revealed by lactate dehydrogenase release from the cells. TCDWF demonstrated a protective effect on EPO-dependent and not EPO-independent progenitor cells. TCDWF induced a modest increase in caspase 3 activity in EPO-independent erythroid progenitor stem cell cultures compared with a significantly higher (P˂0.05) caspase 3 activity in EPO-dependent ones. The results demonstrate that TCDWF may hold promising HbF-inducing compounds, which work synergistically, and suggest a dual modulatory effect on erythropoiesis inherent in this active fraction.

Isolation of peripheral blood mononuclear cells using glycerol density gradient.

Isolation of peripheral blood mononuclear cells (PBMCs) is fraught with challenges including, but not limited to, the cost of limited gradients available for the isolation of PBMCs. Glycerol gradient (1.077 g/ml) was used to isolate PBMCs from adult peripheral blood. The differentiation potential of the isolated cells was assessed by culturing the cells in MEM at 37 °C in 5 % CO2. The results demonstrated that the isolated cells could differentiate into committed linages of the erythroid progeny. LDH assay revealed that glycerol was not cytotoxic to the cells. The use of glycerol density as an alternative could be significant in cell culture experiments.

Anemia amelioration by lactose infusion during trypanosomosis could be associated with erythrocytes membrane de-galactosylation.

African trypanosomosis is a potentially fatal disease that is caused by extracellular parasitic protists known as African trypanosomes. These parasites inhabit the blood stream of their mammalian hosts and produce a number of pathological features, amongst which is anemia. Etiology of the anemia has been partly attributed to an autoimmunity-like mediated erythrophagocytosis of de-sialylated red blood cells (dsRBCs) by macrophages. Lactose infusion to infected animals has proven effective at delaying progression of the anemia. However, the mechanism of this anemia prevention is yet to be well characterized. Here, the hypothesis of a likely induced further modification of the dsRBCs was investigated. RBC membrane galactose (RBC m-GAL) and packed cell volume (PCV) were measured during the course of experimental trypanosomosis in mice infected with Trypanosoma congolense (stb 212). Intriguingly, while the membrane galactose on the RBCs of infected and lactose-treated mice (group D) decreased as a function of parasitemia, that of the lactose-untreated infected group (group C) remained relatively constant, as was recorded for the uninfected lactose-treated control (group B) animals. At the peak of infection, the respective cumulative percent decrease in PCV and membrane galactose were 30 and 185 for group D, and 84 and 13 for group C. From this observed inverse relationship between RBCs membrane galactose and PCV, it is logical to rationalize that the delay of anemia progression during trypanosomosis produced by lactose might have resulted from an induction of galactose depletion from dsRBCs, thereby preventing their recognition by the macrophages.

'Prevalence and antimicrobial susceptibility of Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains from raw meat and meat products in Zaria, Nigeria.

UNLABELLED: The bacterial genera Listeria and Staphylococcus have been frequently isolated from food products and are responsible for a number of animal and human diseases. The aim of the study was to simultaneously isolate and characterize L. monocytogenes and Staphylococcus species from 300 samples of raw meat and meat products, to determine the susceptibility of the organisms to commonly used antimicrobial agents and to determine the presence of haemolysin A (hyl) virulence gene in L. monocytogenes and staphylococcal cassette chromosome mecA (SCCmec) gene in the Staph. aureus isolates using PCR. Of the 85 Listeria isolates tested, 12 L. monocytogenes were identified and tested for their sensitivity to 14 antimicrobial agents. All the 12 isolates (100%) were resistant to nine antimicrobial agents, but however sensitive to gentamicin. Only one isolate was found to harbour the hylA gene. Twenty-nine isolates were confirmed as Staph. aureus by the Microbact 12S identification system and were all presumptively identified as methicillin-resistant Staph. aureus species using oxacillin-resistant Staph. aureus basal medium (ORSAB). The 29 Staph. aureus isolates were tested for their sensitivity to 16 antimicrobial agents, and 11 were resistant to methicillin. None of the 11 Staph. aureus isolates harboured the methicillin resistance, mecA gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Listeria monocytogenes and Staphylococcus aureus are important agents of foodborne diseases. Occurrence of these infectious agents was established in meat and meat products in Zaria, Nigeria. Majority of isolates obtained from this study, displayed multidrug resistance to commonly used antimicrobial agents, including methicillin resistance among the Staph. aureus isolates. The potential virulence of L. monocytogenes found in ready-to-eat food was documented by the carriage of hly A gene by one of the isolates. A different mechanism of methicillin resistance or different homologue of mec A gene may be circulating among Nigerian isolates.

Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability.

Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10 h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K(M) values of 0.60 mM and 0.65 mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12 h with a half-life of 5.80 h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.

Isolation and characterization of a mannose-binding insecticidal lectin gene from Allium sativum (garlic) and its putative role in insect resistance using bioinformatics tools.

Monocot mannose-binding lectins (MMBLs) or agglutinins are an extended superfamily of structurally and evolutionarily related proteins. They play important roles in plant defenses. Here we describe the synthesis of full-length cDNA of monocot mannose-binding insecticidal agglutinin isolated from Allium sativum, a traditional herb known to be of great applications in Africa, using reverse transcription polymerase chain reaction (RT-PCR) with specific primers designed based on the insecticidal sequence (NCBI primary accession no. AY866499.1). Sequence analysis revealed a 327bp open reading frame (ORF) encoding a putative 108-aa agglutinin precursor with a C-terminal domain. Multiple alignments of BLEC1 amino acids with those of eight other MMBLs revealed three highly conserved domains among them, indicating BLEC1 belongs to a member of the MMBL superfamily. Tertiary structure analysis showed that BLEC1 had three potential equal mannose-binding sites. Phylogenetic analysis indicated that 20 MMBLs including BLEC1 belonged to an extended superfamily. Gene ontology analyses indicate one biological process with GO ID: 0006952 representing defense response, with two secondary IDs GO: 0002217 GO: 0042829. The child terms has both negative and positive regulation some of which include GO: 0002242 defense response to parasitic plant and GO: 0002213 defense response to insect. The cloning and characterization of BLEC1 will enable us to study its potential use in plant genetic engineering in the development of insect resistance plant.

Comparative assessment of a DNA and protein Leishmania donovani gamma glutamyl cysteine synthetase vaccine to cross-protect against murine cutaneous leishmaniasis caused by L. major or L. mexicana infection.

Leishmaniasis is a major health problem and it is estimated that 12 million people are currently infected. A vaccine which could cross-protect people against different Leishmania spp. would facilitate control of this disease as more than one species of Leishmania may be present. In this study the ability of a DNA vaccine, using the full gene sequence for L. donovani gamma glutamyl cysteine synthetase (γGCS) incorporated in the pVAX vector (pVAXγGCS), and a protein vaccine, using the corresponding recombinant L. donovani γGCS protein (LdγGCS), to protect against L. major or L. mexicana infection was evaluated. DNA vaccination gave transient protection against L. major and no protection against L. mexicana despite significantly enhancing specific antibody titres in vaccinated infected mice compared to infected controls. Vaccination with the LdγGCS protected against both species but only if the protein was incorporated into non-ionic surfactant vesicles for L. mexicana. The results of this study indicate that a L. donovani γGCS vaccine could be used to vaccinate against more than one Leishmania species but only if the recombinant protein is used.

Enhancing comparative rabies DNA vaccine effectiveness through glycoprotein gene modifications.

Enhancing DNA vaccine effectiveness remains a challenge, especially if the desired goal is immunization efficacy after a single dose. The glycoprotein gene from the rabies virus Evelyn-Rokitnicki-Abelseth (ERA) strain was modified by mutation at amino acid residue 333 from arginine to glutamine. The modified and original unmodified glycoprotein genes were cloned separately and developed as DNA vaccines for immunization in mice. The intramuscular (IM) route using a single dose (100 microg) of a modified DNA vaccine showed virus neutralizing antibody induction by d30, and 80% of the mice survived a challenge in which 100% of unvaccinated controls succumbed. Similar results were obtained using a single dose (10 microg) by the intradermal (ID) route with one-tenth amount of the DNA administered. Administration of single dose of DNA vaccine with unmodified G did not result in the production of detectable levels of virus neutralizing antibody by d30. The results of the IM and the ID routes of administration were statistically significant (P<0.01). Based on these preliminary results, a modified glycoprotein gene from the ERA rabies virus strain may be an ideal candidate for DNA vaccine efficacy enhancement.

Purification and biochemical characterization of lysosomal acid phosphatases (EC 3.1.3.2) from blood stream forms, Trypanosoma brucei brucei.

