Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences

The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences. In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil. This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322. [(3)H]Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug. Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil. We have also found that berenil induces DNA degradation in plasmids containing the homopolymer. Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids. Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect. Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected.     

Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting

Imprinting of the linked and oppositely expressed mouse H19 and Igf2 genes requires a 2-kb differentially methylated domain (DMD) that is located 2 kb upstream of H19. This element is postulated to function as a methylation-sensitive insulator. Here we test whether an additional sequence 5' of H19 is required for H19 and Igf2 imprinting. Because repetitive elements have been suggested to be important for genomic imprinting, the requirement of a G-rich repetitive element that is located immediately 3' to the DMD was first tested in two targeted deletions: a 2.9-kb deletion (Delta D MD Delta G) that removes the DMD and G-rich repeat and a 1.3-kb deletion (Delta G) removing only the latter. There are also four 21-bp GC-rich repetitive elements within the DMD that bind the insulator-associated CTCF (CCCTC-binding factor) protein and are implicated in mediating methylation-sensitive insulator activity. As three of the four repeats of the 2-kb DMD were deleted in the initial 1.6-kb Delta DMD allele, we analyzed a 3.8-kb targeted allele (Delta 3.8kb-5'H19), which deletes the entire DMD, to test the function of the fourth repeat. Comparative analysis of the 5' deletion alleles reveals that (i) the G-rich repeat element is dispensable for imprinting, (ii) the Delta DMD and Delta DMD Delta G alleles exhibit slightly more methylation upon paternal transmission, (iii) removal of the 5' CTCF site does not further perturb H19 and Igf2 imprinting, suggesting that one CTCF-binding site is insufficient to generate insulator activity in vivo, (iv) the DMD sequence is required for full activation of H19 and Igf2, and (v) deletion of the DMD disrupts H19 and Igf2 expression in a tissue-specific manner.     

Morinda lucida reduces contractility of isolated uterine smooth muscle of pregnant and non-pregnant mice.

The present work investigated the effect of Morinda lucida (M. lucida) extract on isolated uterine smooth muscle of pregnant and non-pregnant mice. Pregnant and non-pregnant mice were pretreated with oral stilboesterol (0.1 mg/kg body weight) and killed by cervical dislocation. Thin strips of the uterus were cut and mounted in a 20ml organ bath containing De Jalon solution bubbled with 95%O2-5% CO2 gas mixture. The strips were connected to a force transducer coupled to a Grass 7D Polygraph for the recording of isometric tension. Effects of graded concentrations of oxytocin (OXY; 10-5-10-2 mol/L), acetylcholine (ACh; 10-9-10-5 mol/L) and M. lucida extract (0.015-1.5 mg/ml) were recorded. Fresh uterine strips were then incubated with M. lucida extract for 5mins and cumulative response to OXY was repeated. Another set of fresh strips was incubated in L-NAME for 15mins and the cumulative responses to M.lucida extract were repeated. OXY resulted in increased contractile responses in both pregnant and non-pregnant uterine muscles. M. lucida resulted in relaxation of the uterine smooth muscle in both pregnant and non-pregnant mice at all doses. However, at 1.5mg/ml, M. lucida completely blocked spontaneous uterine contractions. Following incubation with L-NAME, M. lucida extract led to a slightly greater relaxation of the uterine strips. In conclusion, M. lucida reduced contractility of uterine smooth muscle in both pregnant and non-pregnant mice as well as blocking contractile responses to OXY and Ach in uterine smooth muscle of pregnant and non-pregnant mice. There was no significant alteration of M. lucida activity by L-NAME suggesting that the action of the compound on uterine muscle is not associated with impaired nitric oxide synthase.     

