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PubMed1999

S-myristoylation of a glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei.

Covalent modification with lipid can target cytosolic proteins to biological membranes. With intrinsic membrane proteins, the role of acylation can be elusive. Herein, we describe covalent lipid modification of an integral membrane glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from the kinetoplastid Trypanosoma brucei. Myristic acid was detected on cysteine residue(s) (i.e. thiomyristoylation). Thiomyristoylation occurred both co- and post-translationally. Acylated GPI-PLC was active against variant surface glycoprotein (VSG). The half-life of fatty acid on GPI-PLC was 45 min, signifying the dynamic nature of the modification. Deacylation in vitro decreased activity of GPI-PLC 18-30-fold. Thioacylation, from kinetic analysis, activated GPI-PLC by accelerating the conversion of a GPI-PLC.VSG complex to product. Reversible thioacylation is a novel mechanism for regulating the activity of a phospholipase C.

Acylation↗
PubMed1993

An integrated, dynamic model of feed hydration and digestion, and subsequent bacterial mass accumulation in the rumen.

Hydration of feeds and bacterial attachment to feed particles are thought to play major roles in rumen digestion of fibrous feedstuffs. The objective of the present study was to integrate these phenomena in a mechanistic model that could be used for data analysis. The proposed model was based on the conversion of biomass, where digestion end-products can be used for the synthesis of bacterial mass. Digestion of the potentially digestible fraction and subsequent accumulation of bacterial mass was based on a sequential, three-compartment model. These compartments represented substrate undergoing hydration, digestion, and bacterial mass accumulation. A fraction of the substrate was used for synthesis of bacterial mass. It was assumed that these bacteria associate either temporarily or permanently with the remaining substrate. Dacron bags containing either dry or fully-hydrated lucerne (Medicago sativa), maize (Zea mays) cobs, orchard grass (Dactylis glomerata), and wheat straw were incubated in the rumen of a steer that was infused continuously with (15NH4)2SO4. The 15N-enrichments of isolated particle-associated bacteria and residue remaining in the bags were used to estimate bacterial attachment. Substrate remaining and microbial mass accumulation were analysed simultaneously. Hydration did not appear to limit digestion. Fractional rate of digestion and appearance of attached bacterial mass was fastest for lucerne. For lucerne, 5% of the digestion end-products were used for synthesis of bacteria that associated with the substrate, whereas for maize cobs, orchard grass, and wheat straw this was 16, 14, and 19% respectively. Less than 2% of digestion end-products were used for synthesis of bacteria that permanently remained associated with the substrate. Permanent association can occur only with the indigestible fraction, and probably represents bacterial debris. Lysis and/or detachment of bacterial cells was highest for lucerne, and was indicative of the rapid dynamics of lucerne digestion.

Animal Feed↗
PubMed2002

Two-color flow cytometry with a CD19 gate for the evaluation of bone marrow involvement of B-cell lymphoma.

The usefulness of two-color flow cytometry with a CD19 gate (CD19-FCM) for detecting clonal B-cells in the bone marrow was evaluated. Since kappa and lambda ratios analyzed in the bone marrow from healthy adults were within the range of 0.5-3.0, outside of this range was regarded as the existence of clonal B-cells. Mixing experiments in vitro showed that 10% or more of clonal B-cells contaminating normal bone marrow cells could be detected using CD19-FCM. Ninety-nine bone marrow samples from 60 patients with B-cell lymphoma were simultaneously analyzed using CD19-FCM and histologic examination of both bone marrow aspiration and core needle biopsy samples. The agreement of results obtained by both examinations was 82% (81/99). Discrepant results were found in 18 samples of 10 patients. All except one sample were positive for CD19-FCM analysis but negative for histologic examination and the conversion of CD19-FCM-positivity to CD19-FCM-negativity was associated with chemotherapy. These results suggest that CD19-FCM is useful for the evaluation of bone marrow involvement of B-cell lymphoma.

Antigens, CD19↗
PubMed1990

Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates.

Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.

Amino Acid Sequence↗
PubMed1996

Analysis of infection control surveillance data in a long-term-care facility: use of threshold testing.

