Cloning vectors for Streptococcus thermophilus derived from a native plasmid

Marine sponges frequently contain a complex mixture of bacteria, fungi, unicellular algae and cyanobacteria. Epifluorescent microscopy showed that Mycale (Carmia) hentscheli contained coccoid cyanobacteria. The 16S rRNA gene was amplified, fragments cloned and analysed using amplified rRNA gene restriction analysis. The nearly complete 16S rRNA gene of distinct clones was sequenced and aligned using ARB. The phylogenetic analysis indicated the presence of four closely related clones which have a high (8%) sequence divergence from known cyanobacteria, Cyanobacterium stanieri being the closest, followed by Prochloron sp. and Synechocystis sp. All belong to the order Chroococcales. The lack of non-molecular evidence prevents us from proposing a new genus.     

Bias in estimates of quantitative-trait-locus effect in genome scans: demonstration of the phenomenon and a method-of-moments procedure for reducing bias

An attractive feature of variance-components methods (including the Haseman-Elston tests) for the detection of quantitative-trait loci (QTL) is that these methods provide estimates of the QTL effect. However, estimates that are obtained by commonly used methods can be biased for several reasons. Perhaps the largest source of bias is the selection process. Generally, QTL effects are reported only at locations where statistically significant results are obtained. This conditional reporting can lead to a marked upward bias. In this article, we demonstrate this bias and show that its magnitude can be large. We then present a simple method-of-moments (MOM)-based procedure to obtain more-accurate estimates, and we demonstrate its validity via Monte Carlo simulation. Finally, limitations of the MOM approach are noted, and we discuss some alternative procedures that may also reduce bias.     

Truncation mutations in ABCA1 suppress normal upregulation of full-length ABCA1 by 9-cis-retinoic acid and 22-R-hydroxycholesterol

Mutations in ABCA1 uniformly decrease plasma HDL-cholesterol (HDL-C) and reduce cholesterol efflux, yet different mutations in ABCA1 result in different phenotypic effects in heterozygotes. For example, truncation mutations result in significantly lower HDL-C and apoliprotein A-I (apoA-I) levels in heterozygotes compared with nontruncation mutations, suggesting that truncation mutations may negatively affect the wild-type allele. To specifically test this hypothesis, we examined ABCA1 protein expression in response to 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-R-OH-Chol) in a collection of human fibroblasts representing eight different mutations and observed that truncation mutations blunted the response to oxysterol stimulation and dominantly suppressed induction of the remaining full-length allele to 5-10% of wild-type levels. mRNA levels between truncation and nontruncation mutations were comparable, suggesting that ABCA1 expression was suppressed at the protein level. Dominant negative activity of truncated ABCA1 was recapitulated in an in vitro model using transfected Cos-7 cells. Our results suggest that the severe reduction of HDL-C in patients with truncation mutations may be at least partly explained by dominant negative suppression of expression and activity of the remaining full-length ABCA1 allele. These data suggest that ABCA1 requires a physical association with itself or other molecules for normal function and has important pharmacogenetic implications for individuals with truncation mutations.     

Iron detoxification properties of Escherichia coli bacterioferritin. Attenuation of oxyradical chemistry

Bacterioferritin (EcBFR) of Escherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the "ferroxidase center." The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H(2)O(2) as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H(2)O(2) at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H(2)O(2) corresponds to [Fe(II)(2)-P](Z) + H(2)O(2) --> [Fe(III)(2)O-P](Z) + H(2)O, where [Fe(II)(2)-P](Z) represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)(2)O-P](Z) a micro-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2FeOOH((core)) + 4H(+), where two Fe(II) are again oxidized by one H(2)O(2). Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O(2) is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H(2)O(2) produced from O(2) is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H(2)O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H(2)O(2), consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H(2)O(2), thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.     

Molecular phylogenetic analysis of the avian malarial parasite Plasmodium (Novyella) juxtanucleare

Plasmodium (Novyella) juxtanucleare is a widely distributed parasite that primarily infects chickens (Gallus gallus domesticus). All species of Novyella are characterized by very small schizonts, which in the case of P. juxtanucleare are always found juxtaposed to the erythrocyte nucleus, hence its name. Nearly complete small-subunit ribosomal RNA sequences have been obtained from 2 isolates of this species, and comparisons with other Plasmodium species have been made. Phylogenetic analysis reveals that this parasite is closely related to other avian-infecting Plasmodium species and that molecular relationships among the avian-infecting plasmodia do not correspond to their morphology-based subgeneric classifications.     

Effects of cytotoxic T lymphocytes (CTL) directed against a single simian immunodeficiency virus (SIV) Gag CTL epitope on the course of SIVmac239 infection

Vaccine-induced cytotoxic T lymphocytes (CTL) have been implicated in the control of virus replication in simian immunodeficiency virus (SIV)-challenged and simian-human immunodeficiency virus-challenged macaques. Therefore, we wanted to test the impact that vaccine-induced CTL responses against an immunodominant Gag epitope might have in the absence of other immune responses. By themselves, these strong CTL responses failed to control SIVmac239 replication.     

Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum

Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins.     

Tat-vaccinated macaques do not control simian immunodeficiency virus SIVmac239 replication

The regulatory proteins of human immunodeficiency virus may represent important vaccine targets. Here we assessed the role of Tat-specific cytotoxic T lymphocytes (CTL) in controlling pathogenic simian immunodeficiency virus SIVmac239 replication after using a DNA-prime, vaccinia virus Ankara-boost vaccine regimen. Despite the induction of Tat-specific CTL, there was no significant reduction in either peak or viral set point compared to that of controls.     

A kinesin heavy chain (KIF5A) mutation in hereditary spastic paraplegia (SPG10)

We have identified a missense mutation in the motor domain of the neuronal kinesin heavy chain gene KIF5A, in a family with hereditary spastic paraplegia. The mutation occurs in the family in which the SPG10 locus was originally identified, at an invariant asparagine residue that, when mutated in orthologous kinesin heavy chain motor proteins, prevents stimulation of the motor ATPase by microtubule-binding. Mutation of kinesin orthologues in various species leads to phenotypes resembling hereditary spastic paraplegia. The conventional kinesin motor powers intracellular movement of membranous organelles and other macromolecular cargo from the neuronal cell body to the distal tip of the axon. This finding suggests that the underlying pathology of SPG10 and possibly of other forms of hereditary spastic paraplegia may involve perturbation of neuronal anterograde (or retrograde) axoplasmic flow, leading to axonal degeneration, especially in the longest axons of the central nervous system.