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L M Smith

Publications and source records attributed to L M Smith.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1996

Rapid restriction mapping of cosmids by sequence-specific triple-helix-mediated affinity capture.

A simple and rapid strategy for restriction mapping based on sequence-specific triple-helix affinity capture (TAC) was developed. The strategy was applied to the analysis of cosmid clones by the construction of a new cosmid vector, ScosTriplex-II, containing two different triple-helix-forming sequences flanking the cloning site of the original SuperCos-1 cosmid vector. For restriction mapping, the recombinant cosmid DNA is digested with NotI restriction enzyme or with one of four intron-encoded endonucleases for excision of intact inserts followed by controlled partial digestion with a mapping enzyme used in conjunction with the corresponding methyltransferase. The partial digestion products are combined with biotinylated triple-helix-forming oligonucleotides to form a triple-helical complex. The triple-helix complexes are immobilized on streptavidin-coated magnetic beads, washed, and eluted with pH 9 buffer solution. The fragments are separated and directly sized by agarose gel electrophoresis. Bidirectional maps are obtained simultaneously by binding to the two different triple-helix-forming oligonucleotides. No probe labeling, gel drying, blotting to membranes, hybridization, or autoradiography is necessary. Also, TAC conditions that permit gel-free isolation of the terminal restriction fragments from cosmid inserts were found. These advantages afforded by ScosTriplex-II should facilitate the automation of cosmid restriction site fingerprinting needed for large-scale mapping and sequencing projects.

Base Sequence↗
PubMed1996

Analyzing sequencing reactions from bacteriophage M13 by matrix-assisted laser desorption/ionization mass spectrometry.

The current demand for improved DNA sequencing methodologies posed by the Human Genome Project has spurred the investigation of alternatives to gel electrophoresis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has great potential for the rapid analysis of DNA fragments. Mock Sanger sequencing mixtures have been successfully analyzed by MALDI by pooling synthesized oligonucleotides corresponding to the M13 bacteriophage sequence. More recently, analyses of Sanger sequencing fragments enzymatically generated from synthetic templates of 45 or 50 bases were reported. In the present study, these feasibility demonstrations are extended to show MALDI sequencing from the M13 bacteriophage DNA template commonly used in actual Sanger sequencing. The results show sequence determination for extension products up to 35 bases in length. Different desalting and purification procedures were investigated and it was found that salt could be efficiently reduced by removal of the template in a post-reaction step. Work in progress to stabilize DNA by chemical modification, employed in conjunction with the methods described here, should enable significant extension of the length of readable sequence.

Bacteriophage M13↗
PubMed1996

Activation and inhibition of the AP-1 complex in human breast cancer cells.

We have studied the expression and activity of the jun and fos families of transcription factors in a panel of human breast cancer cells. Numerous breast-cancer cell lines showed variable levels of expression of jun and fos family-member RNA, activator protein-1 (AP-1) DNA binding, and transcriptional-activating activities during exponential growth. In all of the breast-cancer lines tested, c-jun RNA and AP-1 DNA-binding activity correlated. In addition, in most breast cancer cell lines AP-1 DNA-binding activity also correlates with AP-1-transactivating activity. However, some breast cancer cell lines have high c-jun RNA expression, high AP-1 DNA-binding activity, and low AP-1-transactivating activity. Such results suggest that in these breast cancer cell lines there exist AP-1 complexes that can bind DNA but cannot activate transcription. Multiple peptide growth factors as well as the tumor promoter 12-0-tetradecanoylphorbol-13-acetate induced the expression of jun and fos family-member RNAs and also increased AP-1 DNA-binding activity and functional AP-1-transcriptional activating activity in MCF7 breast cancer cells. However, treatment with estrogen, a steroid growth factor, failed to increase jun and fos RNA expression and induced minimal increases in AP-1 DNA binding and AP-1-induced transcriptional-activating activity in comparison with that seen after peptide hormone treatment. Thus, mitogenic peptide hormones and the tumor promoter 12-0-tetradecanoylphorbol-13-acetate, but not estrogen, strongly activate the AP-1 transcription factor in breast cancer cells. A dominant-negative mutant of c-jun that specifically inhibits AP-1- transactivating activity in rat fibroblasts inhibited AP-1 transactivating activity in breast-cancer cells and blocked the increase in AP-1-mediated transcription induced by serum or specific growth factors. This dominant-negative mutant also inhibited MCF7 colony formation, indicating that expression of this AP-1 inhibitor suppressed the proliferation of these breast cancer cells. Such results suggest that growth factor-induced proliferation of breast cancer cells can possibly be blocked by inhibiting AP-1-transactivating activity.

