Biomedical subjects

L M Smith

Publications and source records attributed to L M Smith.

At least 19 recordsLinked to original sources

PubMed1998

The immunophilin FKBP65 forms an association with the serine/threonine kinase c-Raf-1.

FKBP65 is a member of the FK506-binding protein class of immunophilins and is the only member reported to contain four peptidylprolyl cis-trans isomerase domains and an unrelated COOH-terminal domain. In this report, we show that the heat shock protein hsp90 and the serine/threonine protein kinase c-Raf-1 are components of FKBP65 immune complexes. The NH2-terminal regulatory domain of c-Raf-1 appears to be required for its interaction with FKBP65. Using GST-FKBP65 fusion protein and purified Raf proteins, we show that full-length FKBP65 can interact with c-Raf-1 but not B-Raf. The activation kinetics of c-Raf-1 after v-H-RasV12 injection of Xenopus oocytes appear to correlate with FKBP65/c-Raf-1 interaction, suggesting that FKBP65 may preferentially associate with forms of c-Raf-1 that are more posttranslationally modified. The interaction of FKBP65 with the c-Raf-heat shock protein 90 heterocomplex implicates this immunophilin in signal-transduction processes.

Animals↗

PubMed1997

Demonstration of a word design strategy for DNA computing on surfaces.

A strategy for DNA computing on surfaces using linked sets of 'DNA words' that are short oligonucleotides (16mers) is proposed. The 16mer words have the format 5'-FFFFvvvvvvvvFFFF-3' in which 4-8 bits of data are stored in 8 variable ('v') base locations, and the remaining fixed ('F') base locations are used as a word label. Using a template and map strategy, a set of 108 8mers each of which possesses at least a 4 base mismatch with the complements to all the other members of the set (4bm complements) are identified for use as a variable base sequence set. In addition, sets of 4 and 12 word labels of the form ABCD....DCBA that are respectively 8bm and 6bm complements with each other are identified. The 16mers are chosen to have a G/C content of 50% in order to make the thermodynamic stability of the perfectly matched hybridized DNA duplexes similar; a simple pairwise additive method is used to estimate the perfect match and mismatch hybridization thermodynamics. A series of preliminary experiments are presented that use small arrays of 16mers attached to chemically modified gold surfaces and fluorescently labeled complements to study the hybridization adsorption and enzymatic manipulation of the oligonucleotides.

Base Sequence↗

PubMed1997

2'-Fluoro modified nucleic acids: polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry.

Fragmentation is a major factor limiting mass range and resolution in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protonation of the nucleobase leads to base loss and backbone cleavage by a mechanism similar to the depurination reactions employed in the chemical degradation method of DNA sequencing. In a previous study [Tang,W., Zhu,L. and Smith,L.M. (1997) Anal. Chem ., 69, 302-312], the stabilizing effect of substituting the 24 hydrogen with an electronegative group such as hydroxyl or fluorine was investigated. These 24 substitutions stabilized the N-glycosidic linkage, blocking base loss and subsequent backbone cleavage. For such chemical modifications to be of practical significance, it would be useful to be able to employ the corresponding 24-modified nucleoside triphosphates in the polymerase-directed synthesis of DNA. This would provide an avenue to the preparation of 24-modified PCR fragments and dideoxy sequencing ladders stabilized for MALDI analysis. In this paper methods are described for the polymerase-directed synthesis of 24-fluoro modified DNA, using commercially available 24-fluoronucleoside triphosphates. The ability of a number of DNA and RNA polymerases to incorporate the 24-fluoro analogs was tested. Four thermostable DNA polymerases [Pfu (exo-), Vent (exo-), Deep Vent (exo-) and UlTma] were found that were able to incorporate 24-fluoronucleotides with reasonable efficiency. In order to perform Sanger sequencing reactions, the enzymes' ability to incorporate dideoxy terminators in conjunction with the 24-fluoronucleotides was evaluated. UlTma DNA polymerase was found to be the best of the enzymes tested for this purpose. MALDI analysis of enzymatically produced 24-fluoro modified DNA using the matrix 2,5-dihydroxy benzoic acid showed no base loss or backbone fragmentation, in contrast to the extensive fragmentation evident with unmodified DNA of the same sequence.

