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Biomedical subjects

W Miller

Publications and source records attributed to W Miller.

At least 37 records · Page 2Linked to original sources
PubMed2001

Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement.

The major distal regulatory sequence for the beta-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.

Binding Sites↗
PubMed2001

Bacteremia, central catheters, and neonates: when to pull the line.

OBJECTIVES: Physicians who treat neonates who become bacteremic while dependent on central venous catheters face a serious and common dilemma. We sought 1) to evaluate the relationship between central venous catheter removal and outcome in bacteremic neonates, 2) to determine species of bacteria that are associated with an increased risk of infectious complications if the central catheter is not removed promptly, and 3) to provide evidence-based recommendations for central catheter management. METHOD: A retrospective cohort study of all neonates who had central venous access and developed bacteremia between July 1, 1995, and July 31, 1999, was conducted in the Duke University neonatal intensive care unit. RESULTS: The outcome for patients in whom the central catheter was not removed within 24 hours of organism identification was significantly worse (odds ratio = 9.8) than it was for those whose catheters were removed promptly. For patients who were infected with Staphylococcus aureus or with nonenteric Gram-negative rods, delayed removal of the central catheter was associated with complicated bacteremia. Catheter sterilization was attempted in 27 neonates who were infected with enteric Gram-negative rods; only 10 of these infants retained their catheters without infection-related complications. Infants who had 4 consecutive blood cultures that were positive for coagulase-negative staphylococcus (CoNS) were at significantly increased risk for end-organ damage and death, compared with infants who had 3 or fewer positive blood culture for CoNS (odds ratio = 29.58). CONCLUSIONS: Bacteremic infants experienced fewer infection-related complications when the central catheter was removed promptly. One positive blood culture for S aureus or a Gram-negative rod warrants central line removal in a neonate. Clinicians who are faced with a neonate who has 1 positive culture for CoNS may attempt medical management without central catheter removal, but documentation of subsequent negative blood cultures is crucial. Once a neonate has 3 positive blood cultures for CoNS, the central catheter should be removed.central line, neonate, bacteremia, bacteria, umbilical catheter, Broviac, percutaneous.

Bacteremia↗
PubMed2001

Discovery of the first non-peptide antagonist of the motilin receptor.

A first-in-class non-peptide antagonist of the motilin receptor was identified through electronic screening of our corporate database against a 3D pharmacophore. The pharmacophore was developed from the motilin 22 residue endogenous peptide using NMR structural data, principles of peptide folding, and peptide structure activity relationships. The NMR data supported helical content within the peptide, and both the hydrophobic staple and N-capping box motifs were identified in the motilin sequence. The conformational features of these motifs were imposed on the peptide structure, providing a constrained conformer as a starting point for database searching. A trisubstituted cyclopentene lead was identified directly from the electronic search. Compounds in this series inhibit the binding of 125I-motilin to human antral smooth muscle membrane and antagonize motilin-induced intracellular calcium mobilization in cells expressing the human motilin receptor. A potent compound developed through optimization, RWJ 68023, is active in binding and cell-based functional assays and is also effective in inhibiting motilin-induced contractility in segments of rabbit duodenum. This orally active compound is currently undergoing clinical evaluation for the treatment of gastrointestinal disorders associated with altered motility.

Amino Acid Sequence↗
PubMed2001

Promising strategies help employers integrate pharmacy and medical programs--and reap cost, quality advantages.

Providing quality pharmacy benefits while managing costs is an escalating challenge for employers. Easy solutions such as higher copays and standard three-tier systems will not provide effective, long-lasting results that include improved clinical outcomes. Employers must concentrate on educating employees on the real cost and value of their pharmacy benefits and they must also take an integrated view of medical and pharmacy benefits.

Cost-Benefit Analysis↗
PubMed2001

Early-phase adaptations of traditional-speed vs. superslow resistance training on strength and aerobic capacity in sedentary individuals.

We performed a randomized exercise training study to assess the effects of traditional Nautilus-style (TR) or superslow (SS) strength training on muscular strength, body composition, aerobic capacity, and cardiovascular endurance. Subjects were 14 healthy, sedentary women, 19-45 years of age (mean +/- SD age, 32.7 +/- 8.9 years), randomized to either the SS or TR training protocols and trained 3 times per week for 10 weeks. Measurements were taken both before and after training, which included a maximal incremental exercise test on a cycle ergometer, body composition, and 1 repetition maximum (1RM) tests on 8 Nautilus machines. Both groups increased their strength significantly on all 8 exercises, whereas the TR group increased significantly more than the SS group on bench press (34% vs. 11%), torso arm (anterior lateral pull-down) (27% vs. 12%), leg press (33% vs. 7%), leg extension (56% vs. 24%), and leg curl (40% vs. 15%). Thus, the TR group's improvement in total exercise weight lifted was significantly greater than that of the SS group after testing (39% vs. 15%). Exercise duration on the cycle ergometer and work rate significantly improved for both groups, but there was no group-by-training interaction. No significant differences were found for body composition or additional aerobic variables measured. Both strength training protocols produced a significant improvement in strength during a 10-week training period, but the TR protocol produced better gains in the absence of changes in percentage of body fat, body mass index, lean body mass, and body weight. In addition, strength training alone did not improve Vo2max, yet short-term endurance increased.

