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Biomedical subjects

L A Lewis

Publications and source records attributed to L A Lewis.

At least 37 records · Page 2Linked to original sources
PubMed1997

The Lck SH2 phosphotyrosine binding site is critical for efficient TCR-induced processive tyrosine phosphorylation of the zeta-chain and IL-2 production.

The lymphocyte-specific tyrosine kinase Lck is essential for TCR-mediated signal transduction. This is in part due to its enzymatic activity as a tyrosine kinase responsible for TCR-induced tyrosine phosphorylation of zeta and CD3 receptor subunits. In addition to its catalytic domain, the Lck protein contains SH3 and SH2 domains capable of associating with other signaling molecules. It has been proposed that phosphotyrosine binding by the Lck SH2 domain may enhance substrate tyrosine phosphorylation by facilitating the processive phosphorylation of multiple sites within the TCR complex. Alternatively or additionally, it may function in adapter activity for facilitating required protein-protein interactions. Previous experiments demonstrate that overexpression of a constitutively activated form of Lck (F505) in the BI-141 T cell hybridoma leads to the Lck kinase activity-dependent enhancement of TCR-mediated signals. Here we demonstrate that mutation of amino acids important for SH2 phosphotyrosine binding significantly compromises the ability of F505 to enhance TCR-mediated protein tyrosine phosphorylation and Ag-induced IL-2 production in BI-141. Examination of the effects of TCR-regulated phosphorylation of the Lck substrate zeta provides in vivo evidence for a role for the Lck SH2 domain in the processive phosphorylation of a multiply phosphorylated substrate.

Binding Sites↗
PubMed1997

T cell antigen receptor-induced IL-2 production and apoptosis have different requirements for Lck activities.

The T cell hybridoma BI-141 has been previously used to dissect the roles of Lck in Ag-induced IL-2 production. Here we demonstrate that BI-141 undergoes apoptosis in response to TCR stimulation using Ag or anti-TCR Abs. Using a panel of BI-141 transfectants expressing constitutively activated Lck (F505) or phosphotyrosine-binding (K154F505 and C156F505) or kinase-impaired (R273F505) mutants, we assess the relative requirements for Lck in TCR-mediated IL-2 production and apoptosis. While BI-141 transfectants expressing F505 are dramatically enhanced in their ability to produce IL-2 in response to Ag relative to K154F505-, C156F505-, or R273F505-expressing transfectants, no differences between these transfectants are observed in their ability to undergo TCR-induced apoptosis. TCR-induced Fas ligand (FasL) expression is demonstrated to be dependent on Lck SH2 and kinase activities, although FasL expression cannot be correlated with apoptosis. Low levels of Fas are constitutively expressed at equal levels on transfectants and are not increased in response to TCR ligation. Together, these data indicate that TCR-induced apoptosis in BI-141 is regulated through a mechanism(s) distinct from the Lck-induced expression of Fas, FasL, or IL-2 production. This TCR signal may be independent of Lck kinase and SH2 activities, or may require lower threshold activity. The identification of differential requirements for particular T cell functions is crucial to understanding how TCR engagement affects downstream T cell functions and may aid in the rational design of therapeutics aimed at specifically modulating particular T cell responses.

Animals↗
PubMed1997

Phylogenetic relationships of the liverworts (Hepaticae), a basal embryophyte lineage, inferred from nucleotide sequence data of the chloroplast gene rbcL.

Sequence data from the chloroplast-encoded gene rbcL were obtained for 24 liverworts, a basal group of embryophytes. Maximum likelihood and parsimony analyses of these data, along with data from other major green plant lineages, confirm hypotheses based on morphological data, such as the paraphyly of bryophytes, and the basal position of liverworts. Molecular data corroborate the deep separation between the complex thalloid and leafy/simple thalloid liverworts implied by morphological data, but the monophyly of liverworts could not be rejected. The effects of accounting for site-to-site rate heterogeneity in these data were examined using maximum likelihood methods. Comparison of trees obtained with and without rate heterogeneity showed that simply allowing for heterogeneity had a greater improvement on likelihood score than optimization of transition/transversion bias. Incorporation of site-to-site rate heterogeneity in the larger analysis, however, did not necessarily change which topology was favored. Properties of rbcL sequences from the two liverwort groups were compared. Significantly different substitution rates were found between leafy/simple thalloid and complex thalloid liverwort taxa, with rates of rbcL sequence evolution in leafy/simple thalloid taxa being higher and more indicative of those of vascular plants, and with those of complex thalloid taxa (such as Marchantia) being slower. Codon usage in rbcL in complex thalloid liverworts was biased toward NNU and NNA, compared to the leafy/simple thalloid liverworts. Although base composition and relative substitution rates differed between the two groups, no significant differences were detected within each of the two groups of liverworts. The signal present in first and second codon sites versus third codon sites was compared. While the third codon positions in rbcL across this taxon sampling are highly variable (with only 15 constant sites of 439), the trees obtained were in general agreement with trees from the entire data set and with trees obtained from independent sources of data. The presence of signal in third codon positions across greater than 400 MY of plant evolution means that definitions of saturation based on pair-wise comparisons of sequences inadequately assess phylogenetic signal.

