Search PubMed⌕ Search

Biomedical subjects

L M Smith

Publications and source records attributed to L M Smith.

At least 55 records · Page 3Linked to original sources
PubMed1999

Mutational analysis of a heterogeneous nuclear ribonucleoprotein A2 response element for RNA trafficking.

Cytoplasmic transport and localization of mRNA has been reported for a range of oocytes and somatic cells. The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE) is a 21-nucleotide segment of the myelin basic protein mRNA that is necessary and sufficient for cytoplasmic transport of this message in oligodendrocytes. The predominant A2RE-binding protein in rat brain has previously been identified as hnRNP A2. Here we report that an 11-nucleotide subsegment of the A2RE (A2RE11) was as effective as the full-length A2RE in binding hnRNP A2 and mediating transport of heterologous RNA in oligodendrocytes. Point mutations of the A2RE11 that eliminated binding to hnRNP A2 also markedly reduced the ability of these oligoribonucleotides to support RNA transport. Oligodendrocytes treated with antisense oligonucleotides directed against the translation start site of hnRNP A2 had reduced levels of this protein and disrupted transport of microinjected myelin basic protein RNA. Several A2RE-like sequences from localized neuronal RNAs also bound hnRNP A2 and promoted RNA transport in oligodendrocytes. These data demonstrate the specificity of A2RE recognition by hnRNP A2, provide direct evidence for the involvement of hnRNP A2 in cytoplasmic RNA transport, and suggest that this protein may interact with a wide variety of localized messages that possess A2RE-like sequences.

Animals↗
PubMed1999

cJun overexpression in MCF-7 breast cancer cells produces a tumorigenic, invasive and hormone resistant phenotype.

We have previously demonstrated decreased Jun/AP-1 activity in the breast cancer cell line MCF-7 when compared to normal or immortalized mammary epithelial cells. In this paper, we overexpress Jun in MCF-7 cells (MCF7Jun) and demonstrate that it results in diverse biologic and biochemical changes, which mimic those seen clinically in breast cancer. Overexpression of Jun causes significant alterations in the composition of AP-1, decreased junB and increased fra-1 expression and results in an increased biologic aggressiveness. MCF7Jun cells exhibit increased cellular motility, increased expression of a matrix degrading enzyme MMP-9, increased in vitro chemoinvasion and tumor formation in nude mice in the absence of exogenous estrogens. Furthermore, MCF7Jun cells are unresponsive to the growth stimulating effects of estrogen and growth inhibitory effects of tamoxifen. Analysis of the estrogen receptor (ER) expression and activity showed that the MCF7Jun cells have no detectable ER. MCF-7 cells overexpressing mutant forms of cJun were responsive to the growth stimulatory effects of estrogen indicating that full-length cJun is required to acquire the estrogen-independent phenotype in breast cancer cells.

Animals↗
PubMed1999

Stable-isotope-assisted MALDI-TOF mass spectrometry for accurate determination of nucleotide compositions of PCR products.

In parallel with a large-scale sequencing effort, the human genome project will need next-generation tools for accurate and efficient analyses of the enormous pool of DNA sequences. Such analyses are required for (a) validation of DNA sequences, (b) comparison of a parent (known) sequence with a related (unknown) sequence, and (c) characterization of sequence polymorphisms in various genes, especially those associated with genetically inherited human diseases. Here, we report a novel method that combines stable isotope 13C/15N labeling of PCR products of the target sequences with analysis of the mass shifts by mass spectrometry (MS). The mass shift due to the labeling of a single type of nucleotide (i.e., A, T, G, or C) reveals the number of that type of nucleotide in a given DNA fragment. Using this technique, we have accurately determined nucleotide compositions of DNA fragments. The method has also been applied to score a known single-nucleotide polymorphism (SNP). The comparisons of nucleotide compositions determined by our method among homologous sequences are useful in sequence validation, sequence comparison, and characterizations of sequence polymorphisms.

Cadherins↗
PubMed1999

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry.