Three Acid phosphatases (ACP) were isolated and characterized from the lysosomes of blood stream forms of Trypanosoma brucei by a combination of isopynic and differential centrifugation through Ficoll, organic solvent precipitation, ion exchange on DEAE cellulose 52 and size exclusion chromatography on Sephadex G-75 columns. The purified ACP emerged as three distinct peaks (ACP I, ACP II and ACP III) with high specific activities and they moved homogeneously on 12% SDS-PAGE each as a single band with relative molecular weight of 36 kDa, 25 kDa and 45 kDa respectively. The purified enzymes were active at an optimum pH and temperature of 5.5 and 40 degrees C respectively. The enzyme activities appeared to be ACP because their activities were enhanced at low pH values and inhibited by the acid phosphatase inhibitor, sodium fluoride. ACP I and ACP II were sensitive to l-tartrate while ACP III was insensitive to l tartrate. The kinetic analysis of the purified enzyme (ACP I, ACP II and ACP III) determined using para-nitrophenylphosphate as substrate gave KM values of 0.2 mM, 0.15 mM and 0.5 mM. Monofunctional group sulfhydryl group inhibitors; HgCl2, and AgCl2 strongly inhibited the activity of ACP III and millimolar concentrations of dithiothreitol and iodoacetamide activated and inhibited the activity of the ACP III respectively, suggesting the involvement of thiol groups at the active site of the enzyme. Thus, differentiating it from ACP I and ACP II. The implication of these findings in relation to the pathology of trypanosomosis is discussed.

DNA vaccination against the parasite enzyme gamma-glutamylcysteine synthetase confers protection against Leishmania donovani infection.

In this study the potential of using Leishmania donovani gamma-glutamylcysteine synthetase (glutamate-cysteine ligase, gamma-GCS) as a rational target for vaccine development was determined. Mice, immunised with plasmid containing the full gene sequence for gamma-GCS (pVAXgammaGCS) or plasmid alone (pVAX control), were challenged with a high dose of L. donovani amastigotes to give a stringent test of the ability of the vaccine to protect against infection. Vaccination with pVAXgammaGCS resulted in the production of specific IgG1 and IgG2a antibodies and resulted in significantly lower liver parasite burdens compared to controls. Protection was also associated with a significant increase in cell-mediated immunity, demonstrated as an increase in nitrite production by ConA stimulated splenocytes, an increase in the percentage of splenic CD3+CD4+ cells, and enhanced granuloma maturation, compared to control values.

Characterization of a sialidase (neuraminidase) isolated from Clostridium chauvoei (Jakari strain).

A sialidase from Clostridium chauvoei (Jakari strain), an indigenous bacterial strain that causes blackleg in Nigerian cattle and other ruminants was isolated and partially purified by chromatography on DEAE cellulose, hydroxyapatite and phenyl agarose columns. The enzyme migrated as a 65-kDa protein after electrophoresis on sodium dodecyl sulphate polyacrylamide gels. It was optimally active at pH 4.5 and 40 degrees C with an activation energy (Ea) of 13.40 kJ mol(-1). It had Km and Vmax values of 170 microM and 200 micromole h(-1) mg(-1) respectively with fetuin as substrate. When sialyllactose (Neu5Ac2,3 lactose) was used as substrate the Km and Vmax values were 8 microM and 5 micromoles min(-1) mg(-1) respectively. The Clostridium chauvoei sialidase cleaved sialic acids from RBC ghosts of sheep, horse, goat, cattle, pig and mice as well as mouse brain cells, albeit at different rates. The enzyme was activated by Ca2+ and Mg2+ and inhibited by the group-specific reagents diethylpyrocarbonate (DEP) and N-ethylmalemide (NEM). The sialidase inhibitors, 2,3 didehydroneuraminic acid (Neu5Ac2,3en) and paranitrophenyl oxamic acid (pNPO) inhibited the enzyme competitively with Ki values of 40 and 30 microM respectively.

Characterization of sialidase from bloodstream forms of Trypanosoma vivax.

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.

Comparative studies on erythrocyte sialic acid levels in apparently healthy indigenous Nigerian poultry species.

