Crystallization and a preliminary X-ray diffraction study of isozyme 3-3 of glutathione S-transferase from rat liver

Crystals of the homodimeric isozyme 3-3 of glutathione S-transferase from rat liver have been obtained with the hanging drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of the enzyme required the presence of both the enzyme inhibitor (9R, 10R)-9, 10-dihydro-9-(S-glutathionyl)-10-hydroxyphenanthrene and the detergent beta-octylglucopyranoside. The crystals belong to the monoclinic space group C2, with cell dimensions of a = 88.24(8) A, b = 69.44(4) A, c = 81.28(5) A, beta = 106.01(6) degrees, and contain four dimeric enzyme molecules per unit cell. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination at high resolution.     

Effects of KCN and Salicylhydroxamic Acid on Respiration of Soybean Leaves at Different Ages

Measurements of respiration were made on leaf discs from glasshouse-grown soybean (Glycine max [L.] Merr. cv `Corsoy') plants in the presence and absence of cyanide (KCN) and salicylhydroxamic acid (SHAM). O₂ uptake by mature leaves measured at 25°C was stimulated by 1 millimolar KCN (63%) and also by 5 millimolar azide (79%). SHAM, an inhibitor of the alternative oxidase and a selection of other enzymes, also stimulated O₂ uptake by itself at concentration of 10 millimolar. However, in combination, KCN and SHAM were inhibitory. The rate of O₂ uptake declined consistently with leaf age. The stimulation of O₂ uptake by KCN and by SHAM occurred only after a certain stage of leaf development had been reached and was more pronounced in fully expanded leaves. In young leaves, O₂ uptake was inhibited by both KCN and SHAM individually. The uncoupler, p-trifluoromethoxy carbonylcyanide phenylhydrazone, stimulated leaf respiration at all ages studied, the stimulation being more pronounced in fully expanded leaves. The uncoupled rate was inhibited by KCN and SHAM individually. The capacity of the cytochrome path declined with leaf age, paralleling the decline in total respiration. However, the capacity of the alternative path peaked at about full leaf expansion, exceeding the cytochrome capacity and remaining relatively constant. These results are consistent with the presence in soybean leaves of an alternative path capacity that seems to increase with age, and they suggest that the stimulation of O₂ uptake by KCN and NaN₃ in mature leaves was mainly by the SHAM-sensitive alternative path. The stimulation of O₂ uptake by SHAM was not expected, and the reason for it is not clear.     

Correction to: Application of a multiphase microreactor chemostat for the determination of reaction kinetics of Staphylococcus carnosus

Bioprocess and Biosystems Engineering - Unfortunately, in the “How to Cite as” section, the given and the family name of the author was incorrectly published, the correct name is...     

Impact of Three Rounds of Mass Drug Administration on Lymphatic Filariasis in Areas Previously Treated for Onchocerciasis in Sierra Leone

Background

1974–2005 studies across Sierra Leone showed onchocerciasis endemicity in 12 of 14 health districts (HDs) and baseline studies 2005–2008 showed lymphatic filariasis (LF) endemicity in all 14 HDs. Three integrated annual mass drug administration (MDA) were conducted in the 12 co-endemic districts 2008–2010 with good geographic, programme and drug coverage. Midterm assessment was conducted 2011 to determine impact of these MDAs on LF in these districts.

Methodology/Principal Findings

The mf prevalence and intensity in the 12 districts were determined using the thick blood film method and results compared with baseline data from 2007–2008. Overall mf prevalence fell from 2.6% (95% CI: 2.3%–3.0%) to 0.3% (95% CI: 0.19%–0.47%), a decrease of 88.5% (p = 0.000); prevalence was 0.0% (100.0% decrease) in four districts: Bo, Moyamba, Kenema and Kono (p = 0.001, 0.025, 0.085 and 0.000 respectively); and seven districts had reductions in mf prevalence of between 70.0% and 95.0% (p = 0.000, 0.060, 0.001, 0.014, 0.000, 0.000 and 0.002 for Bombali, Bonthe, Kailahun, Kambia, Koinadugu, Port Loko and Tonkolili districts respectively). Pujehun had baseline mf prevalence of 0.0%, which was maintained. Only Bombali still had an mf prevalence ≥1.0% (1.58%, 95% CI: 0.80%–3.09%)), and this is the district that had the highest baseline mf prevalence: 6.9% (95% CI: 5.3%–8.8%). Overall arithmetic mean mf density after three MDAs was 17.59 mf/ml (95% CI: 15.64 mf/ml–19.55 mf/ml) among mf positive individuals (65.4% decrease from baseline of 50.9 mf/ml (95% CI: 40.25 mf/ml–61.62 mf/ml; p = 0.001) and 0.05 mf/ml (95% CI: 0.03 mf/ml–0.08 mf/ml) for the entire population examined (96.2% decrease from baseline of 1.32 mf/ml (95% CI: 1.00 mf/ml–1.65 mf/ml; p = 0.000)).

