Discovery of an ebolavirus-like filovirus in europe

Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain.     

The role of sulfhydryl groups in the functioning of DNA-dependent RNA-polymerase

Sulfhydryl groups of Escherichia coli DNA-dependent RNA polymerase were chemically modified with alkylating and mercuric-containing compounds. Iodoacetic acid and iodoacetamide were shown not to affect the enzymatic activity, whereas N-ethylmaleimide and mercuric-containing compounds completely inhibit the RNA synthesis. RNA polymerase modified with mercuric ions looses the ability of binding with promoter--containing DNA fragments. Moreover, mercuric ions inhibit the RNA elongation stage. Suggestion is made the Cys residues of RNA polymerase play a key role in double-stranded DNA unwinding. It is shown that SH-groups of beta- and beta'-subunits participate in the binding with double-stranded fragments of DNA.     

Equilibrium binding of insulin to rat white fat cells at 15 degrees C

Equilibrium binding of insulin to isolated rat epididymal fat cells was investigated. A temperature of 15 degrees C was chosen for the study to minimize lysosomal degradation of insulin. Indeed, medium insulin lost only 1% of its precipitability in trichloroacetic acid during the 4-h incubation required to approach equilibrium. Binding was measured by a method that did not perturb the equilibrium of the system. A new formalism for analyzing binding data in general was introduced. A correction for trapping of insulin in the interstitial space of cell pellets was both necessary and sufficient to derive specific binding data from raw observations. Thus, so-called "nonspecific binding" was unmasked as a misnomer, and the expression "correction for trapping" was proposed as a substitute. Equations for one and two independent classes of binding sites were fit to the data by the method of maximum likelihood, and the best fit was selected based on Akaike's information criterion, as adapted for a constant fractional error. More than 99.7% of the binding sites were found to be describable by a simple binding isotherm with Kd,app = 8.8 multiplied by over divided by 1.3 nM. Less than 0.3% sites had a higher affinity (Kd approximately equal to 8 multiplied by over divided by 3 pM). There were 99,000 x/divided by 1.6 binding sites/cell. These equilibrium parameters are in agreement with values derived from a kinetic analysis, presented in the subsequent paper (Lipkin, E. W., Teller, D. C., and de Ha?n, C. (1986) J. Biol. Chem. 260, 1702-1711).     

Recoverin is a zinc-binding protein

Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its alpha-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett. 1998, 440, 116-118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 x 10(5) M(-1) (dissociation constant 7.1 microM) for Ca2+-loaded wild-type recoverin and 3.3 x 10(4) M(-1) (dissociation constant 30 microM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed.     

An interactive technique for the display of nucleic acid secondary structure.

The ability to visualize nucleic acid secondary structure has become quite important since the advent of computer prediction and biochemical techniques that depict such structures. Manually drawing the conformations can be quite time consuming and tedious. Thus, the ability to draw with the aid of a computer the secondary structure of nucleic acid molecules is quite advantageous. This paper describes an interactive algorithm that permits one to generate such drawings which may then be used for further analysis and/or publications.     

Molecular evidence and clinical importance of β‐arrestins expression in patients with acromegaly

β‐arrestins seem to have a role in endocytosis and desensitization of somatostatin receptor subtype 2 (sst2) and could be associated with the responsiveness to somatostatin receptor ligands (SRL) in patients with acromegaly. To investigate the in vivo correlation between β‐arrestins 1 and 2 with sst2, sst5 and dopamine receptor subtype 2 (D2) expressions, and the association of β‐arrestins with response to first‐generation SRL and invasiveness in somatotropinomas. β‐arrestins 1 and 2, sst2, sst5 and D2 mRNA expressions were evaluated by quantitative real‐time RT‐PCR on tumoral tissue of 96 patients. Moreover, sst2 and sst5 protein expressions were also evaluated in 40 somatotropinomas by immunohistochemistry. Response to SRL, defined as GH <1 μg/l and normal IGF‐I levels, was assessed in 40 patients. The Knosp‐Steiner criteria were used to define invasiveness. Median β‐arrestin 1, β‐arrestin 2, sst2, sst5 and D2 mRNA copy numbers were 478; 9375; 731; 156 ; and 3989, respectively. There was a positive correlation between β‐arrestins 1 and 2 (R = 0.444, P < 0.001). However, no correlation between β‐arrestins and sst2, sst5 (mRNA and protein levels) or D2 was found. No association was found between β‐arrestins expression and SRL responsiveness or tumour invasiveness. Although previous data suggest a putative correlation between β‐arrestins and sst2, our data clearly indicated that no association existed between β‐arrestins and sst2, sst5 or D2 expression, nor with response to SRL or tumour invasiveness. Therefore, further studies are required to clarify whether β‐arrestins have a role in the response to treatment with SRL in acromegaly.     

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.     

Single amino acid substitutions in the Ca2+-binding site of recoverin.II.The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca(2+)-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca2+ ions results in an increase in the mobility of the environment of Trp residues, in higher hydrophobicity, and in elevated thermal stability (its thermal transition shifts by 15 degrees C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca2+ are also characteristic of the -EF2 mutant of recoverin whose second Ca(2+)-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca(2+)-binding sites, respectively, are modified and damaged) are practically indifferent to calcium ions.     

Streptococcus pneumoniae Coinfection Is Correlated with the Severity of H1N1 Pandemic Influenza

Background

Initial reports in May 2009 of the novel influenza strain H1N1pdm estimated a case fatality rate (CFR) of 0.6%, similar to that of seasonal influenza. In July 2009, however, Argentina reported 3056 cases with 137 deaths, representing a CFR of 4.5%. Potential explanations for increased CFR included virus reassortment or genetic drift, or infection of a more vulnerable population. Virus genomic sequencing of 26 Argentinian samples representing both severe and mild disease indicated no evidence of reassortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence. Furthermore, no evidence was found for increased frequency of risk factors for H1N1pdm disease.

Methods/Principal Findings

We examined nasopharyngeal swab samples (NPS) from 199 cases of H1N1pdm infection from Argentina with MassTag PCR, testing for 33 additional microbial agents. The study population consisted of 199 H1N1pdm-infected subjects sampled between 23 June and 4 July 2009. Thirty-nine had severe disease defined as death (n = 20) or hospitalization (n = 19); 160 had mild disease. At least one additional agent of potential pathogenic importance was identified in 152 samples (76%), including Streptococcus pneumoniae (n = 62); Haemophilus influenzae (n = 104); human respiratory syncytial virus A (n = 11) and B (n = 1); human rhinovirus A (n = 1) and B (n = 4); human coronaviruses 229E (n = 1) and OC43 (n = 2); Klebsiella pneumoniae (n = 2); Acinetobacter baumannii (n = 2); Serratia marcescens (n = 1); and Staphylococcus aureus (n = 35) and methicillin-resistant S. aureus (MRSA, n = 6). The presence of S. pneumoniae was strongly correlated with severe disease. S. pneumoniae was present in 56.4% of severe cases versus 25% of mild cases; more than one-third of H1N1pdm NPS with S. pneumoniae were from subjects with severe disease (22 of 62 S. pneumoniae-positive NPS, p = 0.0004). In subjects 6 to 55 years of age, the adjusted odds ratio (OR) of severe disease in the presence of S. pneumoniae was 125.5 (95% confidence interval [CI], 16.95, 928.72; p<0.0001).

Conclusions/Significance

The association of S. pneumoniae with morbidity and mortality is established in the current and previous influenza pandemics. However, this study is the first to demonstrate the prognostic significance of non-invasive antemortem diagnosis of S. pneumoniae infection and may provide insights into clinical management.