DNA↗

PubMed1997

Breast cancer cells have lower activating protein 1 transcription factor activity than normal mammary epithelial cells.

To determine whether normal breast cells have different levels of activating protein 1 (AP-1) expression and activation relative to breast cancer cells, we have compared the level of c-Jun and c-Fos expression and AP-1 activity in human mammary epithelial cells (HMECs) at different stages of transformation (normal proliferating HMECs, immortal HMECs, oncogene-transformed HMECs, and breast cancer cell lines). These studies demonstrated that normal and immortal HMECs have a high basal level of expression of cJun and cFos and higher AP-1 DNA-binding and transcriptional activating activities than do oncogene-transformed HMECs or human breast cancer cells, with a gradual decrease in AP-1 transactivating activity as cells progress through the carcinogenesis pathway (normal > immortal > oncogene-transformed > cancer cell lines). The AP-1 activity in normal or immortal cells was not modulated by growth factor supplementation or oncogene overexpression, as it is in breast cancer cells. However, the addition of suramin, a nonspecific growth factor antagonist, did inhibit AP-1 in these HMECs, suggesting that this high level of AP-1 present in normal HMECs may be due to autocrine stimulation of growth factor pathways. The differences in AP-1 activity in normal and malignant breast cells may indicate that normal cells are more dependent on AP-1-mediated signals for their growth than are breast cancer cells.

Breast↗

PubMed1997

Ligation-mediated PCR amplification of specific fragments from a class-II restriction endonuclease total digest.

A method is described which permits the ligation- mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest. Feasibility was tested using Bcl I and phage lambda DNA as a model enzyme and amplicon system, respectively. Bcl I is one of many widely used restriction enzymes which cleave at palindromic recognition sequences and leave 5'-protruding ends of defined sequence. Using a single pair of universal primers, a given fragment can be specifically amplified after joining the fragments to adaptors consisting of a duplex primer region and a 9-nucleotide protruding single-stranded 5'-end containing the sequence complementary to the cleaved restriction site and a 4-nucleotide 'indexing sequence.' The protruding strand anneals to a restriction fragment by displacing its corresponding strand in the same fragment-specific indexing sequence located juxtaposed to the restriction site. The adaptor is covalently linked to the restriction fragment by T4 DNA ligase, and amplification is carried out under conditions for long-distance PCR using the M13 forward and reverse primers. The technique discriminated robustly between mismatches and perfect matches for the 16 indexing sequences tested to allow individual lambda Bcl I fragments to be amplified from their respective adaptor pairs. A strategy is proposed enabling a non-cloning approach to the accession, physical mapping and sequencing of genomic DNA. The method could also have application in high-throughput genetic mapping and fingerprinting and should expand the enzyme base for ligation- mediated indexing technology which has previously been limited to the Class-IIS and IP restriction endonucleases.

Bacteriophage lambda↗

PubMed1997

Basal nitric oxide synthesis in essential hypertension.