Steroids and triterpenoids from Eastern Nigeria mistletoe, Loranthus micranthus Linn. (Loranthaceae) parasitic on Kola acuminata with immunomodulatory potentials

In search of immunomodulatory constituents from the Eastern Nigeria mistletoe, Loranthus micranthus Linn, two new stigmastane steroids: stigmast-7,20 (21)-diene-3β-hydroxy-6-one (1) and 3β-hydroxy-stigmast-23-ene (2); three (two new and one known) lupeol-based triterpenoid esters: 7β,15α-dihydroxyl-lup-20(29)-ene-3β-palmitate (3), 7β,15α-dihydroxyl-lup-20(29)-ene-3β-stearate (4) and 7β,15α-dihydroxyl-lup-20(29)-ene-3β-eicosanoate (5) were isolated and characterized following bioactivity-guided fractionation. The new compounds, 1, 2, 4 and 5 at concentrations of 10, 25 and 100 μg/ml were subjected to cell proliferation and early activation marker (CD69) expression studies in C57Bl/6 mice splenocytes using flow cytometry techniques against Lipopolysaccharide (LPS; 10 μg/ml) and Concanavalin A (ConA; 2 μg/ml) standards. The stigmastane steroids (1 and 2) at the highest concentration of 100 μg/ml showed statistically significantly (p < 0.05) stimulatory activity on the C57B1/6 splenocytes compared to the controls with values of 46 ± 0.76% and 43 ± 0.46% compared to 7.69 ± 0.41% recorded for the negative control. The novel lupeol esters, 4 and 5 at same concentration of 100 μg/ml exhibited lower stimulations of 30 ± 0.41% and 29 ± 0.17% respectively compared to the controls above. The CD69 expression assay at the above doses showed that all the compounds have minimal stimulation. The present study supports the observed immunomodulatory property of the Eastern Nigeria mistletoe and thus confirms the efficacy of this plant in mitigating against wide array of disease conditions orchestrated by immunodeficiency.     

Enhancing learning objectives by use of simple virtual microscopic slides in cellular physiology and histology: impact and attitudes

The impact and perception of students on the use of a simple, low technology-driven version of a virtual microscope in teaching and assessments in cellular physiology and histology were studied. Its impact on the time and resources of the faculty were also assessed. Simple virtual slides and conventional microscopes were used to conduct the same examinations for the same students. Students performed significantly better in the examination with the virtual slide and also showed a significantly higher preference for virtual slides. The time and cost implications of conducting examinations using the simple virtual slides were reduced by >1,400%. The results reemphasize the need for the design and adoption of simple sustainable technological innovations in developing countries to bridge gaps in purposeful learning environments.     

Rheological behavior of heated starch dispersions in excess water: role of starch granule

Granule size and size distribution, measured by laser diffraction, affected the flow behavior at 20 °C of (2.6% w/w) corn and cowpea starch dispersions heated for various time intervals above their gelatinization temperatures. The standard deviation of the granules' size described the transition of flow behavior from shear thickening in the early stages of gelatinization to shear thinning in the latter stages and influenced the critical shear rate, y_c, for the onset of shear thickening in starch dispersions. The granules swelled to a maximum of about 3.5 times the raw starch granule mean diameter and 65% granule mass fraction. The consistency index of the dispersions increased with granule mean diameter. Modified waxy maize starch dispersions heated at 80 °C exhibited antithixotropic behavior at a shear rate of 200s⁻¹; both dynamic frequency data and Cox-Merz plots revealed their gel-like behavior.     

Mutagenic and physiological responses in the juveniles of African catfish, Clarias gariepinus (Burchell 1822) following short term exposure to praziquantel