The erythrocyte surface sialic acid levels of 200 apparently healthy indigenous Nigerian poultry species (pigeons, guinea fowls, ducks and chickens, n = 50 for each species) presented for slaughter at a poultry abattoir in Zaria, Nigeria was determined. Other parameters determined were packed cell volume (PCV) and plasma total protein (TP) concentration. The mean erythrocyte surface sialic acid concentration of pigeons, guinea fowls, ducks and chickens were 7.88 +/- 2.51, 14.6 +/- 2.51, 17.6 +/- 2.51 and 14.2 +/- 2.51 mg mg(-1) respectively. There was a statistically significant difference between the mean erythrocyte surface sialic acid concentration of all the species of poultry sampled (P < 0.05). The high erythrocyte surface sialic acid concentration in the indigenous Nigerian poultry species could be responsible for their resistance to infectious diseases, whose aetiologic agents produce neuraminidases. The mean PCV of the pigeons, guinea fowls, ducks and chickens were 46.22 +/- 6.91, 38.24 +/- 6.91, 36.50 +/- 6.91 and 36.46 +/- 6.91% respectively. The difference between the mean PCV values of pigeons on the one hand and guinea fowls, ducks and chickens on the other was statistically significant (P < 0.05). A negative correlation (r = -0.36, P < 0.05) between mean erythrocyte surface sialic acid concentrations and PCV was observed, i.e. the birds with the highest mean PCV values had the lowest levels of erythrocyte surface sialic acid. There was no correlation between TP concentration and either erythrocyte surface sialic acid concentration or PCV values. It is suggested, based on this study, that erythrocyte sialic acid types in these species should be determined, as the results may be vital in selective breeding.

Columbin inhibits cholesterol uptake in bloodstream forms of Trypanosoma brucei-A possible trypanocidal mechanism.

The diterpenoid furanolactone (columbin) from Aristolochia albida inhibited growth of culture forms of Trypanosoma brucei. In vitro analysis of the compound at 5-250 microg/ml showed complete lysis of the parasites within 10-20 minutes post incubation. At 50 microg/ml, columbin killed about 50% of the parasites which initially appeared swollen under phase contrast microscopy. Also the total amount of cholesterol diminished dose-dependently in the presence of 10-100 microg/ml of columbin after a 3-day incubation period. In vivo analysis of the compound in T. brucei-infected mice revealed that 25 mg/kg administered for 3 consecutive days, completely cleared the parasites from the peripheral circulation. However, columbin could not clear parasites in the cerebrospinal fluid.

Haemagglutination inhibition antibodies, rectal temperature and total protein of chickens infected with a local Nigerian isolate of velogenic Newcastle disease virus.

Changes in values of haemagglutination inhibition (HI) antibody titre, rectal temperature (RT) and total protein (TP) were determined for Shaver Brown chickens infected with Newcastle disease virus (NDV) Kudu 113. The infected chickens came down with Newcastle disease by day 3 post infection (PI). The major clinical signs were depression, greenish diarrhoea, paralysis of legs and wings, opisthotonus and torticolis. Mortality and morbidity were 52% and 1000%, respectively. There were haemorrhagic lesions in the wall of the intestine, proventricular mucosa and caecal tonsils. There were necrosis and mononuclear cell infiltration of the liver, kidney and spleen. There was a significant increase in daily mean HI antibody titres from days 3 to 9 PI. Similarly, significant rise in daily mean RTs were noticed in the infected chickens from days 1 to 13 PI. On the other hand, there was a decrease in daily mean TP concentrations of infected chickens, beginning from day 3 PI, and the lowest concentration of 2.60 +/- 0.15 g/dl was obtained by days 7 and 11 PI. The values of HI, RT and TP for the control chickens were relatively constant during the experiment. The correlation coefficient (r) between HI and RT was positive and highly significant (r = 0.725, p<0.001), while the relationship between HI and TP was negative but highly significant (r = -0.712, p<0.001). It was concluded that NDV Kudu 113 induced increases in values of HI and RT, which occurred concurrently with a decrease in TP concentrations of infected chickens.

The inhibition of Clostridium chauvoei (jakari strain) neuraminidase activity by methanolic extracts of the stem barks of Tamarindus indicus and Combretum fragrans.

The inhibition of neuraminidase from Clostridium chauvoei (jakari strain) with partially purified methanolic extracts of some plants used in Ethnopharmacological practice was evaluated. Extracts of two medicinal plants, Tamarindus indicus and Combretum fragrans at 100-1000 microg/ml, both significantly reduced the activity of the enzyme in a dose-dependent fashion (P < 0.001). The estimated IC50 values for Tamarindus indicus and Combretum fragrans were 100 and 150 microg/ml respectively. Initial velocity studies conducted, using fetuin as substrate revealed a non-competitive inhibition with the Vmax significantly altered from 500 micromole min(-1) mg(-1) to 240 micromole min(-1) mg(-1) and 340 micromole min(-1) mg(-1) in the presence of Tamarindus indicus and Combretum fragrans respectively. The KM remained unchanged at 0.42 mM. The computed Index of physiological efficiency was reduced from 1.19min(-1) to 0.57min(-1) and 0.75min(-1) with Tamarindus indicus and Combretum fragrans as inhibitors respectively.