Adult↗
PubMed2000

Levels of GATA-1/GATA-2 transcription factors modulate expression of embryonic and fetal hemoglobins.

GATA transcription factors bind the consensus sequence WGATAR, present in the flanking regions of most erythroid specific genes. GATA-1 and GATA-2, coexpressed in erythroid cells, are important for expression of erythroid genes. To elucidate the role of specific GATA transcription factors on globin gene expression, we examined the human alpha- and beta-globin gene clusters for all GATA sites. Conserved GATA sites were found in each of the hypersensitive sites in both beta-and alpha clusters and in proximal regulatory regions of the zeta-, epsilon- and gamma-globin but not the alpha, delta or beta-globin genes. We then tested the effect of increasing levels of GATA-1 and GATA-2 on the expression of endogenous globin genes in human erythroid cells. Increasing GATA-1 levels in K562 cells decreased the levels of epsilon-globin mRNA but had no effect on the levels of expression of gamma, zeta or alpha-globin genes. Increasing GATA-2 levels increased epsilon-globin and gamma-globin transcripts. Increasing levels of GATA-1 also caused a decrease in the expression of endogenous GATA-2, while increased levels of GATA-2 had no effect on GATA-1 mRNA. Our results indicate a differential role of GATA-1 and -2 transcription factors on globin transcripts and suggest a correlation between the conservation of GATA sites in the regulatory regions and the ability of endogenous globin genes to respond to GATA transcription factors. They also suggest that quantitative changes in the levels of GATA-1 or GATA-2 can result in alterations of globin target gene expression and may participate in the ontogenic control of the globin genes.

Amino Acid Sequence↗
PubMed2000

Comparison of the Escherichia coli K-12 genome with sampled genomes of a Klebsiella pneumoniae and three salmonella enterica serovars, Typhimurium, Typhi and Paratyphi.

The Escherichia coli K-12 genome (ECO) was compared with the sampled genomes of the sibling species Salmonella enterica serovars Typhimurium, Typhi and Paratyphi A (collectively referred to as SAL) and the genome of the close outgroup Klebsiella pneumoniae (KPN). There are at least 160 locations where sequences of >400 bp are absent from ECO but present in the genomes of all three SAL and 394 locations where sequences are present in ECO but close homologs are absent in all SAL genomes. The 394 sequences in ECO that do not occur in SAL contain 1350 (30.6%) of the 4405 ECO genes. Of these, 1165 are missing from both SAL and KPN. Most of the 1165 genes are concentrated within 28 regions of 10-40 kb, which consist almost exclusively of such genes. Among these regions were six that included previously identified cryptic phage. A hypothetical ancestral state of genomic regions that differ between ECO and SAL can be inferred in some cases by reference to the genome structure in KPN and the more distant relative Yersinia pestis. However, many changes between ECO and SAL are concentrated in regions where all four genera have a different structure. The rate of gene insertion and deletion is sufficiently high in these regions that the ancestral state of the ECO/SAL lineage cannot be inferred from the present data. The sequencing of other closely related genomes, such as S.bongori or Citrobacter, may help in this regard.

Databases as Topic↗
PubMed2000

A negative cis-element regulates the level of enhancement by hypersensitive site 2 of the beta-globin locus control region.

The core of DNase hypersensitive site (HS) 2 from the beta-globin locus control region is a potent enhancer of globin gene expression. Although it has been considered to contain only positive cis-regulatory sequences, our study of the enhancement conferred by segments of HS2 in erythroid cells reveals a novel negative element. Individual cis-regulatory elements from HS2 such as E boxes or Maf-response elements produced as great or greater enhancement than the intact core in mouse erythroleukemia (MEL) cells, indicating the presence of negative elements within HS2. A deletion series through HS2 revealed negative elements at the 5' and 3' ends of the core. Analysis of constructs with and without the 5' negative element showed that the effect is exerted on the promoters of globin genes expressed at embryonic, fetal, or adult stages. The negative effect was observed in bipotential human cells (K562 and human erythroleukemia (HEL) cells), proerythroblastic mouse (MEL) cells, and normal adult human erythroid cells. The novel negative element also functions after stable integration into MEL chromosomes. Smaller deletions at the 5' end of the HS2 core map the negative element within a 20-base pair region containing two conserved sequences.