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

Base Sequence↗
PubMed1999

Effects of phosphorylation-related drugs on slow Ca2+ tail current in guinea-pig detrusor cells.

In isolated guinea-pig detrusor cells, large conditioning depolarizations evoke slowly deactivating Ca2+ tail currents, considered to represent the second open state. The possible involvement of channel phosphorylation in this open state was examined. Application of isoprenaline caused a marginal increase in Ca2+ channel current evoked by simple depolarization, while forskolin did not. During application of either drug, slow-tail currents were never observed after simple depolarizations. The conditions necessary to induce slow-tail currents were not changed, even when cyclic AMP, ATP-gamma-S (adenosine 5'-O-(3-thiotriphosphate)), GDP-beta-S (guanosine 5'-O-(2-thiodiphosphate)) (in the pipette) or H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) (to the bathing solution) was applied. The frequent depolarization protocol, known to facilitate Ca2+ current via Ca2+ and cyclic AMP-dependent phosphorylation mechanism(s) in cardiac myocytes, did not induce slow-tail currents. These results suggest that the transition of Ca2+ channels to the second open state during large depolarization is not a result of (voltage-operated) channel phosphorylation itself. Possible underlying mechanisms are discussed.

Adrenergic beta-Agonists↗
PubMed1999

Regulation of protein kinase C delta by estrogen in the MCF-7 human breast cancer cell line.

We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) delta in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC delta isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC delta was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC delta expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC delta protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10(-10)-10(-8) M estrogen for 7 days down-regulated specifically PKC delta mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC delta expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor beta while the MCF-7 cells expressed predominantly estrogen receptor alpha and barely detectable levels of estrogen receptor beta. These results suggest that the differential ability of estrogen to regulate PKC beta expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Animals↗
PubMed1999

Controlling charge states of large ions.

The charge state of ions produced in electrospray ionization (ESI) was reduced in a controlled manner to yield predominantly singly charged species by exposure of the aerosol to a bipolar ionizing gas. Analysis of the resulting ions on an orthogonal time-of-flight mass spectrometer yielded mass spectra greatly simplified compared with conventional ESI spectra. The decreased spectral complexity afforded by the charge reduction facilitates the analysis of mixtures by ESI mass spectrometry.

Aerosols↗
PubMed1999

Progress toward demonstration of a surface based DNA computation: a one word approach to solve a model satisfiability problem.

A multi-base encoding strategy is used in a one word approach to surface-based DNA computation. In this designed DNA model system, a set of 16 oligonucleotides, each a 16mer, is used with the format 5'-FFFFvvvvvvvvFFFF-3' in which 4-8 bits of data are stored in eight central variable ('v') base locations, and the remaining fixed ('F') base locations are used as a word label. The detailed implementations are reported here. In order to achieve perfect discrimination between each oligonucleotide, the efficiency and specificity of hybridization discrimination of the set of 16 oligonucleotides were examined by carrying out the hybridization of each individual fluorescently tagged complement to an array of 16 addressed immobilized oligonucleotides. A series of preliminary hybridization experiments are presented and further studies about hybridization, enzymatic destruction, read out and demonstrations of a SAT problem are forthcoming.

Animals↗
PubMed1999

Surface-based DNA computing operations: DESTROY and READOUT.

DNA computing on surfaces is where complex combinatorial mixtures of DNA molecules are immobilized on a substrate and subsets are tagged and enzymatically modified (DESTROY) in repeated cycles of the DNA computation. A restriction enzyme has been chosen for the surface DESTROY operation. For the READOUT operation, both cycle sequencing and PCR amplification followed by addressed array hybridization were studied to determine the DNA sequences after the computations.