BACKGROUND: There is indirect evidence that nitric oxide (NO) synthesis in vascular endothelium of patients with hypertension is altered. The aim of this study was to estimate more directly NO production in patients with untreated essential hypertension by measurement of synthesis of inorganic nitrate, which is the end product of NO oxidation in humans. Two separate studies were undertaken in patients with hypertension and appropriate healthy controls. METHODS: In the first study, ten patients and 13 controls were given a diet containing 82 mumoles nitrate per day for 2 days, with urinary and plasma nitrate measurement and 24 h ambulatory blood pressure monitoring on the 2nd day. In the second study, 11 patients and 11 controls were studied in the postabsorptive state; a bolus of 200 mg L[15N]2 arginine was administered intravenously over 10 min. 24 h ambulatory blood pressure monitoring was done and complete urine collections were made for the next 36 h. FINDINGS: In the first study, 24 h urinary nitrate excretion was lower in the hypertensive patients than in the control group (mean 450 [SEM 37] vs 760 mumoles [77] per 24 h; p < 0.001). There was an inverse correlation between average mean daytime ambulatory blood pressure and nitrate excretion (p = 0.007; r2 = -0.73). In the second study, mean 36 h urinary 15N nitrate excretion was significantly lower in the hypertensive than in the control group (1313 [50] vs 2133 [142] pmoles; p < 0.001). There was an inverse correlation also between average mean daytime ambulatory blood pressure and 24 h urinary 15N nitrate excretion expressed per mmole of creatinine (p = 0.002, r2 = -0.59). In addition, total urinary 15N nitrate excretion in the hypertensive group was significantly higher in women than in men (285 [16] vs 198 [14] micrograms 15N nitrate per kg; p = 0.026). INTERPRETATION: These data suggest that whole-body NO production in patients with essential hypertension is diminished under basal conditions. The origin of the NO we measured is not known, and we cannot tell whether the impaired synthesis is primary or secondary to a rise in blood pressure.

Adult↗

PubMed1997

DNA analysis using an electrospray scanning mobility particle sizer.

A scanning mobility particle sizer (SMPS) allows size separation of gas phase particles according to their electrophoretic mobilities. The addition of an electrospray source (ES) recently allowed extension of SMPS analysis to the macromolecular range. We demonstrate here the application of ES-SMPS to nucleic acids analysis. Single- and double-stranded DNA molecules ranging from 6.1 kDa (single-stranded DNA 20 nucleotides in length) to 300 kDa (500 base-pair double-stranded DNA) were separated and detected by ES-SMPS at the picomole to femtomole levels. The measured electrophoretic mobility diameters were found to correlate with the analytes' molecular weights, while the peak areas could yield quantitative information. No fragmentation of DNA was observed under the conditions employed. Different apparent densities were observed for single-stranded and double-stranded DNAs, showing a different behavior for each type of biomolecule. The total analysis time was about 3 min/spectrum. Further optimization of ES-SMPS is expected to make it a fast and sensitive technique for biopolymer characterization.

DNA↗

PubMed1997

Enhanced discrimination of single nucleotide polymorphisms by artificial mismatch hybridization.

In order to increase the discrimination of single nucleotide polymorphisms in DNA hybridization, artificial mismatches are inserted into probe oligonucleotides using the base analog 3-nitropyrrole. Differences in thermal stability (delta Tm) between hybrids formed with normal and single-nucleotide-variant DNA targets are increased by as much as 200% over conventional hybridization, and are strongly dependent upon the spacing between mismatches. The increased specificity is demonstrated by hybridization analysis and allele-specific amplification within the HLA-DRB locus.

Alleles↗

PubMed1997

Genetic analysis by peptide nucleic acid affinity MALDI-TOF mass spectrometry.

The ability to analyze multiple polymorphic sites rapidly and accurately is crucial in all areas of genetic analysis. We have developed an approach for the detection of multiple point mutations, using allele-specific, mass-labeled, peptide nucleic acid (PNA) hybridization probes, and direct analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The composite mass spectra produced contain peaks of distinct masses corresponding to each allele present, resulting in a mass spectral "fingerprint" for each DNA sample. The hybridization characteristics of PNA:DNA duplexes were found to be highly dependent on both base content and sequence. Results from the analysis of four polymorphic sites contained in exon 4 of the human tyrosinase gene show that this approach is simple, rapid, and accurate with potential applications in many areas of genetic analysis.

Alleles↗

PubMed1997

Comparison of traps and development of a two-stage sampling plan for smokybrown cockroaches (Dictyoptera: Blattidae) outdoors.