OBJECTIVE: To describe long-term trends in nosocomial infection rates and the threshold testing method for evaluating nosocomial surveillance data in a long-term-care facility (LTCF). DESIGN: Descriptive epidemiology of prospectively collected infection control surveillance data and application of threshold testing for detecting possible outbreaks. Threshold testing uses the binomial distribution to calculate probabilities of infection frequency at selected endemic levels (mean number of infections per month) and compares these probabilities to observed infection frequency. SETTING: One hundred twenty-bed LTCF located within a public, university-affiliated hospital. PATIENTS AND METHODS: The study period was 1987 through 1994. Yearly endemic levels of specific types of infection were calculated and threshold levels were determined using a previously published method. In this study, a probability of P = .01 was chosen to determine the threshold at a specific endemic level; if the observed number of infections in a month reached or exceeded the threshold level, the likelihood that this occurred by chance alone was 1% or less. INTERVENTIONS: None. RESULTS: The overall mean nosocomial infection rate ranged from three to five episodes per 1,000 resident care days per year; mean yearly rates from 1990 onward were higher and more stable than those from 1987 to 1989. The most common infections identified were lower respiratory tract, skin and soft tissue, urinary tract, and conjunctivitis. For each of these infections, threshold levels were calculated periodically, using only the monthly frequency of infection. Despite variations in the yearly mean endemic level of various infections, threshold levels were stable except for skin and soft-tissue infection. CONCLUSIONS: Threshold testing for analysis of infection control surveillance data in the LTCF setting is straightforward and does not require knowledge of statistics, special computer software, or calculation of rates; given the stable population in the typical LTCF, threshold testing can be based on variations in the monthly count of infections and provides an objective evaluation of surveillance data and a method to identify when outbreaks may be occurring.

Analysis of Variance↗
PubMed2002

Multistage carcinogenesis and radiation.

We will briefly review the concept of multistage carcinogenesis and the mathematical models built on this idea, including a class of models recently developed to describe the pathogenesis of colon cancer. The simplest model that explicitly accounts for cell proliferation kinetics is the two-stage clonal expansion model. It has been applied to a number of experimental and epidemiological data sets and has generated several thought-provoking hypotheses, especially as they relate to the role of promotion in radiation-induced carcinogenesis. The model generates hazard functions (used to estimate age-specific cancer incidence) that are not always intuitive because of the stochastic nature of the clonal expansion process involved. However, the model does make specific predictions depending on whether initiation, promotion or malignant conversion is affected by a carcinogen. A recent analysis of a large cohort of radiation workers exemplifies this behaviour.

Animals↗
PubMed1992

Fast, statistically based alignment of amino acid sequences on the base of diagonal fragments of DOT-matrices.

We present a new pairwise alignment algorithm that uses iterative statistical analysis of homologous subsequences. Apart from the classical conversion of the DOT-matrix characteristic of the Needleman-Wunsch algorithm (NW), we used only those matrix elements that corresponded to the most non-random subsequence homologies. The most reliable elements of the DOT-matrix are written to the compact competition matrices. The algorithm then searches for alignment on the base of only these matrix elements. Our algorithm has low storage and memory requirements, but provides a reliable alignment for the sequences of weak homology (or, at least for the homology regions). In such cases classical NW algorithms often produce unreliable results on the level of statistical noise due to accumulation of random matchings throughout the aligned sequences.

Algorithms↗
PubMed1987

Time dependent conversion of creatine kinase MM isoforms in man.

Subforms of creatine kinase MM isoenzyme (isoforms, pI: MMA = 7.95, MMB = 7.76, MMC = 7.54) in human myocardium and serum were quantified by chromatofocusing. Creatine kinase MMA was a dominant isoform (greater than 94% of MM) in normal (n = 3) and in both infarcted and non-infarcted myocardium (n = 2). To investigate isoform conversion in vitro partially purified MMA was incubated with human plasma at 37 degrees C for 24 h (n = 5). Creatine kinase MMB (0%, 47%, 44% of MM at 0, 12, 24 h respectively) and MMC (0%, 22%, 42%) sequentially appeared in incubation media whereas MMA (100%, 31%, 14%) disappeared rapidly with a mean disappearance rate of -0.00169(0.00021)(SD) min-1. Individual differences in conversion velocity were small (SD less than 5%). To investigate isoform conversion in vivo serum isoforms were analysed in patients with acute myocardial infarction (n = 7). MMA was first dominant (A:B:C = 54:34:12%) in the early stage (6-9 h after the onset of chest pain) followed by MMB dominant (19:42:39%) in the middle stage (24-35 h), and MMC dominant (6:22:72%) in the late stage (54-60 h). Changes in isoform proportion were time dependent regardless of serum creatine kinase activity. These findings are consistent with canine isoform conversion reported previously except that in man the velocity of conversion was slower than in the dog. Thus analysis of serum creatine kinase MM isoforms may allow the onset of acute myocardial infarction to be precisely dated. Moreover, determination of MMA, the isoform native to myocardium with a short serum half life, may be useful in the prompt diagnosis of myocardial infarction.

