A comprehensive whole genome bacterial phylogeny using correlated peptide motifs defined in a high dimensional vector space

As whole genome sequences continue to expand in number and complexity, effective methods for comparing and categorizing both genes and species represented within extremely large datasets are required. Methods introduced to date have generally utilized incomplete and likely insufficient subsets of the available data. We have developed an accurate and efficient method for producing robust gene and species phylogenies using very large whole genome protein datasets. This method relies on multidimensional protein vector definitions supplied by the singular value decomposition (SVD) of a large sparse data matrix in which each protein is uniquely represented as a vector of overlapping tetrapeptide frequencies. Quantitative pairwise estimates of species similarity were obtained by summing the protein vectors to form species vectors, then determining the cosines of the angles between species vectors. Evolutionary trees produced using this method confirmed many accepted prokaryotic relationships. However, several unconventional relationships were also noted. In addition, we demonstrate that many of the SVD-derived right basis vectors represent particular conserved protein families, while many of the corresponding left basis vectors describe conserved motifs within these families as sets of correlated peptides (copeps). This analysis represents the most detailed simultaneous comparison of prokaryotic genes and species available to date.     

CO2 concentrations in perennially ice-covered lakes of Taylor Valley, Antarctica

Lakes in Taylor Valley, southern Victoria Land,Antarctica, are unusual in that they areperennially covered by a 3–5 m thick ice layer.Previous work on gas concentrations in theselakes has shown that the surface waters aresupersaturated with respect to O₂,N₂O, as well as the noble gases. Our datashow that the dissolved CO₂ (CO_2(aq))concentrations, calculated from pH andCO₂, can be highly undersaturatedat shallow depths of the lakes. CO₂partial pressure values (pCO₂) are as lowas 10^–4.3 atm and 10^–4.2 atm in theeast and west lobes of Lake Bonney,respectively, and 10^–3.8 atm in LakeHoare. CO_2(aq) depletion occurred only inthe uppermost part of the water column, inassociation with elevated primary productivity(PPR). The upward diffusion of CO_2(aq)from the aphotic zone, and the annual input ofCO₂ via glacial meltwater can notreplenish the amount of CO_2(aq) annuallylost to primary productivity in the uppermostmeters of the water column. Calcification is alimited source of CO_2(aq), since the lakesare undersaturated with respect to calcitethrough portions of the austral summer.Preliminary respiration rates have been used toobtain an annual inorganic carbon balance.Further down in the water column, at the sitesof the deep-water maximum in primary production(PPR_max), which in Lakes Bonney andFryxell is associated with nutrient gradients,CO_2(aq) is not undersaturated. A largeupward flux from CO₂-supersaturatedaphotic waters provides a surplus ofCO_2(aq) at the PPR_max. Lake Fryxell,unlike the other lakes, is supersaturated withCO_2(aq) throughout the entire water column.     

Phosphorylation status of the SCR homeodomain determines its functional activity: essential role for protein phosphatase 2A,B'

Sex combs reduced (SCR) is a DROSOPHILA: Hox protein that determines the identity of the labial and prothoracic segments. In search of factors that might associate with SCR to control its activity and/or specificity, we performed a yeast two-hybrid screen. A DROSOPHILA: homologue of the regulatory subunit (B'/PR61) of serine-threonine protein phosphatase 2A (dPP2A,B') specifically interacted with the SCR homeodomain. The N-terminal arm within the SCR homeodomain was shown to be a target of phosphorylation/dephosphorylation by cAMP-dependent protein kinase A and protein phosphatase 2A, respectively. In vivo analyses revealed that mutant forms of SCR mimicking constitutively dephosphorylated or phosphorylated states of the homeodomain were active or inactive, respectively. Inactivity of the phosphorylated mimic form was attributed to impaired DNA binding. Specific ablation of dPP2A,B' gene activity by double-stranded RNA-mediated genetic interference resulted in embryos without salivary glands, an SCR null phenotype. Our data demonstrate an essential role for DROSOPHILA: PP2A,B' in positively modulating SCR function.     