Conclusions/Significance

The results show that mf prevalence decreased to <1.0% in all but one of the 12 districts after three MDAs. Overall mf density reduced by 65.0% among mf-positive individuals, and 95.8% for the entire population.     

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.     

Cyanide-resistant respiration in light- and dark-grown soybean cotyledons

Measurements of respiration were made on intact tissue and mitochondria isolated from soybean (Glycine max [L.] Merr. cv `Corsoy') cotyledons from seedlings of different ages grown in light and darkness. Effects of cyanide (KCN) and salicylhydroxamic acid (SHAM) on O₂ uptake rates were determined. O₂ uptake was faster in light-grown tissue and was inhibited by both KCN and SHAM in all except light-grown tissue older than 9 days. Both inhibitors stimulated O₂ uptake in tissues more than 9 days old. Mitochondria in which O₂ uptake was coupled to ATP synthesis were isolated from all tissues. O₂ uptake by mitochondrial preparations from light- and dark-grown cotyledons was equally sensitive to KCN. Similarly, age did not affect KCN sensitivity, but sensitivity to SHAM declined with age both in the presence and absence of KCN. Estimated capacities of the cytochrome and alternative pathways of the mitochondrial preparations indicated considerably larger cytochrome than alternative pathway capacities. The cytochrome pathway capacities paralleled the state 3 mitochondrial respiration rates, which increased from day 5 to day 7 then declined thereafter. The alternative pathway capacities were not affected by light. The uncoupler, p-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), increased the flow of electrons through the cytochrome pathway at the expense of flow through the alternative pathway in isolated mitochondria. However, the combined capacities did not exceed the rate in the presence of FCCP. The results are interpreted to indicate that the stimulation of respiration by KCN and SHAM observed in the 12-day-old green cotyledons and previously observed in older soybean leaves is not explained by characteristics of the mitochondria.     

Formation of the 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate anion at the active site of glutathione S-transferase: evidence for enzymic stabilization of sigma-complex intermediates in nucleophilic aromatic substitution reactions

Formation of the Meisenheimer complex or sigma-complex [1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate] between glutathione (GSH) and 1,3,5-trinitrobenzene (TNB) can be observed at the active sites of isoenzymes 3-3 and 4-4 of rat liver GSH transferase. The spectroscopic properties (UV-visible and CD) of the enzyme-bound sigma-complex are consistent with a 1:1 complex in an asymmetric environment. Competitive inhibitors which occupy the GSH binding site (e.g., gamma-L-glutamyl-D,L-2-aminomalonylglycine) inhibit sigma-complex formation. The apparent formation constants of the sigma-complex (M) with enzyme-bound GSH (E.GS- + TNB in equilibrium E.M) at pH 7.5 are 5 x 10(4) M-1 and 7 x 10(2) M-1 for isoenzymes 3-3 and 4-4, respectively. Both values are much greater than that in aqueous solution (GS- + TNB in equilibrium M), where Kf = 28 M-1. Isoenzyme 3-3 is roughly an order of magnitude more efficient than 4-4 in catalyzing nucleophilic aromatic substitutions, a fact that appears to correlate with the ability of each enzyme to stabilize the sigma-complex. The pH dependence of Kf(app) for isoenzyme 3-3 is used to probe the ionization behavior of enzyme-bound GSH. The results are consistent with a double-ionization scheme (e.g., H+E.GSH in equilibrium H+E.GS- in equilibrium E.GS-) with pK's of 5.7 and 7.6, which are assigned to the thiol pK and the pK of a protonated base in the active site, respectively. Formation of the sigma-complex is also observed in single crystals of isoenzyme 3-3, providing a clear demonstration of the chemical competence of the crystallized enzyme. The results are discussed with respect to catalytic efficiency and the ability of the enzyme to stabilize sigma-complex intermediates in nucleophilic aromatic substitution reactions.     

