Biomedical subjects

W Blackstock

Publications and source records attributed to W Blackstock.

10 recordsLinked to original sources

PubMed2003

Two-component systems in Haemophilus influenzae: a regulatory role for ArcA in serum resistance.

Knockout mutations were constructed in the arcA gene of a virulent type b strain of Haemophilus influenzae, and the behavior of the resulting mutants was investigated in a number of conditions that mimicked distinct steps in the natural infection pathway. In arcA mutants, synthesis of capsule and lipooligosaccharide (LOS) and growth in synthetic media were unaltered compared to synthesis of capsule and LOS and growth in synthetic media in the wild-type H. influenzae type b parent strain. However, the virulence of the arcA mutants for BALB/c mice was significantly reduced. Upon exposure to human blood or serum, the arcA mutants showed markedly reduced survival compared with the survival of its wild-type parent. Serum resistance could be fully restored by complementation in cis with the H. influenzae arcA gene but not by complementation in cis with the homologous gene from Escherichia coli. The proteomes of wild-type and mutant bacteria were markedly different, especially under anaerobic conditions, underscoring the global regulatory role of ArcAB in H. influenzae. Evaluation of antibody titers and classical complement activities in various serum samples pointed to complement-mediated bactericidal activity as the factor that distinguishes between the arcA mutant and wild-type phenotypes. Comparative analysis of the membrane fractions of the arcA mutants and the wild-type strain revealed several ArcA-regulated proteins, some of which may be implicated in the serum hypersensitivity phenotype.

Animals↗

PubMed2001

Carboplatin/paclitaxel or carboplatin/vinorelbine followed by accelerated hyperfractionated conformal radiation therapy: a preliminary report of a phase I dose escalation trial from the Carolina Conformal Therapy Consortium.

The maximum tolerated dose of conformal radiation therapy delivered at 1.6 Gy bid is being assessed in patients with unresectable stage IIB-IIIB non-small cell lung cancer who have been treated with induction regimens consisting of carboplatin plus paclitaxel or carboplatin plus vinorelbine. Data from the early stages of this parallel phase I study show that the two induction regimens are similar in toxicity and that both induce partial responses in 45% of patients. Both regimens can be followed by conformal radiotherapy using an accelerated hyperfractionated schedule to a dose of at least 80 Gy without experiencing unacceptable toxicity. Key morbidity observed thus far has involved the esophagus. Further cohorts of patients will receive higher doses of conformal radiotherapy (in 6.4 Gy increments) until the maximum tolerated dose is reached.

Aged↗

PubMed2000

Proteomics: new perspectives, new biomedical opportunities.

Proteomics-based approaches, which examine the expressed proteins of a tissue or cell type, complement the genome initiatives and are increasingly being used to address biomedical questions. Proteins are the main functional output, and the genetic code cannot always indicate which proteins are expressed, in what quantity, and in what form. For example, post-translational modifications of proteins, such as phosphorylation or glycosylation, are very important in determining protein function. Similarly, the effects of environmental factors or multigenic processes such as ageing or disease cannot be assessed simply by examination of the genome alone. This review describes the underlying technology and illustrates several areas of biomedical research, ranging from pathogenesis of neurological disorders to drug and vaccine design, in which potential clinical applications are being explored.

Animals↗

PubMed2000

Activation of lytic Epstein-Barr virus (EBV) infection by radiation and sodium butyrate in vitro and in vivo: a potential method for treating EBV-positive malignancies.

The consistent presence of the EBV genome in certain tumors offers the potential for novel EBV-directed therapies. Switching the latent form of EBV infection present in most EBV-positive tumor cells into the cytolytic form may be clinically useful because lytic EBV infection leads to host cell destruction, and very few normal cells contain the EBV genome. It would also be therapeutically advantageous to induce expression of EBV-encoded lytic proteins that convert the nucleoside analogues ganciclovir (GCV) and 3'-azido-3'deoxythymidine (AZT) into their active, cytotoxic forms. In this report, we have explored two different approaches for activating the lytic form of EBV infection in tumors. We show that gamma-irradiation at clinically relevant doses induces lytic EBV infection in lymphoblastoid cell lines in vitro as well as in EBV-positive B-cell tumors in SCID mice. In addition, sodium butyrate (given as a single i.p. dose) is effective for activating lytic viral infection in some EBV tumor types in SCID mice. We also examined whether low-dose gamma-irradiation treatment of EBV-positive lymphoblastoid cells in vitro promotes GCV or AZT susceptibility. The combination of radiation with either GCV or AZT induced significantly more cell killing in vitro than either radiation or prodrug treatment alone. Most importantly, we found that the combination of gamma-irradiation and GCV was much more effective in treating EBV-positive lymphoblastoid tumors in SCID mice than either agent alone. Thus, GCV or AZT treatment could potentially enhance the therapeutic efficacy of radiation therapy for EBV-positive lymphomas in patients.