Four commercial sticky traps were compared with jar traps for sampling smokybrown cockroaches, Periplaneta fuliginosa (Serville), outdoors. Lo-Line and Raid Roach Traps caught more cockroaches than jar traps, whereas the Mr. Sticky and Roach Motel caught < 40% of the cockroaches caught in jar traps. Sampling properties of traps (data collected over 4 yr and 3 cities) were examined retrospectively. Taylor's a and b coefficients were both significantly > 1 for jar traps, Raid Roach Trap, and Lo-Line trap. Estimating cockroach catch typical of Alabama (< 3 per trap) at single houses would require > 20 traps with a precision of 0.25 (i.e., standard error as a proportion of the mean). Taylor's power law was also used as a predictor for within-house (i.e., pooled within-house) and between-house catch variances. Coefficients for these predictors are presented that can be used in a fixed-precision 2-stage sampling plan to estimate cockroach catch from large areas.

Animals↗

PubMed1997

Evaluation of methods of insecticide application for control of smokybrown cockroaches (Dictyoptera: Blattidae).

The insecticide components of a previously developed integrated pest management system for smokybrown cockroaches, Periplaneta fuliginosa (Serville), were tested individually and at reduced application rates in 4 separate trials over 2 yr. A 2-bait combination (pellet and gel baits) did not significantly reduce cockroach abundance more than a single constituent bait alone. A targeted spray of trelamethrin was significantly less effective than a 2-bait combination. Residual activity of insecticides was determined with American cockroaches, P. americana (L.), in a laboratory bioassay. Chlorpyrifos wettable powder remained active < 12 d in the field, after which time its activity was not different from an experimental control (no insecticide residues), whereas chlorpyrifos pellet and hydramethylnon paste baits retained initial activity for > 27 d. Nematodes [Steinernema carpocapsae (Weiser)] applied as a spray solution did not effect the abundance of smokybrown cockroaches, and no cockroaches were recovered that exhibited nematode infection. Time needed to apply various insecticide treatments did not differ substantially (15-25 min per house), even for lower application rates.

Animals↗

PubMed1997

Evidence for signaling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P. ultimum represses the expression of genes in P. fluorescens F113, resulting in altered ecological fitness.

There is increasing evidence that communication between members of the same species, as well as members of different species, is important for the survival of microorganisms in diverse ecological niches, such as the rhizosphere. To investigate whether the phytopathogen Pythium ultimum could alter gene expression in the biocontrol strain Pseudomonas fluorescens F113, which protects the roots of sugar beet from the fungus, a screening system was developed to detect differential expression of bacterial genes in the presence of P. ultimum. The transposon Tn5, containing a promoterless lacZ reporter gene, was used to generate a library of transcriptional gene fusions in P. fluorescens F113. By this screening procedure, five P. fluorescens F113 gene clusters were identified and shown to be repressed in the presence of P. ultimum. The ecological fitness of three of the five reporter mutants in the rhizosphere of seed-inoculated sugar beet was lower than that of the wild type. Furthermore, all five mutants were impaired in their ability to subsequently colonize the rhizosphere of uninoculated sugar beet sown repeatedly in the same soil. With the exception of reporter mutant SF10, which was impaired in nitrogen metabolism, the reporter mutants had growth requirements and biocontrol abilities similar to those of the wild type. This is the first reported case of a fungus repressing the expressing of bacterial genes.

Ecology↗

PubMed1997

Nitrate-reducing bacteria on rat tongues.

Nitrite-producing bacteria (NPB) were isolated from tongues of laboratory rats. The most commonly found nitrite-producing organism was Staphylococcus sciuri, followed by Staphylococcus intermedius, Pasteurella spp., and finally Streptococcus spp. Both morphometric quantification of bacteria on tongue sections and enumeration of culturable bacteria (CFU) showed an increase in the density of bacteria towards the posterior tongue. Up to 65% of bacteria were located in the deep clefts on the posterior tongue. The proportion of culturable NPB in the total culturable microbial population increased from 6% (10(5) CFU cm-2) on the anterior tongue to 65% (10(7) CFU cm-2) on the posterior tongue. Different species compositions of NPB were found on different tongue sections with S. intermedius populations decreasing and S. sciuri and Pasteurella populations increasing towards the posterior tongue. Nitrite production was sensitive to oxygen, and significant nitrite production was only detected on the posterior tongue where the majority of bacteria are situated in deep clefts in the tongue surface. This study suggests the importance of bacteria in nitrite production, from nitrate, on the tongue. Nitrite produced on the tongue may subsequently form nitric oxide in the acidic environment of the stomach. Because of the antimicrobial properties of nitric oxide, a key role for nitrate-reducing tongue bacteria in host animal defense against food-borne pathogens in proposed.