Adult↗
PubMed2000

Web-based visualization tools for bacterial genome alignments.

With the increase in the flow of sequence data, both in contigs and whole genomes, visual aids for comparison and analysis studies are becoming imperative. We describe three web-based tools for visualizing alignments of bacterial genomes. The first, called Enteric, produces a graphical, hypertext view of pairwise alignments between a reference genome and sequences from each of several related organisms, covering 20 kb around a user-specified position. Insertions, deletions and rearrangements relative to the reference genome are color-coded, which reveals many intriguing differences among genomes. The second, Menteric, computes and displays nucleotide-level multiple alignments of the same sequences, together with annotations of ORFs and regulatory sites, in a 1 kb region surrounding a given address. The third, a Java-based viewer called Maj, combines some features of the previous tools, and adds a zoom-in mechanism. We compare the Escherichia coli K-12 genome with the partially sequenced genomes of Klebsiella pneumoniae, Yersinia pestis, Vibrio cholerae, and the Salmonella enterica serovars Typhimurium, Typhi and Paratyphi A. Examination of the pairwise and multiple alignments in a region allows one to draw inferences about regulatory patterns and functional assignments. For example, these tools revealed that rffH, a gene involved in enterobacterial common antigen (ECA) biosynthesis, is partly deleted in one of the genomes. We used PCR to show that this deletion occurs sporadically in some strains of some serovars of S.enterica subspecies I but not in any strains tested from six other subspecies. The resulting cell surface diversity may be associated with selection by the host immune response.

DNA, Bacterial↗
PubMed2000

Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons.

Long-range regulatory elements are difficult to discover experimentally; however, they tend to be conserved among mammals, suggesting that cross-species sequence comparisons should identify them. To search for regulatory sequences, we examined about 1 megabase of orthologous human and mouse sequences for conserved noncoding elements with greater than or equal to 70% identity over at least 100 base pairs. Ninety noncoding sequences meeting these criteria were discovered, and the analysis of 15 of these elements found that about 70% were conserved across mammals. Characterization of the largest element in yeast artificial chromosome transgenic mice revealed it to be a coordinate regulator of three genes, interleukin-4, interleukin-13, and interleukin-5, spread over 120 kilobases.

Animals↗
PubMed2000

Hox cluster genomics in the horn shark, Heterodontus francisci.

Reconstructing the evolutionary history of Hox cluster origins will lead to insights into the developmental and evolutionary significance of Hox gene clusters in vertebrate phylogeny and to their role in the origins of various vertebrate body plans. We have isolated two Hox clusters from the horn shark, Heterodontus francisci. These have been sequenced and compared with one another and with other chordate Hox clusters. The results show that one of the horn shark clusters (HoxM) is orthologous to the mammalian HoxA cluster and shows a structural similarity to the amphioxus cluster, whereas the other shark cluster (HoxN) is orthologous to the mammalian HoxD cluster based on cluster organization and a comparison with noncoding and Hox gene-coding sequences. The persistence of an identifiable HoxA cluster over an 800-million-year divergence time demonstrates that the Hox gene clusters are highly integrated and structured genetic entities. The data presented herein identify many noncoding sequence motifs conserved over 800 million years that may function as genetic control motifs essential to the developmental process.

Animals↗
PubMed2000

Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes.

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding approximately 1.6 Mb and approximately 358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the approximately 189-kb and approximately 152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.

Animals↗
PubMed2000

The human and mouse MHC class III region: a parade of 21 genes at the centromeric segment.

The human major histocompatibility complex (MHC) class III region contains 57-60 structural genes spanning 654-759 kb of genomic DNA. Analysis of the sequence identities of the human and mouse genomic regions between NOTCH4 and complement C2 yields important information on the locations of the coding and regulatory sequences. It also provides insights into the relationship between protein function and level of sequence conservation, and on the clustering of genes with related functions.

Animals↗
PubMed2000

Findings from case studies of state and local immunization programs.