Creatine Kinase↗
PubMed2006

Conversational skills in a semistructured interview and self-concept in deaf students.

The starting point for this study is the importance of linguistic competence in deaf students as part of their process of socialization and the formation of their self-concept. With the 56 deaf students who participated in the research, we consider the following sociodemographic variables: age, sex and degree of hearing loss, and the educational factor with respect to the mode of mainstream schooling. Self-concept was explored using the Spanish version of the Self Development Questionnaire (SDQ; I. Elexpuru, 1992) and the TST-Who Am I? test, adapted from M. H. Kuhn and T. S. McPartland (1954). To obtain the data for conversational competence, a conversation was held with a hearing adult. An explanation is given of the criteria for pragmatic analysis. The main results highlight the relationship between positive self-concept and most aspects of conversational competence. The study concludes with pedagogical procedures for integration, including specific strategies for teaching conversational skills to deaf pupils through nondeaf pupils and vice versa.

Cognition↗
PubMed1998

Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing.

Among the spliceosomal snRNAs, U2 has the most extensive modifications, including a 5' trimethyl guanosine (TMG) cap, ten 2'-O-methylated residues and 13 pseudouridines. At short times after injection, cellularly derived (modified) U2 but not synthetic (unmodified) U2 rescues splicing in Xenopus oocytes depleted of endogenous U2 by RNase H targeting. After prolonged reconstitution, synthetic U2 regenerates splicing activity; a correlation between the extent of U2 modification and U2 function in splicing is observed. Moreover, 5-fluorouridine-containing U2 RNA, a potent inhibitor of U2 pseudouridylation, specifically abolishes rescue by synthetic U2, while rescue by cellularly derived U2 is not affected. By creating chimeric U2 molecules in which some sequences are from cellularly derived U2 and others are from in vitro transcribed U2, we demonstrate that the functionally important modifications reside within the 27 nucleotides at the 5' end of U2. We further show that 2'-O-methylation and pseudouridylation activities reside in the nucleus and that the 5' TMG cap is not necessary for internal modification but is crucial for splicing activity. Native gel analysis reveals that unmodified U2 is not incorporated into the spliceosome. Examination of the U2 protein profile and glycerol-gradient analysis argue that U2 modifications directly contribute to conversion of the 12S to the 17S U2 snRNP particle, which is essential for spliceosome assembly.

Animals↗
PubMed1986

Induction of intrachromosomal recombination in yeast by inhibition of thymidylate biosynthesis.

The biosynthesis of thymidylate in the yeast Saccharomyces cerevisiae can be inhibited by antifolate drugs. We have found that antifolate treatment enhances the formation of leucine prototrophs in a haploid strain of yeast carrying, on the same chromosome, two different mutant leu2 alleles separated by Escherichia coli plasmid sequences. That this effect is a consequence of thymine nucleotide depletion was verified by the finding that provision of exogenous thymidylate eliminates the increased production of Leu+ colonies. DNA hybridization analysis revealed that recombination, including reciprocal exchange, gene conversion and unequal sister-chromatid crossing over, between the duplicated genes gave rise to the induced Leu+ segregants. Although gene conversion unaccompanied by crossing over was responsible for the major fraction of leucine prototrophs, events involving reciprocal exchange exhibited the largest increase in frequency. These data show that recombination is induced between directly repeated DNA sequences under conditions of thymine nucleotide depletion. In addition, the results of this and previous studies are consistent with the possibility that inhibition of thymidylate biosynthesis in yeast may create a metabolic condition that provokes all forms of mitotic recombination.

Genotype↗
PubMed1995

Evolution of immunoglobulin VH pseudogenes in chickens.

In chickens, there is a single functional gene (VH1) coding for the heavy chain variable region of immunoglobulins, and immunoglobulin diversity is generated by gene conversion of the VH1 gene by many variable region pseudogenes (psi VH's) that exist on the 5' side of the VH1 gene. To understand the evolution of this unique genetic system, we conducted statistical analyses of VH1 and psi VH genes together with functional VH genes from other higher vertebrate species. The results indicate, first, that chicken VH genes are all closely related to one another and were derived relatively recently from an ancestral gene belonging to one of the three major groups of VH genes in higher vertebrates. Second, the rate of nonsynonymous substitution is slightly higher than that of synonymous substitution in the complementarity-determining regions (CDRs), which suggests that diversity-enhancing selection has operated in the CDRs even for pseudogenes. However, both the rates of synonymous and nonsynonymous substitution are higher in the CDRs than in the framework regions (FRs), apparently because of an interaction between positive selection and meiotic gene conversion in the CDRs. Third, a dot matrix analysis of the psi VH genes and genomic diversity (D) genes has indicated that the 3' end of psi VH genes is attached by D-gene-like sequences, and this region of psi VH genes has high similarity with D gene sequences. This suggests that V and D genes were fused at some point of evolutionary time and this fused element multiplied by gene duplication. Finally, two alternative hypotheses of explaining the evolution of the chicken VH gene system are presented.

