Immobilization of Cassava Linamarase on Kankara Kaolinite Clay

The enzyme, linamarase was successfully extracted and immobilized on pretreated Kankara kaolinite clay, serving as substitute for other expensive catalysts supports. Acid treatment of the clay was observed to affect the activity of linamarase. The activity of the enzyme was higher in the cortex compared with both in the stem and the leaves. Enzyme activity was observed to increase with increase in the silica contents of the treated clay. Operational stability/activity of the immobilized enzyme was found to reduce by about 50% after 18days at room temperature, following first order denaturation reaction, with rate constant k obtained to be 0.03day-1 with R2 0.957. The Km and Vmax were determined to 0.1986mM-1 and 10.01mM/min respectively, which is an indication of the enzyme’s affinity to the support used. The specific surface area and pore size of kaolin were also found to decrease with rate of enzyme anchoring, pointing to occupation and/or blockage of the available pore. Kankara kaolinite clay is a promising cheaper support material for linamarase immobilization. Keywords: linamarase, immobilization, kaolinite clay, cassava, metakaolin, cyanoge

Development of paleoseismic trench logging and dating techniques: a case study on the Central North Anatolian Fault

The North Anatolian Fault (NAF) is a dextral strike slip fault zone extending ~1400km in an arc across northern Turkey. This study seeks to further constrain the timing of ground rupturing earthquakes of the NAF while developing the techniques used in paleoseismology. A paleoseismic trench was opened ~2.7km NW of Destek on a segment which ruptured (for ~280km) in the 1943 Tosya Earthquake (Mw:7.7). The trench site comprises a pop-up structure formed by a small releasing step-over at a restraining bend which has caused progressive growth of an upslope facing scarp. The trench is situated across the main fault trace and a trapped sedimentary sequence that includes several paleosoils. The stratigraphy is expected to be Late Holocene and historic in age due to the high level of activity on the NAF, although this has yet to be confirmed by radiometric dating. Preliminary interpretation of the trench stratigraphy indicates a record of up to 6 paleoearthquake events, the presence of an angular unconformity suggests the record may be incomplete beyond the 3 most recent events on this strand.Subtle contrasts in stratigraphy made conventional face logging difficult and was therefore augmented by mapping the magnetic susceptibility (MS) of the west wall. Approximately 6000 measurements were made using a Bartington MS2 Magnetic Susceptibility Meter with a MS2E (point) Sensor with a 5cm vertical spacing and a 20cm horizontal spacing predominantly on one side of the trench. A pilot test led to development of a strategy of moving the sensor to the nearest exposure of coarse sand or finer grained material where possible to minimize the noise generated by individual clasts. To negate the sensitivity of the MS logging method to variations in temperature the survey was conducted at night. Plotted data clearly shows the contact between rock units, the rock-soil interface (reflecting fault juxtaposition), anthropogenic influence and some soil stratigraphy. Other paleoseismic investigations on this section of the NAF (Hartleb R. et al 2003 and Yoshioka T. et al 2000) have encountered out-of-stratigraphic-order ranges in 14C ages. They attributed this to reworking, in addition to which the effects of long term human occupation are likely to be similar. The trench yielded a large amount of datable material including 158 charcoal and 140 minute gastropod samples, and some ceramic, bone and slag samples. Unlike charcoal and bone fragments, fragile minute gastropods are unlikely to have been transported, reworked or used by humans, ultimately providing improved accuracy of temporal constraints on paleoearthquakes. Using both charcoal and gastropod samples, the trench chronology can be established and the use of minute gastropods for dating paleoearthquakes can be critiqued

Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5–RNA complexes

AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N6-methyladenosine (m6A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m6A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m6A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m6A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5′-3′ orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m6AC consensus sequence motif. The results support a mechanism involving formation of an m6A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139

Acute Exposure to Artesunate and its Effect on the Hematological Indices, Hepatotoxicity and Histology of the Liver of Adult Wistar Rats

Abstract: The effect of artesunate on the hematological indices, hepatotoxicity and histology of liver was investigated in 20 adult male wistar rats. The animals were divided into 4 groups of 5 each and group 1 which served as control were administered normal saline while groups 2, 3 and 4 were administered 1, 2 and 5 mg/kg/day respectively for a period of 5 days. The animals were humanely sacrificed on the sixth day and blood samples were obtained for hematological indices and serum enzyme analysis. The liver were excised and processed for light microscopy using the H & E stain. Hematological indices indicated insignificant difference in the RBC, WBC and DC counts, while a significant dose dependent increase in PCV and hemoglobin were observed (p<0.05). No changes were observed in the serum levels of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT) and Alkaline Phosphatase (ALP) among the groups. Histological examination of the liver revealed points of focal necrosis among the treated groups. The mild liver tissue damage was more evident among the over dosed group. Artesunate is thus safe, when administered within the therapeutic range

Mechanisms controlling anaemia in Trypanosoma congolense infected mice.

Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia. The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng. The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis