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G D Roberts

Publications and source records attributed to G D Roberts.

At least 73 records · Page 4Linked to original sources
PubMed1989

Preliminary X-ray crystallography studies of recombinant human interleukin-1 alpha. Purification and structural characterization.

Human interleukin-1 alpha, cloned and expressed in E. coli, has been purified and structurally characterized by various physiochemical methods, including mass spectrometry. The recombinant protein has been crystallized by the hanging drop vapor diffusion method using dimethyl sulfoxide as the precipitating agent. The space group is P2(1)2(1)2(1). Unit cell dimensions are a = 44.1, b = 57.1, and c = 61.7 A and alpha = beta = gamma = 90 degrees. The crystals diffract to beyond 1.7 A and are suitable for high resolution data collection. Native diffraction data were collected. Screens for heavy atom derivatives have been initiated.

Amino Acid Sequence↗
PubMed1989

Fungal blood cultures.

It is generally accepted that fungemia is an important and often life-threatening event. Unfortunately, it often remains undetected due to the lack of physician and/or laboratory awareness of the usefulness of fungal blood cultures. Many laboratories do not offer the possibility of performing fungal blood cultures and, if offered, some use methods that are not optimal for recovery or require extended incubation for recovery. Of the methods considered satisfactory, the lysis-centrifugation method (Isolator) has the highest recovery rate and the shortest recovery time. The Bactec and Septi-Chek systems are alternative methods, but have limitations.

Blood↗
PubMed1989

Disseminated Aspergillus terreus infection in immunocompromised hosts.

Aspergillus terreus is ubiquitous in the environment but has rarely been found to be pathogenic. When recovered from clinical specimens, it is commonly considered a saprophyte. We report two cases of fatal disseminated A. terreus infection. The first patient was receiving corticosteroid therapy for immune thrombocytopenia when the condition developed, and the second patient was receiving immunosuppressive therapy after bone marrow transplantation for myelodysplasia. We also describe the frequency of recovery of A. terreus in our laboratory. The serious pathogenic potential of A. terreus in immunocompromised hosts should be recognized.

Adrenal Cortex Hormones↗
PubMed1988

Inactivation of dopamine beta-hydroxylase by p-cresol: isolation and characterization of covalently modified active site peptides.

Recently, p-cresol has been shown to be a mechanism-based inhibitor of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) [Goodhart, P. J., DeWolf, W. E., Jr., & Kruse, L. I. (1987) Biochemistry 26, 2576-2583]. This inactivation was suggested to result from alkylation of an active site residue by an aberrant 4-hydroxybenzyl radical intermediate. In support of this hypothesis, we report here the isolation and characterization of two modified tryptic peptides from DBH inactivated by p-cresol. Using a combination of automated Edman sequencing, mass spectroscopy (MS), and tandem MS, we have determined the sequence of the putative active site peptides, identified the site of attachment of p-cresol, and defined the chemical nature of the adduct formed. Both modified peptides are the same primary sequence: Ala-Pro-Asp-Val-Leu-Ile-Pro-Gly-Gln-Gln-Thr-Thr-Tyc-Trp-Cys-Tyr-Va l-Thr-Glu- Leu-Pro-Asp-Gly-Phe-Pro-Arg, where Tyc is an amino acid residue with the in-chain mass of a cresol-Tyr adduct (106 + 163 Da). Gas-phase deuterium exchange studies (employing N2H3-DCI MS) of the isolated phenylthiohydantoin (Pth) derivatives of modified residue 13 demonstrate that p-cresol forms two chemically distinct covalent adducts and support the hypothesis that a (4-hydroxyphenyl)methyl radical is generated during catalysis. Rearrangement to a (4-methylphenyl)oxy radical may also occur prior to inactivation.

Alkylation↗
PubMed1988

Structural fingerprinting of Asn-linked carbohydrates from specific attachment sites in glycoproteins by mass spectrometry: application to tissue plasminogen activator.

A sensitive and specific strategy has been developed for determining the sites of attachment of Asn-linked carbohydrates in glycoproteins, and defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site. In this carbohydrate 'fingerprinting' strategy, potential glycopeptides are identified by comparing the high pressure liquid chromatography (HPLC) chromatograms of proteolytic digests of a glycoprotein obtained before and after digestion with a glycosidase, usually peptide:N-glycosidase F (PNGase F). The glycopeptide-containing HPLC fractions are analyzed by fast atom bombardment mass spectrometry (FAB MS) prior to and after digestion with PNGase F to identify the former glycosylation site peptide and its sequence location (Carr and Roberts, (1986) Anal. Biochem. 157, 396-406). Carbohydrates are extracted from these fractions as the peracetates which are then permethylated and analyzed by FAB MS. The spectra exhibit molecular weight-related ions for each of the parent oligosaccharides present in the fraction which provide composition in terms of hexose, deoxyhexose, N-acetylhexosamine and sialic acid. The relative ratios of these peaks reflect the relative abundances of the various carbohydrate homologs present in the mixture. The derivatives formed are directly amenable to methylation analysis for determination of linkage. This strategy enables the structural classes of carbohydrates at specific attachment sites to be determined using only a few nmol of glycoprotein. The carbohydrate fingerprinting strategy has been applied to a number of glycoproteins including tissue plasminogen activator, the results for which are described herein.