Randomized trial of safety and effectiveness of chlorproguanil-dapsone and lumefantrine-artemether for uncomplicated malaria in children in the Gambia

Background

Chlorproguanil-dapsone (Lapdap), developed as a low-cost antimalarial, was withdrawn in 2008 after concerns about safety in G6PD deficient patients. This trial was conducted in 2004 to evaluate the safety and effectiveness of CD and comparison with artemether-lumefantrine (AL) under conditions of routine use in G6PD normal and G6PD deficient patients with uncomplicated malaria in The Gambia. We also examined the effects of a common genetic variant that affects chlorproguanil metabolism on risk of treatment failure.

Methods

1238 children aged 6 months to 10 years with uncomplicated malaria were randomized to receive CD or artemether-lumefantrine (AL) and followed for 28 days. The first dose was supervised, subsequent doses given unsupervised at home. G6PD genotype was determined to assess the interaction between treatment and G6PD status in their effects on anaemia. The main endpoints were clinical treatment failure by day 28, incidence of severe anaemia (Hb<5 g/dL), and haemoglobin concentration on day 3.

Findings

One third of patients treated with AL, and 6% of patients treated with CD, did not complete their course of medication. 18% (109/595) of children treated with CD and 6.1% (36/587) with AL required rescue medication within 4 weeks, risk difference 12% (95%CI 8.9%–16%). 23 children developed severe anaemia (17 (2.9%) treated with CD and 6 (1.0%) with AL, risk difference 1.8%, 95%CI 0.3%–3.4%, P = 0.02). Haemoglobin concentration on day 3 was lower among children treated with CD than AL (difference 0.43 g/dL, 95% CI 0.24 to 0.62), and within the CD group was lower among those children who had higher parasite density at enrolment. Only 17 out of 1069 children who were typed were G6PD A- deficient, of these 2/9 treated with CD and 1/8 treated with AL developed severe anaemia. 5/9 treated with CD had a fall of 2 g/dL or more in haemoglobin concentration by day 3.

Interpretation

AL was well tolerated and highly effective and when given under operational conditions despite poor adherence to the six-dose regimen. There were more cases of severe malaria and anaemia after CD treatment although G6PD deficiency was uncommon.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00118794","term_id":"NCT00118794"}}NCT00118794     

No Evidence for Lymphatic Filariasis Transmission in Big Cities Affected by Conflict Related Rural-Urban Migration in Sierra Leone and Liberia

Background

In West Africa, the principal vectors of lymphatic filariasis (LF) are Anopheles species with Culex species playing only a minor role in transmission, if any. Being a predominantly rural disease, the question remains whether conflict-related migration of rural populations into urban areas would be sufficient for active transmission of the parasite.

Methodology/Principal Findings

We examined LF transmission in urban areas in post-conflict Sierra Leone and Liberia that experienced significant rural-urban migration. Mosquitoes from Freetown and Monrovia, were analyzed for infection with Wuchereria bancrofti. We also undertook a transmission assessment survey (TAS) in Bo and Pujehun districts in Sierra Leone. The majority of the mosquitoes collected were Culex species, while Anopheles species were present in low numbers. The mosquitoes were analyzed in pools, with a maximum of 20 mosquitoes per pool. In both countries, a total of 1731 An. gambiae and 14342 Culex were analyzed for W. bancrofti, using the PCR. Two pools of Culex mosquitoes and 1 pool of An. gambiae were found infected from one community in Freetown. Pool screening analysis indicated a maximum likelihood of infection of 0.004 (95% CI of 0.00012–0.021) and 0.015 (95% CI of 0.0018–0.052) for the An. gambiae and Culex respectively. The results indicate that An. gambiae is present in low numbers, with a microfilaria prevalence breaking threshold value not sufficient to maintain transmission. The results of the TAS in Bo and Pujehun also indicated an antigen prevalence of 0.19% and 0.67% in children, respectively. This is well below the recommended 2% level for stopping MDA in Anopheles transmission areas, according to WHO guidelines.

Conclusions

We found no evidence for active transmission of LF in cities, where internally displaced persons from rural areas lived for many years during the more than 10 years conflict in Sierra Leone and Liberia.