Animals↗
PubMed2006

Cytochrome P450 1A4 and 1A5 in common cormorant (Phalacrocorax carbo): evolutionary relationships and functional implications associated with dioxin and related compounds.

The present study characterized cytochrome P4501A (CYP1A) isoforms from common cormorant (Phalacrocorax carbo) with regard to their evolutionary relationships and their roles in disposition of dioxin and related compounds (DRCs). Two clones isolated from a cormorant liver cDNA library were named CYP1A4 and CYP1A5 on the basis of greatest overall amino acid identity shared with chicken (Gallus gallus) CYP1A4 (78%) and CYP1A5 (78%), respectively. Spatial heterogeneity in phylogenetic signal along the sequences strongly indicated that cormorant CYP1A4 and CYP1A5 have undergone partial interparalog gene conversion, similar to chicken and mammalian CYP1As. Phylogenetic analysis of a putatively unconverted region produced a tree topology consistent with the orthology of avian CYP1A5s with mammalian CYP1A2s and avian CYP1A4s with mammalian CYP1A1s. Hepatic CYP1A4 and CYP1A5 mRNA levels in wild cormorants from Lake Biwa, Japan, were quantified to examine the effects of DRCs on isoform-specific expression and to evaluate the toxicokinetics of DRCs in which CYP1A expression is involved. Both CYP1A4 and CYP1A5 mRNA levels were positively correlated with total tetrachlorodibenzo-p-dioxin toxic equivalents and concentrations of each congener in most cases in the liver, suggesting the induction of both enzymes through a shared transcriptional mechanism. The lack of correlation of 2,3,7,8-tetrachlorodibenzofuran and 3,3',4,4'-tetrachlorobiphenyl (PCB77) to CYP1A gene expression is likely due to the rapid metabolism of these two congeners. Liver-to-muscle concentration ratios for most DRC congeners except PCB77 and mono-ortho coplanar polychlorinated biphenyls significantly increased with an elevation of CYP1A4 and CYP1A5 mRNA levels. The present data suggest that hepatic sequestration of some DRCs occurs in cormorant via binding to either CYP1A5 or both CYP1A4 and CYP1A5.

Amino Acid Sequence↗
PubMedDate not supplied

Correlational analysis of five commonly used measures of mental status/functional abilities in patients with Alzheimer disease.

There are multiple assessment techniques to help clinicians diagnose, stage, and measure the rate of progression in Alzheimer disease (AD). We analyzed retrospectively the relationship between scores on commonly used scales and tests (Mini-Mental State, the Blessed Information-Memory-Concentration Test, the Alzheimer's Disease Assessment Scale--cognitive portion, the Activities of Daily Living Scale, and the Global Deterioration Scale) of 100 successive admissions to a memory clinic. Patients were included in the study if they were diagnosed subsequently with probable AD and if all five measures were administered in the same day. Regression analysis yielded 20 linear equations that allowed for conversion between test scores on any two instruments. With the exception of the Activities of Daily Living Scale (intercorrelation range with the other four instruments, r = 0.56-0.66), intercorrelations were generally high (r = 0.81-0.87). The results of this study should provide a clinically useful tool for converting test scores on five commonly used dementia screening/rating instruments.

Activities of Daily Living↗
PubMed1983

Oxygen radical dependent lung damage following thermal injury of rat skin.

Acute thermal injury (70 degrees C, 30 sec) to rat skin results in progressive consumptive depletion of the complement system. Individual complement components (C3, C4, C6) each show reductions in hemolytic activity. Crossed immunoelectrophoresis analysis of serum from thermally injured rats reveals conversion of C3 compatible with activation of the complement system. During the first hour following thermal injury, C5a-related chemotactic activity appears in the serum and is temporally related to the development of neutropenia. Lung injury, as revealed by increases in lung permeability, develops progressively during a 6-hour period and parallels changes in complement levels. Morphologically, lung changes include leukoaggregates within pulmonary capillaries and the presence of intra-alveolar hemorrhage. Protection from lung injury following remote thermal injury to skin is afforded by depleting animals of complement or neutrophils, or by systemic treatment of animals with a combination of catalase and superoxide dismutase. Antihistamine drugs have no protective effect. These data suggest that acute thermal injury leads to systemic complement activation, neutrophil activation, and acute lung injury that is related to production of toxic oxygen products by activated blood neutrophils.