Animals↗

PubMed1997

Chemical synthesis of nitric oxide in the stomach from dietary nitrate in humans.

BACKGROUND/AIMS: It has been suggested that dietary nitrate, after concentration in the saliva and reduction to nitrite by tongue surface bacteria, is chemically reduced to nitric oxide (NO) in the acidic conditions of the stomach. This study aimed to quantify this in humans. METHODS: Ten healthy fasting volunteers were studied twice, after oral administration of 2 mmol of potassium nitrate or potassium chloride. Plasma, salivary and gastric nitrate, salivary and gastric nitrite, and gastric headspace NO concentrations were measured over six hours. RESULTS: On the control day the parameters measured varied little from basal values. Gastric nitrate concentration was 105.3 (13) mumol/l (mean (SEM), plasma nitrate concentration was 17.9 (2.4) mumol/l, salivary nitrate concentration 92.6 (31.6) mumol/l, and nitrite concentration 53.9 (22.8) mumol/l. Gastric nitrite concentrations were minimal (< 1 mumol/l). Gastric headspace gas NO concentration was 16.4 (5.8) parts per million (ppm). After nitrate ingestion, gastric nitrate peaked at 20 minutes at 3430 (832) mumol/l, plasma nitrate at 134 (7.2) mumol/l, salivary nitrate at 1516.7 (280.5) mumol/l, and salivary nitrite at 761.5 (187.7) mumol/l after 20-40 minutes. Gastric nitrite concentrations tended to be low, variable, and any rise was non-sustained. Gastric NO concentrations rose considerably from 14.8 (3.1) ppm to 89.4 (28.6) ppm (p < 0.0001) after 60 minutes. All parameters remained increased significantly for the duration of the study. CONCLUSIONS: A very large and sustained increase in chemically derived gastric NO concentrations after an oral nitrate load was shown, which may be important both in host defence against swallowed pathogens and in gastric physiology.

Administration, Oral↗

PubMed1997

Measurement of NO synthesis in humans by L-[15N2]arginine: application to the response to vaccination.

Induction of nitric oxide (NO) synthesis is a key element of the inflammatory response in humans. We describe a sensitive gas isotope ratio mass spectrometric (GIRMS) method for measuring urinary [15N]nitrate production during intravenous infusion of L-[guanidino-15N2]arginine and its application to investigate the effects of a controlled inflammatory stimulus, typhoid vaccination, on NO synthesis in humans. Intravenous infusion of L-[15N2]arginine at 5-12 mumol.kg-1.h-1 for 24 h in three subjects was used to determine arginine and nitrate pool kinetics. Eight subjects received primed constant infusion of 2.5 mumol.kg-1.h-1 of L-[15N2]arginine for 12 h once before and again after typhoid vaccination. NO synthesis was calculated from 15N enrichment of plasma arginine and urinary nitrate, measured by gas chromatography mass spectrometry and GIRMS, respectively, and total urinary nitrate excretion. Baseline NO synthesis was 298 +/- 44 nmol.h-1.kg lean body mass-1, representing 0.41% of arginine flux. After vaccination, NO synthesis (267 +/- 77 nmol.h-1.kg-1) was not increased (P = 0.18), despite demonstration of an acute phase response. Typhoid vaccination is not accompanied by accelerated NO synthesis.

Adult↗

PubMed1997

Tissue tropism of the HPRS-103 strain of J subgroup avian leukosis virus and of a derivative acutely transforming virus.