Base Sequence↗
PubMed1996

Automatic matrix determination in four dye fluorescence-based DNA sequencing.

The four dye fluorescence detection strategy is a widely used approach to automated DNA sequence analysis. An important aspect of data processing in this approach is the multicomponent analysis to deduce the concentrations of four fluorophores from fluorescence emission intensities at four different wavelengths. This requires knowledge of the correct transformation matrix M. The matrix M is a function both of the fluorophores employed and the fluorescence detection system. M is typically determined either by a calibration process with individual dyes, or by choosing four well-separated individual peaks corresponding to the four different dyes. Both are time-consuming and complicated procedures for routine use. An automatic scheme for finding M directly from raw sequence data is presented here. This facilitates data analysis and the underlying algorithm may also find utility in other multispectral applications.

Algorithms↗
PubMed1996

Magnesium transport in magnesium-loaded ferret red blood cells.

Mg efflux from ferret red blood cells is stimulated when cells are Mg loaded, but the properties of efflux depend on the loading method. When cell Mg content is altered using A23187, which is subsequently washed away, Mg efflux is minimal until intracellular ionized [Mg] ([Mg2+]i) is greater than 0.9 mM, it then increases substantially with [Mg2+]i. Efflux from loaded cells falls as external [Na] ([Na]o) is reduced, and net Mg influx (against an electrochemical gradient) is seen when [Na]o is sufficiently low. Both influx and efflux are amiloride sensitive. Mg influx from media containing a normal or low [Na] is not affected by reducing [Mg2+]i to very low levels. When cells are Mg loaded by incubating them in media containing 5 mM Na and Mg, Mg efflux is again minimal until [Mg2+]i is greater than 0.9 mM and then it increases with [Mg2+]i, but at a rate approximately 4 times faster than in cells loaded using A23187. This efflux is little affected by 1 mM amiloride. Thus Mg-loading using A23187 reveals the [Mg2+]i dependence of a transporter which is amiloride sensitive, reversible and can operate against an electrochemical gradient, consistent with Na-Mg antiport. Loading by incubation in low-[Na] media activates a high-capacity Mg transporter which obscures the antiporter.

Amiloride↗
PubMed1996

Morphology and body composition predict ovipositional history of female smokybrown cockroaches (Dictyoptera: Blattidae) in the laboratory.

We tested the hypothesis that presence and color of shed follicles in ovarioles of smokybrown cockroaches, Periplaneta fuliginosa (Serville), were correlated with the number of oothecae deposited in the laboratory. There were no shed follicles in females not having oviposited, whereas all females that had deposited 1-4 oothecae had shed follicles or resorption bodies, and females that had deposited > or = 5 oothecae had only shed follicle cells in the base of ovarioles. Other important correlates of female age (days after adult eclosion) were abdominal fat-free, thoracic fat and fat-free masses, and leg fat mass. Oocyte length could be substituted for oocyte fat and fat-free mass because these variables were correlated with oocyte length. Crop mass and delay in dissection after oothecal deposition were not correlated to female age or number of oothecae. Mass of body fractions (fat and fat-free) increased as females deposited additional oothecae. A generalized linear model predicted that as thoracic fat mass increased the number of oocytes already deposited by a female increased. In addition, the model predicted that red follicles would be found in females having deposited 1-3 oothecae and yellow follicles were predicted to be found in females having deposited > 2 oothecae.