Animals↗
PubMed1999

Signalling by the fungus Pythium ultimum represses expression of two ribosomal RNA operons with key roles in the rhizosphere ecology of Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 produces antifungal metabolites that protect the roots of sugarbeet from the fungus Pythium ultimum. The phytopathogen, in turn, has the ability to downregulate the expression of genes fundamental to the rhizosphere competence of the bacterial strain. This paper describes the characterization of two of these genes, which were isolated by screening a mini-Tn5::lacZ mutant bank for differential expression of beta-galactosidase in the presence of P. ultimum. In order to identify the genes affected in reporter mutants SF3 and SF5, the transposons and flanking regions were cloned. Sequence analysis of the regions flanking the transposons in SF3 revealed that mini-Tn5::lacZ had inserted into a tRNA(Ile) gene, which maps within a ribosomal RNA (rrn) operon. In SF5, the transposon inserted between the promoter of a second rrn operon and a gene encoding a 16S rRNA. Southern blot analysis demonstrated that there are five rrn operons in P. fluorescens F113 and that the transposons in SF3 and SF5 had inserted into two different operons. Further characterization of these mutants suggests that their reduced rhizosphere competence is not the result of reduced viability in the short term but may be accounted for partly by reduced growth rates under conditions that support rapid growth. Analysis of lacZ expression in the reporter mutants indicate that the marked rrn operons are regulated differently, suggesting different physiological roles.

Base Sequence↗
PubMed1999

Renal responses to angiotensin II receptor blockade in ventilated preterm newborn lambs.

UNLABELLED: Renal and cardiovascular immaturity has been linked with poor outcomes in the premature human newborn. Despite extensive study in the fetus, the contribution of the renin-angiotensin system to renal and cardiovascular function in the premature newborn has not been well characterized. To evaluate the angiotensin II contribution to preterm newborn renal and cardiovascular functions, preterm (120-day) and near-term (136-day) lambs were Caesarean delivered and ventilated. One hour following delivery, animals were randomized to receive angiotensin II receptor-blockade (saralasin; 20 microg kg(-1) min(-1)) or saline (CON). Prior to blockade, mean +/- SEM values for urine flow (U(Flow)), urinary sodium excretion (U(NaV)), and fractional excretion of sodium (FENa) were similar in all groups. Angiotensin II receptor-blockade decreased U(Flow), U(NaV) and FENa in the 120-day group with no changes in the 136-day animals. No changes in mean arterial pressure, or plasma angiotensin II, aldosterone, and renin activity levels were noted at either gestational age. CONCLUSIONS: (1) angiotensin II contributes to the regulation of renal function in 120-day preterm lambs without changing blood pressure and (2) angiotensin II-mediated feedback inhibition of renin release is uncoupled in preterm newborns.

Aldosterone↗
PubMed1999

Novel endogenous retroviral sequences in the chicken genome closely related to HPRS-103 (subgroup J) avian leukosis virus.

HPRS-103, the prototype of avian leukosis virus (ALV) subgroup J, is a recently identified retrovirus associated with myeloid leukosis in meat-type chickens. Although this virus shows high sequence identity to other ALV subgroups within the gag and pol genes, its env gene is highly diverged (with only about 40% sequence identity) from other ALV subgroups. On the other hand, the sequence of the env gene of HPRS-103 was 75% identical to that of E51, a member of the EAV family of endogenous avian retroviruses. It is reported here that the chicken genome also contains another EAV-related element, EAV-HP, showing much greater sequence identity (over 97%) to the HPRS-103 env gene. Southern blotting analysis showed that EAV-HP-related sequences were distinct from EAV-O and were present in all lines of chicken examined and in grey jungle fowl, but were absent from several other avian species. The potential role of these endogenous sequences in the evolution of ALV subgroup J viruses is discussed.

Amino Acid Sequence↗
PubMed1999

The natural history of extremity venous repair performed for trauma.

Surgical repair of extremity venous injuries remains controversial. Literature supports both ligation and repair when analyzed for functional recovery. However, few studies review the natural history of venous repair for trauma. Twenty patients were prospectively enrolled in a protocol of immediate repair of major extremity veins. Simple venorraphy and complex reconstructions were performed at the discretion of the operative team. Patients were studied by contrast venogram on postoperative day 3 and 6 weeks after surgery. Patients with occluded repairs at 3 days received a 5-day course of intravenous anticoagulation and were discharged. Overall, patency at 3 days was 55 per cent and increased to 88 per cent at 6 weeks (P < 0.02). Lateral venorraphy and direct reapproximation had higher patency rates than complex repairs at 6 weeks (92% versus 50%; P < 0.05). All veins that were patent at 3 days remained patent (correlation coefficient 1.0). Repair of traumatized extremity veins carries minimal morbidity and has a high rate of early and eventual patency. Long-term anticoagulation in the face of early thrombosis is unnecessary.