Chloroplasts↗
PubMed1997

Mercury accumulation in mink fed fish collected from streams on the Oak Ridge Reservation.

This study evaluates effects of feeding mercury (Hg) contaminated fish collected from streams on the Oak Ridge Reservation (ORR) on mink. Diets composed of 25, 50, or 75% fish collected from streams on the ORR were fed to mink beginning 3 months prior to breeding and ending 6 weeks following whelping. Mercury concentrations in diets, tissues of adult mink and their offspring, and physiological and reproductive effects were recorded and compared to concentrations and effects observed in mink fed diets composed of 75% fish collected from the Clinch River above the ORR or from the ocean. Mercury concentrations in prepared diets and in tissues of adult mink and their offspring increased progressively with increased percentage of ORR fish in the diets. Female mink fed diets containing 75% ORR fish had reduced body weight and a decreased number of kits compared to those fed diets containing 75% fish collected above the ORR or from the ocean. However, based on previously reported Hg concentrations associated with adverse effects in mink, the observed adverse effects are not thought to result from exposure to Hg.

Animals↗
PubMed1997

Molecular characterization of hpuAB, the haemoglobin-haptoglobin-utilization operon of Neisseria meningitidis.

We previously identified HpuB, an 85 kDa Fe-repressible protein required for utilization of Fe from, and binding to, haemoglobin and the haemoglobin-haptoglobin complex. The gene for hpuB was cloned from Neisseria meningitidis strain DNM2 and the predicted amino acid sequence indicates that HpuB is an outer membrane receptor belonging to the TonB family of high-affinity transport proteins. A second open reading frame, predicted to encode a 34.8 kDa lipoprotein, was discovered 5' to hpuB, and was designated hpuA. HpuA was identified in a total-membrane-protein preparation by construction of a mutant lacking HpuA. Acylation of HpuA was confirmed by [3H]-palmitic acid labelling of meningococci. Consensus promoter sequences were not apparent 5' to hpuB. The hpuA insertion mutation exerted a polar effect, abolishing expression of hpuB, suggesting that hpuA and hpuB are co-transcribed. The 3.5 kb polycistronic hpuAB mRNA was identified and shown to be transcriptionally repressed by iron. The transcriptional start site was identified 33 nucleotides 5' to the hpuA translational start site, appropriately positioned around consensus promoter and ferric uptake regulator (Fur)-box sequences. The structure of this operon suggests that HpuA-HpuB is a two-component receptor analogous to the bipartite transferrin receptor TbpB-TbpA.

Amino Acid Sequence↗
PubMed1997

Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway.

The Escherichia coli insertion sequence, IS2, is a member of the IS3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed -1 translational frameshift near to the end of orfA and just after the start of orfB. We have characterized two major products of IS2 intramolecular transposition, which accumulate in cells that express the IS2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS2 with just a single basepair (occasionally 2bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS2 end (IRL). The second product is a figure-eight molecule that contains all the IS2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single-stranded version of the minicircle junction--the precise 3' end of IRR has been cleaved and joined to a target just outside the 5' end of IRL; the remaining vector sequences have a free 5' end, derived from cleavage at the 3' end of IRR, and a free 3' end, released upon cleavage of the target site adjacent to IRL. We propose that figure-eight molecules are the precursor to IS2 minicircles and that the formation of these two products is the initial step in IS2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co-integrates.

Bacterial Proteins↗
PubMed1996

The phylogenetic position of Rhopalura ophiocomae (Orthonectida) based on 18S ribosomal DNA sequence analysis.