Animals↗

PubMed2000

Cross-species identification of novel Candida albicans immunogenic proteins by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

We have previously reported the usefulness of two-dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the detection of the major Candida albicans antigens (Pitarch et al., Electrophoresis 1999, 20, 1001-1010). The identification of these antigens would be useful for the characterization of good markers for the disease, and for the development of efficient diagnostic strategies. In this work we have used nanoelectrospray tandem mass spectrometry to obtain amino acid sequence information from the immunogenic proteins previously detected. We report here the cross-species identification of these antigens by matching of tandem mass spectrometry data to Saccharomyces cerevisiae proteins. Using this approach, we unambiguously identified the four C. albicans immunogenic proteins analyzed, namely aconitase, pyruvate kinase, phosphoglycerate mutase and methionine synthase. Furthermore, we report for the first time that aconitase, methionine synthase and phosphoglycerate mutase have antigenic properties in C. albicans.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2000

A proteomic approach for the study of Saccharomyces cerevisiae cell wall biogenesis.

In fungi, cell shape is determined by the presence of a rigid cell wall which separates the cell from the extracellular medium. This highly dynamic structure is essential for the maintenance of cell integrity and is involved in several phenomena such as flocculation, adherence and pathogenicity. The composition of the fungal cell wall is well known, but issues such as the assembly and remodeling of its components remain poorly understood. In an attempt to study the de novo construction of the yeast cell wall, we have undertaken a large-scale proteomic approach to analyze the proteins secreted by regenerating protoplasts. Upon incubation of protoplasts in regenerating conditions, numerous proteins are secreted into the culture medium. These presumably include proteins destined for the cell wall, comprising both structural proteins as well as enzymes involved in cell wall biogenesis. This work reports the establishment of a reference map of proteins secreted by regenerating protoplasts by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and their identification by mass spectrometry. Thirty-two different proteins have been identified, including known cell wall proteins, glycolytic enzymes, heat shock proteins, and proteins involved in several other processes. Using this approach, novel proteins possibly involved in cell wall construction have also been identified. This reference map will allow comparative analyses to be carried out on a selected collection of mutants affected in the cell wall.

Amino Acid Sequence↗

PubMed1997

Proteome analysis: genomics via the output rather than the input code.

A knowledge of the 'proteome,' total protein output encoded by a genome, provides information on (1) if and when predicted gene products are translated, (2) the relative concentrations of gene products, and (3) the extent of posttranslational modification, none of which can be accurately predicted from the nucleic acid sequence alone. The current status of proteome analysis is reviewed with respect to some of the techniques employed, automation, relevance to genomic studies, mass spectrometry and bioinformatics, limitations, and recent improvements in resolution and sensitivity for the detection of protein expression in whole cells, tissues, or organisms. The concept of 'proteomic contigs' is introduced for the first time. Traditional approaches to genomic analysis call upon a number of strategies to produce contiguous DNA sequence information, while 'proteomic contigs' are derived from multiple molecular mass and isoelectric point windows in order to construct a picture of the total protein expression within living cells. In higher eukaryotes, the latter may require several dozen image subsets of protein spots to be stitched together using advanced image analysis. The utility of both experimental and theoretical peptide-mass fingerprinting (PMF) and associated bioinformatics is outlined. A previously unknown motif within the peptide sequence of Elongation Factor Tu from Thermus aquaticus was discovered using PMF. This motif was shown to possess potential significance in maintaining structural integrity of the entire molecule.

DNA↗

PubMed1996

Proton MR spectroscopy of squamous cell carcinoma of the upper aerodigestive tract: in vitro characteristics.

PURPOSE: To determine the ability of in vitro high-field-strength proton MR spectroscopy to differentiate squamous cell carcinoma of the upper aerodigestive tract from uninvolved muscle. METHODS: Surgical specimens of squamous cell carcinoma arising from the upper aerodigestive tract (n = 18) and from muscle (n = 13) were examined in vitro using high-field (11 T) proton MR spectroscopy. The peak heights of choline and creatine were measured for tumor and muscle at echo times of 136 and 272. The choline/creatine (Cho/Cr) ratio was compared between tumor and normal tissue for each echo time. Student's t test was used to determine whether a significant difference existed between proton MR spectroscopic measurements of the Cho/Cr ratio for tumor and muscle. RESULTS: The mean Cho/Cr ratio was consistently higher in tumor than in muscle at all echo times; however, statistically significant differences between tumor and muscle were identified only at longer echo times (136 and 272). CONCLUSION: The Cho/Cr peak height ratio can be used to differentiate tumor from muscle in vitro (at 11 T).