Quantitative changes in gastric mucosal glycoproteins: effect of cholinergic agonist and vagal nerve stimulation in the rat.

The role of the vagus nerve and cholinergic mechanisms in the control of the rat gastric mucin and protein (PROT) release in vivo was investigated. Under urethane anaesthesia (1.25 g kg(-1)), the rats had their gastric lumen perfused with saline. Mucus secretion was measured as a function of adherent mucus on the mucosa surface and the luminal content of sialic acids (SIA), galactose (GAL), pyruvate and PROT. Electrical stimulation of the vagi significantly increased the levels of mucus (3.23 +/- 025 microg g(-1) tissue, P < 0.05), free sialic acid (FS) (0.18 +/- 0.04 mg mL(-1), P < 0.05) and PROT (0.25 +/- 0.003 mg mL(-1), P < 0.05) when compared with control animals. Bilateral cervical vagotomy had no significant effect on adherent mucus or basal levels of PROT, SIA and GAL (P > 0.05) with respect to the control. In both vagotomized and vagal intact animals, the cholinergic agonist (carbachol, 200 mg kg(-1)) significantly increased PROT, adherent mucus and FS (P < 0.05) and decreased bound sialic acid (P > 0.05). There were no visible haemorrhagic streaks on the gastric mucosa of vagotomized, vagal intact and carbachol-treated animals. The results suggest that vagus nerve does not exert a tonic control on gastric glycoprotein secretion in vivo and that cholinergic effect on the mucus secreting cells may be implemented via the intrinsic nerves of the enteric nervous system.

Mechanism of adsorption of mucin to titanium in vitro.

Mucin is the main salivary protein in the mouth of animals including man. The present study aims at investigating the role of electrostatic interactions in the adsorption of mucin to titanium in vitro. The binding profile of mucin to titanium was analyzed according to an adsorption isotherm. Mucin was dissolved and the solution suspended with native, calcium, magnesium, or potassium treated commercially pure Ti powder, at pH 3.0 and 7.4. The amount of unabsorbed protein in the supernatant fluid was measured. The maximum amount of adsorbed mucin was 0.11 mg/1.0 g of Ti. The mucin-Ti association constant was estimated to be 2.91 ml/mg. Pretreatment of Ti with calcium, or magnesium alone, or combined resulted in increased adsorption of mucin to Ti. No increase in adsorption was recorded following pretreatment of Ti with potassium. The results indicate the involvement of electrostatic interactions in the absorption of mucin to Ti.

Effect of dibutyltin(IV) on the ultrastructure of African Trypanosoma spp.

Diorganotins (R2SnX2) are compounds with a wide variety of biological properties. In an attempt to follow the morphological events and to characterize the toxic effects of diorganotins on in vitro cultured African Trypanosoma spp., the ultrastructural alterations induced on the parasites by dibutyltins (Bu2SnX2) were followed. The data obtained indicate that these compounds induced irreparable damage to the in vitro cultured bloodstream forms of the parasites. Transmission and scanning electron microscopy allowed observations on the perturbation of the kinetoplast, extensive cytoplasmic swellings, disconfiguration around the flagellar pocket and membrane disintegration. Fluorescence microscopy with 4,6-diamidine-2-phenylindole stain was also used to visualize the survival or degeneration of kDNA. Understanding the collateral cellular toxic effect of these compounds on the parasites may shed light on the possible mechanism by which they kill trypanosomes. Agarose gel electrophoresis resolution of isolated kDNAs revealed no fragmentation by these compounds following in vitro incubation at 37 degrees C. However, fragmentation was observed from the gel electrophoresis of kDNA isolated from in vitro cultured Bu2SnX2-exposed parasites. Transmission electron microscopy of the kDNAs revealed the same pattern as observed with gel electrophoresis. These results provide evidence for the possible involvement of the Bu2Sn moiety in the in vivo-induced fragmentation of trypanosomal kDNA and consequent trypanolysis. This observation also underlies the relevance of organometallics in the therapy of African trypanosomiasis.