Selective transport of a new class of purine antimetabolites by the protozoan parasite Trypanosoma brucei

Purine antimetabolites have been very successful therapeutic agents against a host of infectious diseases and malignancies. Success of the treatment relies as much on the efficient accumulation by the target cell or organism as it does on selective action on a vital biochemical pathway of the target cell. Here we compare the ability of a new class of tricyclic purine antimetabolites to interact with transporters from human erythrocytes or Trypanosoma brucei. We show that these compounds display a remarkable selectivity for the parasite's transporters. The adenine analogue showed greater trypanocidal activity than the hypoxanthine or guanine analogues in vitro.     

Cloning of the pig renal organic anion transporter 1 (pOAT1)

A pig kidney cDNA library was screened for the porcine ortholog of the multispecific organic anion transporter 1 (pOAT1). Several positive clones were isolated resulting in two alternatively spliced cDNA clones of pOAT1 (pOAT1 and pOAT1A). pOAT1-cDNAs consist of 2126 or 1895 base pairs (EMBL Acc. No. AJ308234 and AJ308235) encoding 547 or 533 amino acid residue proteins with 89, 87, 83 and 81% homology to the human, rabbit, rat, and mouse OAT1, respectively. Heterologous expression of pOAT1 in Xenopus laevis oocytes revealed an apparent K(m) for [3H]PAH of 3.75 +/- 1.6 microM. [3H]PAH uptake mediated by pOAT1 was abolished by 0.5 mM glutarate or 1 mM probenecid. Functional characterization of pOAT1A did not show any affinity for [3H]PAH. In summary, we cloned two alternative splice variants of the pig ortholog of organic anion transporter 1. One splice form (pOAT1) showed typical functional characteristics of organic anion transporter 1, whereas the second form appears not to transport PAH.     

Unexpected inhibition of S-adenosyl-L-homocysteine hydrolase by a guanosine nucleoside

A series of shape-modified flexible nucleosides ('fleximers', 1, 2, and 3) was modeled, synthesized and subsequently assayed against S-adenosyl-L-homocysteine hydrolase (SAHase). No inhibitory activity was observed for the adenosine fleximer, which served as a substrate, but moderate inhibitory activity was exhibited by the guanosine fleximers. This is the first known report of a guanosine nucleoside analogue possessing activity against SAHase.     

Risk Factors for Maternal Mortality in Rural Tigray,Northern Ethiopia: A Case-Control Study

Background

Maternal mortality continues to have devastating impacts in many societies, where it constitutes a leading cause of death, and thus remains a core issue in international development. Nevertheless, individual determinants of maternal mortality are often unclear and subject to local variation. This study aims to characterise individual risk factors for maternal mortality in Tigray, Ethiopia.

Methods

A community-based case-control study was conducted, with 62 cases and 248 controls from six randomly-selected rural districts. All maternal deaths between May 2012 and September 2013 were recruited as cases and a random sample of mothers who delivered in the same communities within the same time period were taken as controls. Multiple logistic regression was used to identify independent determinants of maternal mortality.

Results

Four independent individual risk factors, significantly associated with maternal death, emerged. Women who were not members of the voluntary Women’s Development Army were more likely to experience maternal death (OR 2.07, 95% CI 1.04–4.11), as were women whose husbands or partners had below-median scores for involvement during pregnancy (OR 2.19, 95% CI 1.14–4.18). Women with a pre-existing history of other illness were also at increased risk (OR 5.58, 95% CI 2.17–14.30), as were those who had never used contraceptives (OR 2.58, 95% CI 1.37–4.85). Previous pregnancy complications, a below-median number of antenatal care visits and a woman’s lack of involvement in health care decision making were significant bivariable risks that were not significant in the multivariable model.

Conclusions

The findings suggest that interventions aimed at reducing maternal mortality need to focus on encouraging membership of the Women’s Development Army, enhancing husbands’ involvement in maternal health services, improving linkages between maternity care and other disease-specific programmes and ensuring that women with previous illnesses or non-users of contraceptive services are identified and followed-up as being at increased risk during pregnancy and childbirth.     

Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish

Background  

The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-β superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.     