BACKGROUND: As part of its examination of federal support for immunization services during the past decade, the Institute of Medicine (IOM) Committee on Immunization Finance Policies and Practices (IFPP) commissioned eight case studies of the states of Alabama, Maine, Michigan, New Jersey, North Carolina, Texas, and Washington; and a two-county study of Los Angeles and San Diego in California. Specifically, the IOM Committee and these studies reviewed the use of Section 317 grants by the states. Section 317 is a discretionary grant program that supports vaccine purchase and other immunization-related program activities. These studies afforded the Committee an in-depth look at local policy choices, the performance of immunization programs, and federal and state spending for immunization during the past decade. METHODS: The case-study reports were developed through interviews with state and local health department officials, including immunization program directors, Medicaid agency staff, budget analysts, and Centers for Disease Control and Prevention public health advisors to the jurisdiction. Other sources included state and federal administrative records and secondary sources on background factors and state-level trends. The case studies were supplemented by site visits to Detroit, Houston, Los Angeles, Newark, and San Diego. OBSERVATIONS: The nature of immunization "infrastructure" supported by the Section 317 program is shifting from primarily service delivery to a broader set of roles that puts the public effort at the head of a broad immunization partnership among public health, health financing, and other entities in both the public and private sectors. The rate and intensity of transition vary across the case-study areas. In the emerging pattern, service delivery increasingly takes place in the private sector and is related to managed care. "Infrastructure" is moving beyond supporting a core state staff and local health department service delivery to include such activities as immunization registries, quality improvement, and coordination with programs outside public health agencies. At the same time, the recent decline in federal Section 317 support is forcing difficult choices between old and new activities at the state and local levels. CONCLUSIONS: Immunization programs function as an organic component of the local health care financing and delivery systems of which they are a part. Immunization efforts are organized and conducted within distinctive state and local fiscal, economic, and health care contexts. Section 317 Financial Assistance grants, while playing a vital role in supporting immunization "infrastructure," have been too unstable and unpredictable to elicit the strategic planning, programming, and own-source spending that would be optimal for state and local programs. The predominant immunization function of state and local public health agencies is becoming assurance of age-appropriate immunization throughout the lifespan. To be successful in this emerging role, the health agencies must be supported with appropriate staffing, interagency collaboration, and clearly articulated authority.

Adult↗
PubMed2000

High-dose chemotherapy and hematopoietic stem cell rescue for breast cancer: experience in California.

The role of high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell rescue in breast cancer is still controversial. We analyzed the outcomes of 1111 consecutive patients with histologically proven breast cancer who underwent HDCT at 5 major California medical centers. The overall treatment-related mortality (TRM) was 2.3%. TRM was not influenced by disease stage or the HDCT regimen delivered, but it was influenced by hematopoietic graft source. The TRM was 6.1% when bone marrow with or without blood stem cells was used, but only 1.4% when blood stem cells alone were used (P < .001). With a median follow-up of 2.8 years (range, 0.1-8.2 years) after HDCT and autologous hematopoietic stem cell rescue, the estimated 5-year event-free survival (EFS) and overall survival (OS) for stage II/IIIA patients with > or =10 involved axillary lymph nodes were 67% and 76%, respectively. Patients with metastatic breast cancer (MBC) (median follow-up, 1.9 years [range, 0.03-8.3 years]) achieving a complete response (CR) to conventional-dose chemotherapy or rendered to a "no evidence of disease" status before HDCT had significantly better estimated 5-year EFS and OS (28% and 57%, respectively) than those achieving a partial response before HDCT (19% and 27%, respectively; P < or = .0001). Our data suggest that HDCT with hematopoietic stem cell rescue is safe and can be beneficial to patients with high-risk primary breast cancer and for those with MBC achieving CR/no evidence of disease.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

A randomized trial comparing prednisone with antithymocyte globulin/prednisone as an initial systemic therapy for moderately severe acute graft-versus-host disease.

Glucocorticoids remain the standard approach to initial systemic management of acute graft-versus-host disease (aGVHD). For patients refractory to steroids, antithymocyte globulin (ATG) is frequently used as salvage therapy. We decided to test whether the combination of corticosteroids and equine ATG would improve the outcome of initial management of aGVHD, especially in high-risk patients such as recipients of unrelated donor (URD) transplants. One hundred patients with grade II to IV aGVHD having undergone a related or URD marrow transplant were enrolled in the study. Of the patients, 46 were randomly assigned to therapy with prednisone (60 mg/m2 per day x 7 days) and 50 received ATG/prednisone (15 mg/kg ATG bid plus 20 mg/m2 prednisone bid x 5 days, each followed by an 8-week prednisone taper). An intent-to-treat analysis of the overall response at day 42 revealed equivalent complete plus partial response rates of 76% in both the prednisone and ATG/prednisone therapy groups (P > .80). In univariate analysis, patient age, donor type, site of involvement, or aGVHD stage did not influence overall response to therapy (all P > .2). When treatment arms were studied separately, no single clinical feature predicted outcome in either group. Complications were more frequent in the ATG/prednisone arm; patients experienced more infections with cytomegalovirus (44% versus 22%; P = .02) and more frequent pneumonitis, both infectious and noninfectious (50% versus 24%; P < .01). Epstein-Barr virus lymphoproliferative disease was uncommon (4 cases) and comparable in both arms (P = .35). There was no significant difference in survival at day 100, 6 months, and 2 years between the 2 treatment arms. The more intensive immunosuppressive combination of ATG/prednisone failed to improve control of aGVHD and may have affected survival by causing more infectious complications. Combination therapy with ATG should thus be reserved as second-line therapy in the management of aGVHD.

Acute Disease↗