The tissue tropism was studied for the HPRS-103 strain of avian leukosis virus, which belongs to a new envelope subgroup, designated J. Studies were conducted in blood monocyte and bone marrow cell cultures and in chickens from six lines that had been shown previously to differ in susceptibility to induction by this virus of myeloid leukosis and other tumors. Using an immunohistochemical technique to detect expression of viral group-specific antigen (Gag) in various tissues, we detected no major differences among the six lines of chickens at 3 and 7 weeks of age following infection as embryos. Thus, Gag expression did not correlate with differences in tumor susceptibility. Of the tissues examined, greatest Gag expression was observed in cells specific to the adrenal gland, heart, kidney, proventriculus and especially in smooth muscle cells and connective tissue. After infection of 1-day-old chicks, greater tissue expression was observed in line 21 chicks, which mostly developed a tolerant viremic infection, than in Brown Leghorn chicks, which developed virus-neutralizing antibodies. An acutely transforming virus, strain 966, derived from HPRS-103-induced myeloid leukosis, showed a tropism similar to HPRS-103. The HPRS-103 strain showed a lower propensity to replicate in the medullary region of the lymphoid follicles of the bursa of Fabricius than did the RAV-1 strain of subgroup A avian leukosis virus. This low bursal tropism may be a factor in why HPRS-103 does not induce lymphoid leukosis. The HPRS-103 and 966 virus replicated in blood monocyte cultures from chickens from the six lines, indicating a tropism for the myelomonocytic cell lineage. In comparison, as previously reported, RAV-1 did not replicate well in the monocyte cultures, whereas RAV-2, a subgroup B avian leukosis virus, did replicate. The tropism of HPRS-103 for monocytes may relate to its ability to cause myeloid leukosis. Monocyte and bone marrow cell cultures from the six lines ranked similarly in differences in susceptibility to transformation by 966 virus and showed evidence that their relative susceptibilities correlated with susceptibility of chickens from these lines to induction of myeloid leukosis by HPRS-103, suggesting common tissue-specific viral and host factors involved in oncogenesis by these two viruses.

Animals↗

PubMed1996

M13-102: a vector for facilitating construction and improving quality of M13 shotgun libraries.

A modified vector, M13-102, is described which utilizes the previously reported M13-100 direct selection strategy for shotgun cloning [Guilfoyle and Smith, Nucleic Acids Res. 22 (1994) 100-107]. In these vectors, direct selection replaces the need for phosphatase treatment of vector DNA and is achieved by insertional inactivation of M13 gene X. When not inactivated, the engineered overproduction of the M13 gene X product mediates phage replication repression. M13-102 contains two new additions: (1) a sequence enabling triple-helix-mediated affinity capture (TAC) for purification of linearized vector DNA, and (2) universal primer sequences for wider compatibility with commercial instruments that support fluorescence-based sequencing. Using a biotinylated homopyrimidine oligodeoxyribonucleotide as third-strand probe, TAC is performed on streptavidin-coated magnetic beads [Ji et al., Genetics Analysis: Techniques and Applications 11 (1994) 43-47], and serves as a rapid and efficient alternative to gel purification. To reduce tandem insertions, phosphatase treatment of insert DNA was easily invoked without sacrificing cloning efficiency. The combined capabilities of direct selection, TAC purification and phosphatase treatment of inserts should facilitate library construction and improve overall library quality.

Bacteriophage M13↗

PubMed1996

Purification of single-stranded M13 DNA by cooperative triple-helix-mediated affinity capture.

A solid-phase triple-helix-mediated affinity capture method is described for the purification of single-stranded M13 DNA for use as template in fluorescence-based DNA sequencing reactions. In this method, a biotinylated polypyrimidine oligonucleotide "loop" bound to streptavidin-coated magnetic beads is used to selectively capture single-stranded M13 DNA from high-titer phage supernatant through the formation of a cooperative triple helix (CTH) complex between the oligonucleotide and a polypurine site previously cloned into the M13 vector. Capture is accomplished at acidic pH to encourage triple-helix formation, while elution is performed at alkaline pH with heating to destroy the CTH complex. The beads can be reused up to three times without probe replenishment. Yields of M13 ssDNA in excess of 1 microgram per milliliter of culture are obtained, sufficient for use as template in fluorescence-based DNA sequencing reactions.

Bacteriophage M13↗