Oxidative regulation of fatty acid-induced tau polymerization

Alzheimer's disease (AD) is characterized by the presence of amyloid-positive senile plaques and tau-positive neurofibrillary tangles. Aside from these two pathological hallmarks, a growing body of evidence indicates that the amount of oxidative alteration of vulnerable molecules such as proteins, DNA, and fatty acids is elevated in the brains of AD patients. It has been hypothesized that the elevated amounts of protein oxidation could lead directly to the formation of neurofibrillary tangles through a cysteine-dependent mechanism. We have tested this hypothesis in an in vitro system in which tau assembly is induced by fatty acids. Using sulfhydryl protective agents and site-directed mutagenesis, we found that cysteine-dependent oxidation of the tau molecule is not required for its polymerization and may even be inhibitory. However, by adjusting the oxidative environment of the polymerization reaction through the addition of a strong antioxidant or through the addition of an oxidizing system consisting of iron, adenosine diphosphate, and ascorbate, we found that oxidation does play a major role in our in vitro paradigm. The results indicated that fatty acid oxidation, the amount of which is found to be elevated in AD patients, can facilitate the polymerization of tau. However, "overoxidation" of the fatty acids can inhibit the process. Therefore, we postulate that specific fatty acid oxidative products could provide a direct link between oxidative stress mechanisms and the formation of neurofibrillary tangles in AD.     

Effects of acid adaptation of Escherichia coli O157:H7 on efficacy of acetic acid spray washes to decontaminate beef carcass tissue

Exposure to low pH and organic acids in the bovine gastrointestinal tract may result in the induced acid resistance of Escherichia coli O157:H7 and other pathogens that may subsequently contaminate beef carcasses. The effect of acid adaptation of E. coli O157:H7 on the ability of acetic acid spray washing to reduce populations of this organism on beef carcass tissue was examined. Stationary-phase acid resistance and the ability to induce acid tolerance were determined for a collection of E. coli O157:H7 strains by testing the survival of acid-adapted and unadapted cells in HCl-acidified tryptic soy broth (pH 2.5). Three E. coli O157:H7 strains that were categorized as acid resistant (ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated inducible acid tolerance (ATCC 43889) were used in spray wash studies. Prerigor beef carcass surface tissue was inoculated with bovine feces containing either acid-adapted or unadapted E. coli O157:H7. The beef tissue was subjected to spray washing treatments with water or 2% acetic acid or left untreated. For strains ATCC 43895 and 43889, larger populations of acid-adapted cells than of unadapted cells remained on beef tissue following 2% acetic acid treatments and these differences remained throughout 14 days of 4 degrees C storage. For both strains, numbers of acid-adapted cells remaining on tissue following 2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For strain ATCC 43890, there was no difference between populations of acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by E. coli O157:H7 can negatively influence the effectiveness of 2% acetic acid spray washing in reducing the numbers of this organism on carcasses.     

The isolated hepatocyte preparation: 30 years on

A method for the preparation of intact rat hepatocytes in high yield was first described in 1969. The procedure involved digestion of hepatic tissue by perfusion of the liver with crude collagenase; later, purified collagenase without other enzymic additions was shown to be ineffective. Recently it has been discovered that the combination of purified collagenase plus elastase is superior to crude collagenase in that it consistently provides high yields of undamaged hepatocytes. The isolated hepatocyte preparation has proved particularly useful for the study of mechanisms responsible for long-range interactions within the cell. These can be studied over prolonged time courses and in the presence of graded concentrations of specific inhibitors. Studies of this kind have demonstrated a close relationship between cytoplasmic metabolic flows and mitochondrial forces and have also revealed that the cytoplasmic and mitochondrial free NAD-linked redox potentials are maintained by energy-dependent reactions.