Praziquantel (PZQ) is an acylated quinoline-pyrazine originally developed for veterinary application but now one of the most used anti-helminthic drugs for treatment of certain trematodes and cestodes in both human and other animals. The present study investigated the mutagenic and physiological responses in the juveniles of African catfish, Clarias gariepinus following short term exposure to praziquantel. Based on the 53.52 mg/l 96 h LC₅₀ of PZQ obtained, two sublethal concentrations of 5.35 and 10.70 mg/l of the drug were selected and fish were exposed to these concentrations and control for 15 days. Micronuclei induction in the peripheral blood of PZQ-exposed fish was highest on day 10 but the fish morphological parameters were not affected. The packed cell volume (PCV) was significantly reduced (p < 0.05) from day 5 while red blood cells (RBC) and hemoglobin (Hb) significantly declined (p < 0.05) on day 15. Macrocytic anemia was observed on day 1 of study and thereafter microcytic anemia developed on day 5 of study. The white blood cell (WBC) was significantly (p < 0.05) elevated from day 10 of exposure while values of mean cellular volume (MCV), mean cellular hemoglobin (MCH) and mean cellular hemoglobin concentration (MCHC) were not significantly different (p > 0.05) from the control. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glucose levels significantly increased while protein reduced (p < 0.05) throughout the exposure period but a mixed trend was observed in the leukocyte differentials. PZQ should be used with caution as sublethal exposure elicited micronucleus induction and alterations of hematological and biochemical parameters in the fish.     

Economic Burden of Heart Failure: Investigating Outpatient and Inpatient Costs in Abeokuta,Southwest Nigeria

Background: Heart failure (HF) is a deadly, disabling and often costly syndrome world-wide. Unfortunately, there is a paucity of data describing its economic impact in sub Saharan Africa; a region in which the number of relatively younger cases will inevitably rise. Methods: Heath economic data were extracted from a prospective HF registry in a tertiary hospital situated in Abeokuta, southwest Nigeria. Outpatient and inpatient costs were computed from a representative cohort of 239 HF cases including personnel, diagnostic and treatment resources used for their management over a 12-month period. Indirect costs were also calculated. The annual cost per person was then calculated. Results: Mean age of the cohort was 58.0±15.1 years and 53.1% were men. The total computed cost of care of HF in Abeokuta was 76, 288,845 Nigerian Naira (US$508, 595) translating to 319,200 Naira (US$2,128 US Dollars) per patient per year. The total cost of in-patient care (46% of total health care expenditure) was estimated as 34,996,477 Naira (about 301,230 US dollars). This comprised of 17,899,977 Naira- 50.9% ($US114,600) and 17,806,500 naira −49.1%($US118,710) for direct and in-direct costs respectively. Out-patient cost was estimated as 41,292,368 Naira ($US 275,282). The relatively high cost of outpatient care was largely due to cost of transportation for monthly follow up visits. Payments were mostly made through out-of-pocket spending. Conclusion: The economic burden of HF in Nigeria is particularly high considering, the relatively young age of affected cases, a minimum wage of 18,000 Naira ($US120) per month and considerable component of out-of-pocket spending for those affected. Health reforms designed to mitigate the individual to societal burden imposed by the syndrome are required.     

Assessment of the Quality of Antenatal Care Services Provided by Health Workers Using a Mobile Phone Decision Support Application in Northern Nigeria: A Pre/Post-Intervention Study

BackgroundGiven the shortage of skilled healthcare providers in Nigeria, frontline community health extension workers (CHEWs) are commonly tasked with providing maternal and child health services at primary health centers. In 2012, we introduced a mobile case management and decision support application in twenty primary health centers in northern Nigeria, and conducted a pre-test/post-test study to assess whether the introduction of the app had an effect on the quality of antenatal care services provided by this lower-level cadre.MethodsUsing the CommCare mobile platform, the app dynamically guides CHEWs through antenatal care protocols and collects client data in real time. Thirteen health education audio clips are also embedded in the app for improving and standardizing client counseling. To detect changes in quality, we developed an evidence-based quality score consisting of 25 indicators, and conducted a total of 266 client exit interviews. We analyzed baseline and endline data to assess changes in the overall quality score as well as changes in the provision of key elements of antenatal care.ResultsOverall, the quality score increased from 13.3 at baseline to 17.2 at endline (p<0.0001), out of a total possible score of 25, with the most significant improvements related to health counseling, technical services provided, and quality of health education.ConclusionThese study results suggest that the introduction of a low-cost mobile case management and decision support application can spur behavior change and improve the quality of services provided by a lower level cadre of healthcare workers. Future research should employ a more rigorous experimental design to explore potential longer-term effects on client health outcomes.