The Orthonectida is a small, poorly known phylum of parasites of marine invertebrates. Their phylogenetic placement is obscure; they have been considered to be multicellular protozoans, primitive animals at a "mesozoan" grade of organization, or secondarily simplified flatworm-like organisms. The best known species in the phylum, Rhopalura ophiocomae, was collected on San Juan Island, Wash. and a complete 18S rDNA sequence was obtained. Using the models of minimum evolution and parsimony, phylogenetic analyses were undertaken and the results lend support to the following hypotheses about orthonectids: (1) orthonectids are more closely aligned with triploblastic metazoan taxa than with the protist or diploblastic metazoan taxa considered in this analysis; (2) orthonectids are not derived members of the phylum Platyhelminthes; and (3) orthonectids and rhombozoans are not each other's closest relatives, thus casting further doubt on the validity of the phylum Mesozoa previously used to encompass both groups.

Animals↗
PubMed1996

Deep-level diagnostic value of the rDNA-ITS region.

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.

Base Sequence↗
PubMed1996

Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae.

The majority of in vitro-grown Neisseria gonorrhoeae strains were unable to use hemoglobin as the sole source of iron for growth (Hgb-), but a minor population was able to do so (Hgb+). The ability of Hgb+ gonococci to utilize hemoglobin as the iron source was associated with the expression of an iron-repressible 89-kDa hemoglobin-binding protein in the outer membrane. The N-terminal amino acid sequence of this protein revealed amino acids, from positions 2 to 16, identical to those of HpuB, an 85 kDa iron-regulated hemoglobin-haptoglobin utilization outer membrane protein of Neisseria meningitidis. Isogenic mutants constructed by allelic replacement with a meningococcal hpu::mini-Tn3erm construct no longer expressed the 89-kDa protein. Mutants could not utilize hemoglobin to support growth but still grew on heme. Thus, the gonococcal HpuB homolog is a functional hemoglobin receptor and is essential for growth with hemoglobin.

Bacterial Outer Membrane Proteins↗
PubMed1995

Identification of an iron-regulated outer membrane protein of Neisseria meningitidis involved in the utilization of hemoglobin complexed to haptoglobin.

Hemoglobin complexed to the plasma protein haptoglobin can be used by Neisseria meningitidis as a source of iron to support growth in vitro. An N meningitidis mutant, DNM2E4, was generated by insertion of the mini-Tn3erm transposon into the gene coding for an 85-kDa iron-regulated outer membrane protein. Membrane proteins prepared from DNM2E4 were identical to those of the wild-type strain except that the 85-kDa protein was not produced. This mutant was unable to use hemoglobin-haptoglobin complexes as an iron source to support growth and was also impaired in the utilization of free hemoglobin. The mutant failed to bind free hemoglobin, hemoglobin-haptoglobin complexes, or apo-haptoglobin in a solid-phase dot blot assay. The 85-kDa protein was affinity purified when hemoglobin-haptoglobin complexes were used as a ligand but was not purified when free hemoglobin was used. We hypothesize that the 85-kDa iron-regulated protein is the hemoglobin-haptoglobin receptor and designate this protein Hpu (for hemoglobin-haptoglobin utilization).

Amino Acid Sequence↗
PubMed1995

The gay dance party culture in Sydney: a qualitative analysis.

We investigated the gay dance party phenomenon in Sydney in terms of its social and historical context by carrying out 16 qualitative interviews with patrons and organisers of these events. Sydney with a population of 3.5 million is both the capital of New South Wales and the Gay capital of Australia. The gay dance party appears to be a response to a number of conditions which co-existed in the mid 1980s, as previously suggested by Wotherspoon (1991). These included the decriminalization of homosexuality and enactment of the anti-discrimination legislation which set the scene for greater expression of a gay lifestyle, the tradition of inner-city gay bars and private parties as meeting places, and the development of an inner-city gay enclave centred around the Kings Cross, Surry Hills, and Darlinghurst areas. In addition, the development of so-called "party drugs" such as Ecstasy which acted as mood-modulators, the availability of appropriately sited venues, with the development of laser-lighting and other computer-generated light shows, and the new threat of HIV infection acted to provide the context for a more ritualized set of social interactions which fulfilled social and psychological needs for many of the gay party patrons.

Adult↗
PubMed1994

Insertional inactivation of the gene for the meningococcal lactoferrin binding protein.