Adolescent↗
PubMed1998

An approach to predicting the stabilities of peptide nucleic acid:DNA duplexes.

An approach is described for predicting peptide nucleic acid (PNA):DNA duplex stability from base sequence by approximating the total free energy of dissociation, delta G degree tot, for these duplexes as the sum of five parameters: (a) a nearest-neighbor interaction summation term, sigma Nj delta G degree j; (b) an initiation term, delta G degree i; (c) a dangling-end stabilization term, delta G degree e; (d) a PNA:DNA stabilization term per nearest-neighbor interaction, delta G degree pna; and (e) an ionic strength term, delta G degree Na. Parameters (a) and (b) are approximated using previously determined values for DNA:DNA duplexes, whereas parameters (c), (d), and (e) are empirically determined. These terms are used to calculated delta G degree tot, which is used in conjunction with a transition enthalpy (delta H degree) value, also approximated from nearest-neighbor values previously derived for DNA:DNA duplexes, to calculate a melting temperature (Tm) for the PNA:DNA duplex. Predicted Tm values calculated by this approach agreed fairly well with measured values for 11 different PNA:DNA duplexes, as well as with literature values. The approach also accurately models ionic strength effects.

Base Sequence↗
PubMed1998

MALDI-TOF mass spectrometric typing of single nucleotide polymorphisms with mass-tagged ddNTPs.

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based method has recently been reported for the typing of single nucleotide polymorphisms using single nucleotide primer extension. This method is limited in some cases by the resolution of the mass determination, as the mass difference between nucleotides can be as little as 9 Da (the difference between A and T). A variation of this method is described here in which a mass-tagged dideoxynucleotide is employed in the primer extension reactions in place of the unmodified dideoxynucleotide. The increased mass difference due to the presence of the mass-tags substantially improves the accuracy and versatility of the procedure.

Dideoxynucleosides↗
PubMed1998

Peripheral primitive neuroectodermal tumor presenting with diffuse cutaneous involvement and 7;22 translocation.

We report an unusual case of peripheral primitive neuroectodermal tumor (pPNET) in an infant presenting with congenital cutaneous nodules and a t(7;22)(p21;q11.2). The biologic behavior of the tumor diverged over time from a slowly growing tumor with multiple cutaneous nodules to a more aggressive neoplasm characterized by pulmonary metastases and a soft tissue mass showing additional cytogenetic alterations.

Chromosomes, Human, Pair 22↗
PubMed1998

Development and application of polymerase chain reaction (PCR) tests for the detection of subgroup J avian leukosis virus.

Subgroup J avian leukosis virus (ALV) is a recently identified avian retrovirus associated with myeloid leukosis in meat-type chickens. The env gene of the HPRS-103 strain of ALV, the prototype of this subgroup, differs considerably from that of other subgroups, but shows close homology to the env-like sequences of members of the EAV family of endogenous retroviruses. Polymerase chain reaction (PCR) tests using two sets of primers were developed for the specific detection of the members of this new subgroup along with another pair of primers for detecting other subgroup viruses. The specificity and sensitivity of this detection system was compared with the conventional detection methods in experimentally and naturally infected samples. The use of PCR was found to be rapid, specific and more sensitive than the conventional diagnostic tests for the detection of ALV. Moreover, the two subgroup J ALV-specific PCR tests were found to be capable of differentiating between 'prototype-like' viruses and more recent isolates which show extensive antigenic and sequence variations. The use of this test as a rapid and sensitive method of detection of viruses in epidemiological studies and eradication programs is discussed.

Animals↗