The structures of frataxin oligomers reveal the mechanism for the delivery and detoxification of iron

Defects in the mitochondrial protein frataxin are responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1:40,000 children. Here, we present the crystal structures of the iron-free and iron-loaded frataxin trimers, and a single-particle electron microscopy reconstruction of a 24 subunit oligomer. The structures reveal fundamental aspects of the frataxin mechanism. The trimer has a central channel in which one atom of iron binds. Two conformations of the channel with different metal-binding affinities suggest that a gating mechanism controls whether the bound iron is delivered to other proteins or transferred to detoxification sites. The trimer constitutes the basic structural unit of the 24 subunit oligomer. The architecture of this oligomer and several features of the trimer structure demonstrate striking similarities to the iron-storage protein ferritin. The data reveal how stepwise assembly provides frataxin with the structural flexibility to perform two functions: metal delivery and detoxification.     

IgG1 is pathogenic in Leishmania mexicana infection

There are >2 million new cases of leishmaniasis annually, and no effective vaccine has been developed to prevent infection. In murine infection, Leishmania mexicana, which lives intracellularly in host macrophages, has developed pathways to hijack host IgG to induce a suppressive IL-10 response through FcγRs, the cell-surface receptors for IgG. To guide vaccine development away from detrimental Ab responses, which can accompany attempts to induce cell-mediated immunity, it is crucial to know which isotypes of IgG are pathogenic in this infection. We found that IgG1 and IgG2a/c induce IL-10 from macrophages in vitro equally well but through different FcγR subtypes: IgG1 through FcγRIII and IgG2a/c through FcγRI primarily, but also through FcγRIII. In sharp contrast, mice lacking IgG1 develop earlier and stronger IgG2a/c, IgG3, and IgM responses to L. mexicana infection and yet are more resistant to the infection. Thus, IgG1, but not IgG2a/c or IgG3, is pathogenic in vivo, in agreement with prior studies indicating that FcγRIII is required for chronic disease. This calls into question the assumption that macrophages, which should secrete IL-10 in response to IgG1 and IgG2a/c immune complexes, are the most important source of IL-10 generated by IgG-FcγR engagement in L. mexicana infection. Further investigations are required to better determine the cell type responsible for this immunosuppressive FcγRIII-induced IL-10 pathway and whether IgG2a/c is protective.     

Occurrence,conformational features and amino acid propensities for the pi-helix

The most abundant helix type in proteins is the alpha-helix, accounting for about 31% of amino acid secondary structure states, while the 3(10)-helix accounts for about 4%. The pi-helix appears to be extremely rare and is considered to be unstable. Existing secondary structure definition methods find very few within the Protein Data Bank. Using an improved pi-helix definition algorithm to search a non-redundant subset of high-resolution and well-refined protein structures, we found that almost every tenth protein contained a pi-helix. This enabled us to show for the first time that the pi-helix has structural parameters that are different from the hypothesized model values. It also has distinctive amino acid preferences and it is conserved within functionally related proteins. Features that may contribute to the stability of the pi-helical structure have also been identified. In addition to hydrogen bonds, several other factors contribute to the stability of pi-helices. The pi-helix may have some functional advantages over other helical structures. Thus, we describe cases where the side chains of functionally important residues at every fourth position within a pi-helix could be aligned and brought close together in a way that would not be allowed by any other helix type.     

A major photoperiod-sensitivity gene tagged with RFLP and isozyme markers in rice

Summary Photoperiod-sensitive rice (Oryza sativa L.) cultivars are widely grown in rainfed lowland areas with unfavorable water regimes. A molecular marker for the trait would be useful in genetic and physiological studies and in developing improved photoperiod-sensitive cultivars. Previous genetic studies identified a major gene for photoperiod sensitivity on chromosome 6. We have tested an isozyme marker and several RFLP probes mapping to chromosome 6 in an attempt to identify marker(s) tightly linked to photoperiod sensitivity in tropical rice cultivars. We report here that the isozyme gene Pgi-2 is linked (23.2±4.7 cM) to the photoperiod-sensitivity gene in the cultivar GEB-24. Although association of duration with Pgi-2 alleles can be used to detect segregation of the photoperiod sensitivity gene in crosses, it will probably not be useful as a marker in selection because of its loose linkage. In contrast, a gene for photoperiod sensitivity in the cultivar Puang Rai 2 was found to be closely linked to the rice genomic clone RG64. Among 15 F₃ lines homozygous for photoperiod insensitivity, no recombinants were detected with RG64. This clone is thus an excellent probe to follow segregation of the major photoperiod-sensitivity gene in rice crosses.     

Synthesis of chloromethyl ketone derivatives of fatty acids. Their use as specific inhibitors of acetoacetyl-coenzyme A thiolase, cholesterol biosynthesis and fatty acid synthesis.