Functional expression of pig renal organic anion transporter 3 (pOAT3)

With the cloning of pig renal organic anion transporter 1 (pOAT1) (Biochimie 84 (2002) 1219) we set up a model system for comparative studies of cloned and natively isolated membrane located transport proteins. Meanwhile, another transport protein involved in p-aminohippurate (PAH) uptake on the basolateral side of the proximal tubule cells was identified, designated organic anion transporter 3 (OAT3). To explore the contribution of pOAT1 to the PAH clearance in comparison to OAT3, it was the aim of this study to extend our model by cloning of the pig ortholog of OAT3. Sequence comparisons of human organic anion transporter 3 (hOAT3) with the expressed sequence tag (EST) database revealed a clone and partial sequence of the pig renal organic anion transporter 3 (pOAT3) ortholog. Sequencing of the entire open reading frame resulted in a protein of 543 amino acid residues encoded by 1632 base pairs (EMBL Acc. No. AJ587003). It showed high homologies of 81%, 80%, 76%, and 77% to the human, rabbit, rat, and mouse OAT3, respectively. A functional characterization of pOAT3 in Xenopus laevis oocytes yielded an apparent Km (Kt) for [3H]estrone sulfate of 7.8 +/- 1.3 microM. Moreover, pOAT3 mediated [3H]estrone sulfate uptake was almost abolished by 0.5 mM of glutarate, dehydroepiandosterone sulfate, or probenecid consistent with the hallmarks of OAT3 function.     

Health Extension Workers' and Mothers' Attitudes to Maternal Health Service Utilization and Acceptance in Adwa Woreda,Tigray Region,Ethiopia

Background

The maternal health system in Ethiopia links health posts in rural communities (kebeles) with district (woreda) health centres, and health centres with primary hospitals. At each health post two Health Extension Workers (HEWs) assist women with birth preparedness, complication readiness, and mobilize communities to facilitate timely referral to mid-level service providers. This study explored HEWs’ and mother’s attitudes to maternal health services in Adwa Woreda, Tigray Region.

Methods

In this qualitative study, we trained 16 HEWs to interview 45 women to gain a better understanding of the social context of maternal health related behaviours. Themes included barriers to health services; women’s social status and mobility; and women’s perceptions of skilled birth attendant’s care. All data were analyzed thematically.

Findings

There have been substantial efforts to improve maternal health and reduce maternal mortality in Adwa Woreda. Women identified barriers to healthcare including distance and lack of transportation due to geographical factors; the absence of many husbands due to off-woreda farming; traditional factors such as zwar (some pregnant women are afraid of meeting other pregnant women), and discouragement from mothers and mothers-in-law who delivered their children at home. Some women experienced disrespectful care at the hospital. Facilitators to skilled birth attendance included: identification of pregnant women through Women’s Development Groups (WDGs), and referral by ambulance to health facilities either before a woman’s Expected Due Date (EDD) or if labour started at home.

Conclusion

With the support of WDGs, HEWs have increased the rate of skilled birth attendance by calling ambulances to transfer women to health centres either before their EDD or when labour starts at home. These findings add to the growing body of evidence that health workers at the community level can work with women’s groups to improve maternal health, thus reducing the need for emergency obstetric care in low-income countries.     

Mutation of amino acid 475 of rat organic cation transporter 2 (rOCT2) impairs organic cation transport

Protein sequence alignments revealed one amino acid position, where organic cation transporters (OCTs, aspartate (D) at position 475 of rOCT2) and organic anion transporters (OATs, arginine (R) at position 466 of rOAT1) are charged oppositely. To address the impact of this amino acid for protein function we cloned rat organic cation transporter 2 (rOCT2), the renal electrogenic cation transporter of the basolateral side of proximal tubule cells. Site-directed mutagenesis was used to generate rOCT2-D475R (rOCT2-mut). Heterologous expression of rOCT2 wild-type (rOCT2-wt) in A6 cells resulted in a significant uptake of the fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP(+)). Accordingly, rOCT2-wt-transfected COS 7 cells showed an almost fourfold uptake of 25 microM [(14)C]-TEA, whereas rOCT2-mut did not exhibit any uptake of [(14)C]-TEA. These data indicate that rOCT2 transports both ASP(+) and TEA and that aspartate at position 475 of rOCT2 plays a critical role in transport function.