Typhoid Fever in Young Children

One hundred and fifty cases of typhoid in infants and young children are reported. In children over 5 years of age the features resembled those in adults, but in those under this age they were often nonspecific including fever, convulsions, diarrhoea, and vomiting. Attention is drawn to the importance of a blood culture in anaemic afebrile children who develop rigors and high fever after blood transfusions.     

Methylation changes at NR3C1 in newborns associate with maternal prenatal stress exposure and newborn birth weight

Early life experiences, including those in utero, have been linked to increased risk for adult-onset chronic disease. The underlying assumption is that there is a critical period of developmental plasticity in utero when selection of the fetal phenotype that is best adapted to the intrauterine environment occurs. The current study is the first to test the idea that extreme maternal psychosocial stressors, as observed in the Democratic Republic of Congo, may modify locus-specific epigenetic marks in the newborn resulting in altered health outcomes. Here we show a significant correlation between culturally relevant measures of maternal prenatal stress, newborn birth weight and newborn methylation in the promoter of the glucocorticoid receptor NR3C1. Increased methylation may constrain plasticity in subsequent gene expression and restrict the range of stress adaptation responses possible in affected individuals, thus increasing their risk for adult-onset diseases.     

Dynamic Strategies for Target-Site Search by DNA-Binding Proteins

Gene regulatory proteins find their target sites on DNA remarkably quickly; the experimental binding rate for lac repressor is orders-of-magnitude higher than predicted by free diffusion alone. It has been proposed that nonspecific binding aids the search by allowing proteins to slide and hop along DNA. We develop a reaction-diffusion theory of protein translocation that accounts for transport both on and off the strand and incorporates the physical conformation of DNA. For linear DNA modeled as a wormlike chain, the distribution of hops available to a protein exhibits long, power-law tails that make the long-time displacement along the strand superdiffusive. Our analysis predicts effective superdiffusion coefficients for given nonspecific binding and unbinding rate parameters. Translocation rate exhibits a maximum at intermediate values of the binding rate constant, while search efficiency is optimized at larger binding rate constant values. Thus, our theory predicts a region of values of the nonspecific binding and unbinding rate parameters that balance the protein translocation rate and the efficiency of the search. Published data for several proteins falls within this predicted region of parameter values.     

An Endoplasmic Reticulum Retrieval Signal Partitions Human Foamy Virus Maturation to Intracytoplasmic Membranes

Among all retroviruses, foamy viruses (FVs) are unique in that they regularly mature at intracytoplasmic membranes. The envelope glycoprotein of FV encodes an endoplasmic reticulum (ER) retrieval signal, the dilysine motif (KKXX), that functions to localize the human FV (HFV) glycoprotein to the ER. This study analyzed the function of the dilysine motif in the context of infectious molecular clones of HFV that encoded mutations in the dilysine motif. Electron microscopy (EM) demonstrated virion budding both intracytoplasmically and at the plasma membrane for the wild-type and mutant viruses. Additionally, mutant viruses retained their infectivity, but viruses lacking the dilysine signal budded at the plasma membrane to a greater extent than did wild-type viruses. Interestingly, this relative increase in budding across the plasma membrane did not increase the overall release of viral particles into cell culture media as measured by protein levels in viral pellets or infectious virus titers. We conclude that the dilysine motif of HFV imposes a partial restriction on the site of viral maturation but is not necessary for viral infectivity.     

Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease

Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m(5)C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21(DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase. The mcrA coding region contains several non-optimal E. coli triplets. Addition of the pACYC-RIL tRNA encoding plasmid to the BL21(DE3) host increased the yield of recombinant McrA (rMcrA) upon induction about 5- to 10-fold. McrA protein expressed at 37 degrees C is insoluble but a significant fraction is recovered as soluble protein after autoinduction at 20 degrees C. rMcrA protein, which is predicted to contain a Cys(4)-Zn(2+) finger and a catalytically important histidine triad in its putative nuclease domain, binds to several metal chelate resins without addition of a poly-histidine affinity tag. This feature was used to develop an efficient protocol for the rapid purification of nearly homogeneous rMcrA. The native protein is a dimer with a high alpha-helical content as measured by circular dichroism analysis. Under all conditions tested purified rMcrA does not have measurable nuclease activity on HpaII methylated (Cm(5)CGG) DNA, although the purified protein does specifically bind HpaII methylated DNA. These results have implications for understanding the in vivo activity of McrA in "restricting" m(5)C-containing DNA and suggest that rMcrA may have utility as a reagent for affinity purification of DNA fragments containing m(5)C residues.     

Fludarabine induces differential expression of proteins in human leukemia and lymphoma cells

The purine analog fludarabine (FdAMP) is widely used for chemotherapy of B-lymphoid malignancies, and multiple mechanisms of action leading to apoptosis have been proposed. We examined changes at the protein level induced in the Raji cell line (Burkitt's lymphoma) by fludarabine nucleoside (FdA). Raji cells are sensitive to FdA. Raji cells treated with FdA (3 micro M, 24 hours), accumulate multiple phosphorylated forms of p53 in the nucleus that in turn degrade to phosphorylated forms of p40. Using CD antibody microarrays to determine surface expression profiles for Raji cells treated with FdA, we found up-regulation of the following CD antigens: CD20, CD54, CD80, CD86, and CD95. FdA thus induces changes in the genetic program of the cells that might be exploited to obtain synergy with therapeutic antibodies.     

Conservation of structure and mechanism in primary and secondary transporters exemplified by SiaP, a sialic acid binding virulence factor from Haemophilus influenzae

Extracytoplasmic solute receptors (ESRs) are important components of solute uptake systems in bacteria, having been studied extensively as parts of ATP binding cassette transporters. Herein we report the first crystal structure of an ESR protein from a functionally characterized electrochemical ion gradient dependent secondary transporter. This protein, SiaP, forms part of a tripartite ATP-independent periplasmic transporter specific for sialic acid in Haemophilus influenzae. Surprisingly, the structure reveals an overall topology similar to ATP binding cassette ESR proteins, which is not apparent from the sequence, demonstrating that primary and secondary transporters can share a common structural component. The structure of SiaP in the presence of the sialic acid analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid reveals the ligand bound in a deep cavity with its carboxylate group forming a salt bridge with a highly conserved Arg residue. Sialic acid binding, which obeys simple bimolecular association kinetics as determined by stopped-flow fluorescence spectroscopy, is accompanied by domain closure about a hinge region and the kinking of an alpha-helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes.     

Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance.

A series of replication-competent Moloney murine leukemia virus vectors was constructed in which each vector contained a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat. Two of the resulting viruses, MLV (murine leukemia virus) DHFR*-5 and MLV DHFR*-7, were able to stably transfer methotrexate resistance to infected fibroblast cells upon multiple rounds of virus replication and in the absence of drug selection. Cell lines producing recombinant virus with high titers were established, which indicated that the insert did not grossly interfere with viral replication functions. These vectors should be useful for introducing and expressing foreign genes in vivo in tissues and whole animals in which virus spread is needed for efficient infection.