A general route for the synthesis of chloromethyl ketone derivatives of fatty acids is described. 5-Chloro-4-oxopentanoic acid, 7-chloro-6-oxoheptanoic acid, 9-chloro-8-oxononanoic acid and 11-chloro-10-oxoundecanoic acid were synthesized by this method and tested as covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The K1 decreased by approx. 20-fold for each pair of methylenes added to the chain length, showing that the initial stage in inhibitor binding occurs at a non-polar region of the protein. This region is probably located at the enzyme active site, since inhibition was prevented by acetoacetyl-CoA or acetyl-CoA but not by CoA. The site of modification by chloromethyl ketone derivatives of fatty acids is restricted to a thiol group, since inactivation of the enzyme was prevented by reversible thiomethylation of the active-site thiol. In contrast, an amino-directed reagent, citraconic anhydride, still inactivated the enzyme, even when the active-site thiol was protected. Evidence that the enzyme thiol was particularly reactive came from studies on the pH-dependence of the alkylation reaction and thiol-competition experiments. Inhibition of the enzyme proceeded suprisingly well at acidic pH values and a 10(5) molar excess of external thiol over active-site thiol was required to prevent inhibition by 0.3 mM-9-chloro-8-oxononanoic acid. In addition to inhibiting isolated acetoacetyl-CoA thiolase, in hepatocytes the chloromethyl ketone derivatives of fatty acids also inhibited chloresterol synthesis, which uses this enzyme as an early step in the biosynthetic pathway. In isolated cells, the chloromethyl ketone derivatives of fatty acids were considerably less specific in their inhibitory action compared with 3-acetylenic derivatives of fatty acids, which act as suicide inhibitors of acetoacetyl-CoA thiolase. However, 9-chloro-8-oxononanoic acid was also an effective inhibitor of both hepatic cholesterol and fatty acid synthesis in mice in vivo, whereas the acetylenic fatty acid derivative, dec-3-ynoic acid, was completely ineffective. The effective inhibitory dose of 9-chloro-8-oxononanoic acid (2.5-5 mg/kg) was substantially lower than the estimated LD50 for the inhibitor (100 mg/kg).     

Effect of dothiepin on gastric ulceration mediated by lipid derived eicosanoids

Dothiepin, a tricyclic antidepressant, significantly inhibited the development of gastric ulcers induced by alcohol, aspirin, indomethacin and Shay's pyloric ligation. Antisecretory studies in pyloric ligated rats revealed that the drug at a dose of 100 mg/kg significantly reduced total acidity, gastric output and protein content. In another set of experiments, dothiepin significantly reduced gastric output, total acidity and protein content in pyloric ligated rats which received 50% alcohol (v/v) 30 minutes after the administration of dothiepin.     

A novel locus (DFNA24) for prelingual nonprogressive autosomal dominant nonsyndromic hearing loss maps to 4q35-qter in a large Swiss German kindred

Nonsyndromic hearing loss is one of the most genetically heterogeneous traits known. A total of 30 autosomal dominant nonsyndromic hearing-loss loci have been mapped, and 11 genes have been isolated. In the majority of cases, autosomal dominant nonsyndromic hearing loss is postlingual and progressive, with the exception of hearing impairment in families in which the impairment is linked to DFNA3, DFNA8/12, and DFNA24, the novel locus described in this report. DFNA24 was identified in a large Swiss German kindred with a history of autosomal dominant hearing loss that dates back to the middle of the 19th century. The hearing-impaired individuals in this kindred have prelingual, nonprogressive, bilateral sensorineural hearing loss affecting mainly mid and high frequencies. The DFNA24 locus maps to 4q35-qter. A maximum multipoint LOD score of 11.6 was obtained at 208.1 cM at marker D4S1652. The 3.0-unit support interval for the map position of this locus ranges from 205.8 cM to 211.7 cM (5.9 cM).     

Sterols of the red alga Rissoella verruculosa

The Mediterranean red alga Rissoella verruculosa contains desmosterol in the esterified form whereas this sterol is not present in the free state.     

Severe Sepsis in Severely Malnourished Young Bangladeshi Children with Pneumonia: A Retrospective Case Control Study

BackgroundIn developing countries, there is no published report on predicting factors of severe sepsis in severely acute malnourished (SAM) children having pneumonia and impact of fluid resuscitation in such children. Thus, we aimed to identify predicting factors for severe sepsis and assess the outcome of fluid resuscitation of such children.MethodsIn this retrospective case-control study SAM children aged 0–59 months, admitted to the Intensive Care Unit (ICU) of the Dhaka Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh from April 2011 through July 2012 with history of cough or difficult breathing and radiologic pneumonia, who were assessed for severe sepsis at admission constituted the study population. We compared the pneumonic SAM children with severe sepsis (cases = 50) with those without severe sepsis (controls = 354). Severe sepsis was defined with objective clinical criteria and managed with fluid resuscitation, in addition to antibiotic and other supportive therapy, following the standard hospital guideline, which is very similar to the WHO guideline.ResultsThe case-fatality-rate was significantly higher among the cases than the controls (40% vs. 4%; p<0.001). In logistic regression analysis after adjusting for potential confounders, lack of BCG vaccination, drowsiness, abdominal distension, acute kidney injury, and metabolic acidosis at admission remained as independent predicting factors for severe sepsis in pneumonic SAM children (p<0.05 for all comparisons).

Conclusion and Significance

We noted a much higher case fatality among under-five SAM children with pneumonia and severe sepsis who required fluid resuscitation in addition to standard antibiotic and other supportive therapy compared to those without severe sepsis. Independent risk factors and outcome of the management of severe sepsis in our study children highlight the importance for defining optimal fluid resuscitation therapy aiming at reducing the case fatality in such children.