Amidohydrolases↗
PubMed1988

Fungal infections of the immunocompromised host: clinical and laboratory aspects.

Fungal infections of the immunocompromised host are being seen with greater frequency than ever before. In addition, a growing list of unusual and unexpected etiologic agents presents a unique and difficult challenge to the clinician and microbiologist. The clinical manifestations of opportunistic fungal infections are often not characteristic and, in many instances, may prevent a rapid diagnosis from being made. Clinical microbiology laboratories should consider any organism as a potential etiologic agent. This requires that all fungi recovered from immunocompromised patients be thoroughly identified and reported so that their clinical significance may be assessed. This review presents a brief discussion of the clinical and laboratory aspects of some fungal infections seen in this important group of patients.

Humans↗
PubMed1988

Identification of mycobacteria from culture by using the Gen-Probe Rapid Diagnostic System for Mycobacterium avium complex and Mycobacterium tuberculosis complex.

Commercially available kits (Mycobacterium avium Complex Rapid Diagnostic System and Mycobacterium tuberculosis Complex Rapid Diagnostic System; Gen-Probe, Inc., San Diego, Calif.) utilizing nucleic acid hybridization for the rapid identification of members of the M. avium-M. intracellulare complex and M. tuberculosis complex were evaluated by using 339 clinical and American Type Culture Collection (Rockville, Md.) isolates. The tests, which can be performed in approximately 2 h, use specific [125I]DNA probes complementary to the rRNAs of M. avium, M. intracellulare, and M. tuberculosis complex, the latter of which includes M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti. The M. avium-M. intracellulare probes correctly identified 99 of 114 M. avium-M. intracellulare isolates, with 7 false-negatives and 8 false-positives, for a sensitivity of 93.4% and a specificity of 96.6%. After repeat testing, 110 of 114 were correctly identified, with 4 false-negatives and no false-positives, for a sensitivity of 96.5% and a specificity of 100%. The M. tuberculosis complex probe correctly identified 99 of 102 M. tuberculosis isolates, with 1 false-negative and 2 false-positives, for a sensitivity of 99% and a specificity of 99.2%. After repeat testing, 100 of 102 isolates were correctly identified, with no false-negatives and 2 false-positives, for a sensitivity of 100% and a specificity of 99.2%. Overall, there were 15 discrepant M. avium-M. intracellulare results, with 4 such results after repeat testing, and 3 discrepant M. tuberculosis complex results, with 2 such results after repeat testing. The Gen-Probe kits are highly sensitive and specific for use in identifying M. avium-M. intracellulare complex and M. tuberculosis complex isolates and will be useful in the clinical laboratory which can use the present radionuclide-containing kits cost effectively.

Cost-Benefit Analysis↗
PubMed1988

Experience with the use of pronase to eliminate interference factors in the latex agglutination test for cryptococcal antigen.

Cryptococcal antigen titers of 70 cerebrospinal fluid and 57 serum specimens from patients suspected of having cryptococcosis were determined both before and after treatment with pronase. Median titers of cerebrospinal fluid specimens before and after treatment were 128 and 128, respectively; mean geometric titers of these specimens before and after treatment were 102 and 204, respectively. Median titers of the serum specimens before and after treatment were 16 and 512, respectively; mean geometric titers of these specimens before and after treatment were 19 and 631, respectively. The modified latex agglutination test did not detect antigen in any of 50 cerebrospinal fluid and 51 serum specimens from patients not suspected of having cryptococcosis. These results suggest that the pronase modification increases the sensitivity of the latex agglutination test and that the modification be routinely incorporated into it.

Agglutination Tests↗
PubMed1988

Laboratory diagnosis of systemic fungal diseases.

The increase in the number of fungal infections seen in debilitated and immunocompromised patients in the last several years makes it necessary to consider all fungi as potential pathogens. Clinical microbiology laboratories are playing increasingly important roles in the recovery, isolation, and identification of these fungi. This article contains specific recommendations and references concerning a practical approach to the laboratory identification of systemic fungi. The proper and timely selection, collection, and transport of specimens is imperative, and clinicians are responsible for appropriate specimen selection to ensure optimal chances of recovery of pathogens. Respiratory tract secretions and blood are excellent sources for detection of disseminated fungal infection. Specimens should be placed into transport media if the sample size is small or if only a small number of organisms are thought to be present. Direct microscopic examination of specimens can provide valuable information, often allowing a clinician to initiate immediate therapy. Specimens that are more likely than others to contain systemic fungi and that should be examined routinely include the following: pulmonary biopsy material, bronchial washes and lavages, specimens from immunocompromised patients, purulent specimens, and specimens suspected of containing a specific fungus. Valuable methods of examining specimens directly include treatment with KOH and calcofluor white. Use of media to recover fungi is the basis of making a laboratory diagnosis of a fungal disease, and the use of proper recovery and subculture media is imperative. Noninhibitory media allow contaminants to grow readily and should be used only to recover fungi from normally sterile body sites or for subculture. Blood-enriched media allow almost all pathogenic and saprophytic fungi to flourish. Therefore, such media, unless they contain antibiotics, should not be used as primary recovery media. Media that contain antibiotics should be used as primary recovery media to prevent overgrowth of pathogenic fungi by contaminants. Yeasts recovered from clinical specimens can be identified by a combination of tests, which include direct microscopic examination, germ tube formation, microscopic morphology of growth on corn meal agar, and ability to utilize certain carbohydrates. Molds recovered from clinical specimens are identified by a combination of growth rate, colonial characteristics, size and shape of hyphae, and microscopic examination of reproductive structures and other fungal elements.(ABSTRACT TRUNCATED AT 400 WORDS)