The lactoferrin binding protein (LBP) of Neisseria meningitidis (the putative meningococcal receptor for human lactoferrin, LF), has been previously characterized as an outer-membrane protein of approximately 105 kDa. Using N-terminal amino acid sequence to generate an oligonucleotide probe, a clone from a lambda gt11 phage library was isolated. This clone was subjected to shuttle mutagenesis, in which an erythromycin mini-transposon was used to interrupt the LBP coding sequence. This insertion mutation was introduced into the meningococcus. A N. meningitidis strain that carried this transposon insertion no longer produced the 105 kDa protein. The absence of this protein was correlated with the inability to bind LF or to use LF as an iron source. The LBP mutant was able to grow with other Fe sources and demonstrated no other visible membrane protein alterations. These data confirm the suggestion that LBP is the meningococcal receptor.

Amino Acid Sequence↗
PubMed1994

Why students choose a primary care or nonprimary care career. The Specialty Choice Study Group.

PURPOSE: To analyze the factors influencing students to choose residency training in primary care (internal medicine, family medicine, and pediatrics) or nonprimary care specialties and study the combined effect of reported responses on their choice. MATERIALS AND METHODS: A 12-item questionnaire using a 7-point Likert scale was mailed to the 1,164 graduating seniors from 9 medical schools in 1991. Responses ranged from 1, very negative influence, to 7, very positive influence. Four indicated no influence. The overall response rate was 69%. Univariate analysis of factors associated with specialty choice was done with the Mantel-Haenzsel chi-square test. Odds ratios were calculated for each significant variable without controlling for other variables. Factors found to have univariate significance were then tested for combined significance with logistic regression analysis. The regression was performed on a randomly chosen training sample, and validated on a test sample. RESULTS: Forty-five percent of respondents chose an internship and planned residency training in a primary care specialty. Factors that remained positively associated with choosing a primary care specialty when controlling for other factors were desire to provide comprehensive care, to keep options open, and to undertake ambulatory care. Desire for monetary reward was negatively associated with choice of a primary care specialty. CONCLUSIONS: Positive educational experiences in the ambulatory setting should be enhanced, and disparity in remuneration among disciplines reduced.

Adult↗
PubMed1994

Learning, experience, and self-assessment of competence of third-year medical students in performing bedside procedures.

BACKGROUND: Third-year medical students are expected to perform common bedside procedures as part of their daily ward activities, but there are few programs to teach these skills or document students' abilities to perform them. PURPOSE: In addition to describing the clinical procedures course at the Medical College of Georgia School of Medicine, the purpose of this paper is to report how often students perform common bedside procedures during the third year of medical school, whether the number of times a procedure has been performed is indicative of self-perceived level of competence, and how useful the students find the procedures course for learning how to perform common procedures. METHOD: In June 1991, the 163 entering third-year students participated in a two-day course in which they were taught universal precautions and provided specific step-by-step instructions for performing 16 clinical procedures. One year later, after the students had completed their third-year clerkships, they were asked to report the numbers of times they had performed the procedures during their clerkships, to provide self-assessments of their competency, and to indicate how helpful the course had been for learning clinical procedures. The association between the number of times the students reported performing a procedure and how competent they felt to perform the procedure was assessed using Kendall's tau coefficient. RESULTS: A total of 100 students returned completed questionnaires. Most of the students reported performing venipuncture, obtaining blood cultures, and scrubbing surgically more than five times, but they reported performing other procedures less frequently. For most procedures, self-assessment of competency correlated with frequency of performance. The procedures course was well received by students and instructors, based on both written evaluations and informal verbal feedback. CONCLUSION: Surprisingly, the students' self-reports indicated that many students performed procedures on patients infrequently. As expected, for most procedures there was a significant association between frequency of performance and self-assessed competency.

Academic Medical Centers↗
PubMed1994

The non-random pattern of insertion of IS2 into the hemB gene of Escherichia coli.

The hemB gene of Escherichia coli has been identified as a hot spot for the insertion of the transposable element IS2. The insertional specificity of IS2 is still unclear. This study reports on the attempt to sequence a statistically significant number of insertions in hemB, in order to determine whether there might be a basis for future studies to determine a molecular basis of IS2 insertional specificity. The results indicate that IS2 inserts in a non-random manner into a 240 bp segment at the 5' end of the gene (region I). Twenty-one of 24 insertions occurred in region I. Three insertions have been identified in the two middle 250 bp segments of the 975 bp gene, and none in the 3' terminal segment. A seventeen bp sequence showing 88.2% identity with a segment of IS2, 221 bp from the 3' terminus has been identified in region I. Four instances of repeated insertion between the same pair of nucleotides have been observed at four different sites.

Bacterial Proteins↗