Carcinoma, Squamous Cell↗

PubMed1993

Local failure and margin status in early-stage breast carcinoma treated with conservation surgery and radiation therapy.

OBJECTIVE: The authors determined whether microscopically positive surgical margins are detrimental to the outcome of early stage breast cancer patients treated with conservation surgery and radiation therapy. SUMMARY BACKGROUND DATA: The optimal extent of breast surgery required for patients treated with conservation surgery and radiation therapy has not been established. To achieve breast preservation with good cosmesis, it is desirable to resect as little normal tissue as possible. However, it is critical that the resection does not leave behind a tumor burden that cannot be adequately managed by moderate doses of radiation. It is not known whether microscopically positive surgical margins are detrimental to patient outcome. METHODS: The records of 259 consecutive women (262 breasts) treated with local excision (complete removal of gross tumor with a margin) and axillary dissection followed by radiation therapy for clinical stage I and II infiltrating ductal breast cancer at Duke University Medical Center and the University of North Carolina between 1983 and 1988 were reviewed. Surgical margins were considered positive if tumor extended to the inked margins; otherwise the margins were considered negative. Margins that could not be determined, either because the original pathology report did not comment on margins, or because the original specimen had not been inked were called indeterminate. RESULTS: Of the 262 tumors, 32 (12%) had positive margins, 132 (50%) had negative margins, and the remaining 98 (38%) had indeterminate margins. There were 11 (4%) local failures; 3/32 (9%) from the positive margin group, 2/132 (1.5%) from the negative margin group, and 6/98 (6%) from the indeterminate group. The actuarial local failure rates at 5 years were 10%, 2%, and 10%, respectively, p = 0.014 positive vs. negative, p = 0.08 positive vs. indeterminate (log rank test). Margin status had no impact on survival or freedom from distant metastasis; 63 patients who originally had positive or indeterminate margins were re-excised. Two of 7 with positive margins after re-excision versus 1/56 rendered margin negative had a local recurrence. CONCLUSIONS: The authors recommend re-excision for patients with positive margins because of improved local control of those rendered margin negative and identification of those patients at high risk for local failure (those who remain positive after re-excision). Because margin status impacts on local control, tumor margins after conservation surgery should be accurately determined in all patients.

Adult↗

PubMedDate not supplied

Proton MR spectroscopy of squamous cell carcinoma of the extracranial head and neck: in vitro and in vivo studies.

PURPOSE: To determine the ability of in vitro one-dimensional and two-dimensional proton MR spectroscopy to help differentiate squamous cell carcinoma of the extracranial head and neck from normal tissues and to correlate the in vitro observations with clinical studies. METHODS: In vitro 1-D and 2-D correlated proton MR spectroscopy (11 T) was performed in tissue specimens of squamous cell carcinoma of the head and neck (n = 19), in normal tissue (n = 13), in metastatic cervical lymph nodes (n = 3), and in a squamous cell carcinoma cell line. In vivo 1-D proton MR spectroscopy (1.5 T) was performed in patients with squamous cell carcinoma (n = 7) and in healthy volunteers (n = 7). The ratio of the areas under the choline (Cho) and creatine (Cr) resonances were calculated for 1-D proton MR spectra for the in vitro tissue studies and correlated with the in vivo studies. Data from in vitro 2-D correlated spectroscopy were analyzed for differences in the presence or absence of various metabolites in samples of tumor and normal tissue. Statistical analysis consisted of 2 x 2 factorial repeated measures analysis of variance (ANOVA), discriminate analysis, and chi2 test. RESULTS: The mean in vitro 1-D proton MR spectroscopic Cho/Cr ratio was significantly higher in tumor than in normal tissue. The difference between the mean ratios appeared to increase with increasing echo time. All in vivo tumor Cho/Cr ratios were greater than the calculated mean in vitro tumor ratio, whereas six of the seven volunteers had no detectable Cho and Cr resonances. Two-dimensional correlated MR spectroscopic data revealed that a variety of amino acids have a significantly greater likelihood of being detected in tumor than in normal tissues. CONCLUSIONS: One-dimensional and 2-D proton MR spectroscopy can help differentiate primary squamous cell carcinoma and nodal metastases containing squamous cell carcinoma from normal tissue both in vitro and in vivo. In addition, 2-D spectroscopy can help identify the presence of certain amino acids in squamous cell carcinoma that are not detected in normal tissue.

Carcinoma, Squamous Cell↗