Animals↗
PubMed2004

Preeclampsia is associated with loss of neuronal nitric oxide synthase expression in vascular smooth muscle cells of the human umbilical cord.

AIMS: Umbilical blood vessels are not innervated and regulation of blood flow to the placenta must depend on structural changes and the effect of vasoactive factors. Failure to achieve these adaptations may result in reduced fetoplacental perfusion. The purpose of this study was to determine whether neuronal nitric oxide synthase (nNOS) is expressed in human vascular smooth muscle cells (VSMCs) of the fetoplacental circulation. nNOS has been described as a non-endothelial NOS counterregulating vasoconstriction only in the VSMCs of animal models. Therefore, we investigated nNOS expression in the fetoplacental unit from preeclamptic and healthy pregnancies. METHODS AND RESULTS: We investigated nNOS regulation by immunohistochemistry, Western blotting and reverse transcriptase-polymerase chain reaction analysis. nNOS activity was determined by measuring the conversion of L-3H-arginine to L-3H-citrulline. nNOS expression was revealed only in VSMCs of the human umbilical veins, but not in umbilical arteries. A more direct assessment of nNOS activity showed that a small, but consistent amount of nNOS is present in the denuded media of the umbilical vein. In VSMCs of the umbilical veins during preeclampsia a total loss of nNOS protein expression and a significant decrease in mRNA expression were seen. CONCLUSIONS: Loss of nNOS expression is associated with preeclampsia. It may alter the regulation of blood flow in the fetal and maternal placental vasculature in preeclampsia. However, the impact of NO produced by nNOS on the vascular tone of umbilical veins remains to be elucidated.

Adult↗
PubMed2006

Molecular basis for control of conjugation by bacterial pheromone and inhibitor peptides.

In many bacteria expression of lateral gene transfer and of virulence factors is controlled by cell-cell signalling systems. Molecular interactions of microbial signal molecules with their cognate receptors are not well understood. For the Enterococcus faecalis conjugative plasmid pCF10, the PrgX protein serves as a molecular switch controlling expression of conjugation and virulence genes encoded by the plasmid. The induction state of a pCF10-carrying donor cell is determined by the ratio of two signalling peptides, cCF10 pheromone and iCF10 inhibitor. Recent analysis of PrgX/cCF10 interactions suggests a mechanism for conversion to the induced state. However, the means by which iCF10 peptide antagonizes cCF10 activity is unclear, and it has been suggested that inhibitor peptides block import of pheromone peptides. We now show that both of these peptides interact with the same binding pocket of PrgX, but they differentially alter the conformation of the protein and its oligomerization state, resulting in opposing biological activities.

Bacterial Proteins↗
PubMed1996

Muconolactone isomerase of the 3-oxoadipate pathway catalyzes dechlorination of 5-chloro-substituted muconolactones.

An enzyme of Alcaligenes eutrophus JMP 134 which catalyzes dechlorination of (4R, 5R)- and (4R,5S)-5-chloro-3-methyl- and (4R, 5S)-5-chloromuconolactone of principally 3-methyl-trans-, 3-methyl-cis-dienelactone and cis-dienelactone, respectively, was purified to homogeneity. The enzyme was identified as muconolactone isomerase on the basis of its high activity with muconolactone and on its high degree of sequence similarity with previously described muconolactone isomerases. Molecular mass determinations of the highly hydrophobic and heat-resistant enzyme indicated a decameric structure involving a single 10.100-kDa subunit similar to that of muconolactone isomerase of Pseudomonas putida. Kinetic analysis showed cooperative effects between the subunits during conversion of (4R, 5S)-5-chloro-3 -methylmuconolactone. (4R, 5S)-5-chloromuconolactone was the preferred substrate, over the natural substrate (4S)-muconolactone. The (4S, 5S)-structure was found to be an inhibitor of (4R, 5R)-5-chloro-3-methylmuconolactone transformation. Methylsubstitution of the substrate results in a higher affinity for the enzyme, but a drastically lower velocity, resulting in a lower specificity constant.

4-Butyrolactone↗