Animals↗
PubMed1996

Causes of gastrointestinal hemorrhage in neonates and children.

The causes of gastrointestinal hemorrhage in children were detailed in 1964 by Spencer. We investigated the causes of gastrointestinal hemorrhage in 165 children seen at our institution over a 13-year period. The most common causes of gastrointestinal hemorrhage in the hospitalized patients were necrotizing enterocolitis (44%), anal fissures (23%), and guaiac-positive stools of unknown cause (16%). The mortality rate was 5.4%. Necrotizing enterocolitis was the most common cause of death. Only 16 patients required surgery. Gastrointestinal hemorrhage is an infrequent cause of mortality and morbidity in hospitalized pediatric patients.

Adolescent↗
PubMed1996

Frequency discrimination and speech recognition by patients who use the Ineraid and continuous interleaved sampling cochlear-implant signal processors.

Patients who use the Ineraid cochlear implant were tested in four experiments with materials which assessed frequency discrimination and speech understanding. In each experiment both frequency discrimination and speech recognition varied among patients. Correlations between the two measures were significant and ranged from 0.60 to 0.83. Most generally, frequency discrimination was better in the frequency domain of F1 than in the domain of F2. In experiment 5, both the Ineraid signal processing strategy and a continuous interleaved sampling (CIS) strategy were implemented for a single patient. The CIS strategy improved frequency discrimination in the domain of F2 and improved speech understanding.

Adult↗
PubMed1996

Induction of the gap-pgk operon encoding glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase of Xanthobacter flavus requires the LysR-type transcriptional activator CbbR.

In a previous study, a gene (pgk) encoding phosphoglycerate kinase was isolated from a genomic library of Xanthobacter flavus. Although this gene is essential for autotrophic growth, it is not located within the cbb operon encoding other Calvin cycle enzymes. An analysis of the nucleotide sequence upstream from pgk showed the presence of a gene encoding glyceraldehyde-3-phosphate dehydrogenase and the 3' end of an open reading frame encoding a protein which is 50% identical to transketolase encoded by cbbT of X. flavus. Gene fusions between pgk and lacZ demonstrated that the gap and pgk genes are organized in an operon. Induction of the Calvin cycle in heterotrophically growing cells resulted in a sixfold increase in phosphoglycerate kinase activity in parallel with the appearance of ribulosebisphosphate carboxylase activity. This superinduction of phosphoglycerate kinase did not occur in an X. flavus strain in which cbbR, encoding the transcriptional activator of the cbb operon, was disrupted. The failure to superinduce the gap-pgk operon is not caused by the absence of a functional Calvin cycle, since the expression of this operon in an X. flavus strain with a defective ribulosebisphosphate carboxylase enzyme was the same as the expression in the wild type. It is therefore concluded that the expression of both the cbb and gap-pgk operons is controlled by CbbR.

Amino Acid Sequence↗
PubMed1996

Differentiation of bacterial 16S rRNA genes and intergenic regions and Mycobacterium tuberculosis katG genes by structure-specific endonuclease cleavage.

We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonuclease, Cleavase I. We have applied this technique to the detection and localization of mutations associated with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species and strains. The technique described here is based on the observation that single strands of DNAs can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase I. The patterns of fragments produced are characteristic of the sequences responsible for the structure, so that each DNA has its own structural fingerprint. Amplicons, containing either a single 5'-fluorescein or 5'-tetramethyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and -sensitive M. tuberculosis, the 5' 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, staphylococcus, hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the structural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Bland patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differentiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple, and sensitive method for analyzing nucleic acid sequences and may find wide utility in microbial analysis.

Bacterial Proteins↗