Cell Envelope Changes in Solvent-Tolerant and Solvent-Sensitive Pseudomonas putida Strains following Exposure to o-Xylene

Solvent-tolerant and -sensitive Pseudomonas putida strains were studied to determine their cell envelope changes following exposure to o-xylene. Both strains produced trans-unsaturated fatty acids. The tolerant strain showed an increase in total fatty acids, an increase in saturated fatty acids, and modified lipopolysaccharide. It is suggested that these envelope modifications aid in survival at high concentrations of organic solvents.     

Autoantibodies to neuronal glutamate receptors in patients with paraneoplastic neurodegenerative syndrome enhance receptor activation.

BACKGROUND: Paraneoplastic syndromes are "remote" complications of cancer characterized clinically by neurological disease. The sera and cerebrospinal fluid (CSF) from patients with paraneoplastic neurological syndromes (PNS) frequently contain autoantibodies to ill-defined neuronal antigens. We report here that neuronal glutamate receptors are targets for autoantibodies found in the serum from some patients with well-characterized PNS. MATERIALS AND METHODS: We have analyzed the serum from seven patients with well-characterized PNS for the presence of autoreactive antibodies to non-NMDA glutamate receptor subunits. Autoantibodies were assessed using Western blot, immunohistochemistry, and immunocytochemistry. Whole-cell electrophysiological recordings were used to examine the effect of antibodies on glutamate receptors expressed by cortical neurons in culture. RESULTS: Six of seven patients' serum contained autoantibodies to the non-NMDA glutamate receptor (GluR) subunits GluR1, GluR4, and/or GluR5/6. No patient had autoantibodies to GluR2, and only one patient exhibited weak immunoreactivity to GluR3. Electrophysiological analysis demonstrated that the serum from four of the six GluR-antibody-positive patients enhanced glutamate-elicited currents on cultured cortical neurons but had no effect on receptor function alone. Enhancement of glutamate-elicited currents was also produced by affinity-purified antibody to GluR5. CONCLUSIONS: The occurrence of autoantibodies to specific neuronal neurotransmitter subunits in the sera of patients with PNS and the ability of these autoantibodies to modulate glutaminergic receptor function suggest that some paraneoplastic neurological injury could result from glutamate-mediated excitotoxicity.     

Influence of redox potential on the anaerobic biotransformation of nitrogen-heterocyclic compounds in anoxic freshwater sediments

The potential for degradation of four nitrogen-heterocyclic compounds was investigated in fresh-water sediment slurries maintained under denitrifying, sulfate-reducing, and methanogenic conditions. Pyridine (10 mg/l) was rapidly transformed within 4 weeks under denitrifying conditions but persisted for up to 3 months under sulfate-reducing and methanogenic conditions. No intermediate biotransformation products of pyridine metabolism were detected under denitrifying conditions. Quinoline (10 mg/l) was completely transformed without a lag phase under methanogenic and sulfate-reducing conditions after incubation for 23 and 45 days, respectively. 2-Hydroxyquinoline was produced concomitantly with quinoline transformation under methanogenic and sulfate-reducing conditions. Under denitrifying conditions, less than 23% of the initial concentration of quinoline was transformed after anaerobic incubation for 83 days. Indole, however, was completely removed from sediment slurries under denitrifying, sulfate-reducing, and methanogenic conditions after anaerobic incubation for 18, 27, and 17 days, respectively. Only low amounts of oxindole (2–4 mg/l) accumulated during indole metabolism under methanogenic and denitrifying conditions, but under sulfate-reducing conditions, oxindole accumulation was stoichiometric with indole transformation. No evidence for biotransformation of carbazole was noted for all anaerobic conditions tested.     

A novel human type I hair keratin gene: evidence for two keratin hHa3 isoforms

We present the nucleotide and amino acid sequence for a novel human type I hair keratin, which could be identified through its high sequence homology and strict carboxyterminal length identity as a human ortholog of the murine hair keratin mHa3. Our hHa3 sequence differs, however, from that of a previously described hHa3 hair keratin (published only as an amino acid sequence; [13]) in 24 amino acid positions, 8 of which occur in the middle of the carboxyterminal domain. PCR of genomic DNA from 25 normal human subjects using a primer pair derived from sequence segments located in the 3-region of our hHa3 clone that encode conserved amino acid sequences in both keratins, resulted in the amplification of two distinct products of 0.38 kbp and 1.0 kbp. DNA sequence analysis of the cloned PCR products allowed identification of the 0.38 kb sequence as that originating from Yuet al. [13] and the 1.0 kb sequence as that being derived from our data. The difference in fragment length was due to unique intron 6 sequences, indicating that these two keratin species are encoded by genes of their own. Moreover, extensive Southern blot analyses with DNA from 25 unrelated individuals of different races using a 3-noncoding sequence from our keratin and the intron 6 sequence of the keratin of Yuet al. [13], as hybridization probes showed that both keratin genes are present as single copy sequences occurring ubiquitously and without gross alterations in the human genome. Collectively, these data demonstrate that the human type I hair keratin described in this paper represents an isoform of the previously described hHa3 keratin. We propose that these hHa3 isoforms be named in chronological order of discovery hHa3-I and hHa3-II.     

Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.     

Polymorphic Admixture Typing in Human Ethnic Populations

A panel of 257 RFLP loci was selected on the basis of high heterozygosity in Caucasian DNA surveys and equivalent spacing throughout the human genome. Probes from each locus were used in a Southern blot survey of allele frequency distribution for four human ethnic groups: Caucasian, African American, Asian (Chinese), and American Indian (Cheyenne). Nearly all RFLP loci were polymorphic in each group, albeit with a broad range of differing allele frequencies (δ). The distribution of frequency differences (δ values) was used for three purposes: (1) to provide estimates for genetic distance (differentiation) among these ethnic groups, (2) to revisit with a large data set the proportion of human genetic variation attributable to differentiation within ethnic groups, and (3) to identify loci with high δ values between recently admixed populations of use in mapping by admixture linkage disequilibrium (MALD). Although most markers display significant allele frequency differences between ethnic groups, the overall genetic distances between ethnic groups were small (.066–.098), and <10% of the measured overall molecular genetic diversity in these human samples can be attributed to “racial” differentiation. The median δ values for pairwise comparisons between groups fell between .15 and .20, permitting identification of highly informative RFLP loci for MALD disease association studies.     

Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems

A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing Legionella pneumophila along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions. The materials varied in their abilities to support biofilm development and the growth of L. pneumophila. Elastomeric surfaces had the most abundant biofilms supporting the highest numbers of L. pneumophila CFU; this was attributed to the leaching of nutrients for bacterial growth from the materials. No direct relationship existed between total biofouling and the numbers of L. pneumophila CFU.     

CENTRAL MOMENTS AND PROBABILITY DISTRIBUTION OF COLLESS'S COEFFICIENT OF TREE IMBALANCE

The great increase in the number of phylogenetic studies of a wide variety of organisms in recent decades has focused considerable attention on the balance of phylogenetic trees—the degree to which sister clades within a tree tend to be of equal size—for at least two reasons: (1) the degree of balance of a tree may affect the accuracy of estimates of it; (2) the degree of balance, or imbalance, of a tree may reveal something about the macroevolutionary processes that produced it. In particular, variation among lineages in rates of speciation or extinction is expected to produce trees that are less balanced than those that result from phylogenetic evolution in which each extant species of a group has the same probability of speciation or extinction. Several coefficients for measuring the balance or imbalance of phylogenetic trees have been proposed. I focused on Colless's coefficient of imbalance (7) for its mathematical tractability and ease of interpretation. Earlier work on this statistic produced exact methods only for calculating the expected value. In those studies, the variance and confidence limits, which are necessary for testing the departure of observed values of I from the expected, were estimated by Monte Carlo simulation. I developed recursion equations that allow exact calculation of the mean, variance, skewness, and complete probability distribution of I for two different probability-generating models for bifurcating tree shapes. The Equal-Rates Markov (ERM) model assumes that trees grow by the random speciation and extinction of extant species, with all species that are extant at a given time having the same probability of speciation or extinction. The Equal Probability (EP) model assumes that all possible labeled trees for a given number of terminal taxa have the same probability of occurring. Examples illustrate how these theoretically derived probabilities and parameters may be used to test whether the evolution of a monophyletic group or set of monophyletic groups has proceeded according to a Markov model with equal rates of speciation and extinction among species, that is, whether there has been significant variation among lineages in expected rates of speciation or extinction.     

Modification of isolated subunit c of the F1F0-ATPase from Propionigenium modestum by dicyclohexylcarbodiimide

Subunit c of the F₁F₀-ATPase from Propionigenium modestum was extracted from the particulate cell fraction with chloroform/methanol. The protein was further purified by carboxymethyl cellulose chromatography and anion exchange HPLC in the organic solvent. SDS-PAGE of the purified protein indicated a single stained protein band migrating as expected for the c-subunit. Incubation of isolated subunit c in chlorform/methanol or aqueous buffer containing dodecyl-β- -maltoside with [¹⁴C]dicyclohexylcarbodiimide (DCCD) resulted in the incorporation of radioactivity into the protein. The rate of this reaction depended on the external pH; it was significantly faster in the more acidic than in the alkaline pH range. In the presence of Na⁺ subunit c was partially protected from labeling with [¹⁴C]DCCD at pH 6.1 and at pH 7.5, whereas no protection was evident at pH 5.5. At pH 7.5, the rate of subunit c labeling by [¹⁴C]DCCD in the presence of 20 mM NaCl was about 50% lower than in the absence of Na⁺ ions. The isolated c-subunit therefore apparently retains in part the Na⁺ binding site which, when occupied, diminishes the reactivity of the protein towards DCCD.