Culture Media↗
PubMed1987

In vitro activity of ciprofloxacin and ofloxacin against the Mycobacterium avium-intracellulare complex.

The in vitro activity of ciprofloxacin and ofloxacin against strains of Mycobacterium avium-intracellulare complex were studied by the standard 1% proportion method. Thirty-eight of 100 strains (30%) were inhibited by ciprofloxacin at 2 micrograms/ml and 11 of 30 strains (37%) were inhibited by ofloxacin at 8 microm/ml. Patients having these more susceptible isolates may potentially benefit from therapy with these agents [corrected].

Anti-Bacterial Agents↗
PubMed1987

Laboratory methods for the diagnosis of disseminated histoplasmosis: clinical importance of the lysis-centrifugation blood culture technique.

Disseminated histoplasmosis, an uncommon disease that is usually fatal if not treated, is being recognized with increasing frequency in immunosuppressed patients. We retrospectively reviewed the laboratory methods used to establish the diagnosis of disseminated histoplasmosis in 28 patients examined at our institution. Tissue stains provided the initial diagnosis in 13 of the patients (46%). Serologic tests (complement fixation or immunodiffusion) were positive in 25 patients (89%); the frequencies of positive serologic results were similar in immunocompetent and in immunosuppressed patients. Blood cultures were positive in 20 of the patients (71%) and provided the initial diagnosis in 7 (25%); 5 of these 7 cases (71%) were detected with use of the lysis-centrifugation method. Furthermore, the recovery time for this blood culture method was less than that for a conventional blood culture method (brain-heart infusion biphasic technique). The lysis-centrifugation blood culture technique is an important laboratory method for the diagnosis of disseminated histoplasmosis.

Adult↗
PubMed1987

Transient fungemia in acute pulmonary histoplasmosis: detection by new blood-culturing techniques.

Transient fungemia has been thought to occur with acute pulmonary histoplasmosis in humans, but this has never been confirmed. We report two cases of acute pulmonary histoplasmosis in which transient fungemia was detected by the lysis-centrifugation blood-culturing technique. Both patients had self-limited acute pulmonary histoplasmosis, and neither was treated with antifungal therapy. Follow-up evaluation at three years and at 17 months disclosed no evidence of active pulmonary or disseminated disease. Transient Histoplasma capsulatum var. capsulatum fungemia may occur with acute pulmonary histoplasmosis. Fungemia may be prevented or arrested by an intact immune system or by other factors not yet known.

Acute Disease↗
PubMed1987

Saksenaea vasiformis osteomyelitis.

A 24-year-old man sustained a crush injury to the tibia, which subsequently became infected with Saksenaea vasiformis. He was treated with debridement and a free myocutaneous flap, but amputation was necessary because of mycotic osteomyelitis. S. vasiformis was recovered and identified on the basis of its characteristic morphology on cornmeal agar.

Adult↗
PubMed1987

Comparison of Du Pont Isolator and Roche Septi-Chek for detection of fungemia.

The rapid detection of fungemia in hospitalized patients is imperative, particularly for those who are immunocompromised. Our laboratory compared the Roche Septi-Chek with the Du Pont Isolator for the recovery of fungi from blood. Of 23,586 matched pairs of blood cultures, 199 were positive. The Isolator detected 178 (89.4%) and the Septi-Chek detected 119 (59.7%) of all positive isolates. The mean recovery time for the Isolator and Septi-Chek was 2.2 and 4.9 days, respectively. The Isolator detected fungemia earlier than the Septi-Chek did and was the only culture system positive in 83% of 53 patients, whereas the Septi-Chek system yielded the same results in only 13% of the patients. The Isolator provides a more rapid and sensitive method for the recovery of fungi from blood.

Blood↗
PubMed1987

Actinoidin A2, a novel glycopeptide: production, preparative HPLC separation and characterization.

An unidentified Nocardia sp. (SK&F-AAJ-193) was isolated and found to produce actinoidin A and a novel analog which we have named actinoidin A2. This new glycopeptide antibiotic differs from actinoidin A by the presence of rhamnose instead of acosamine. This analog was isolated using Dianion HP-20 resin followed by a specific glycopeptide affinity column (Affigel-10-D-Ala-D-Ala). The purification was accomplished using preparative ion-pairing chromatography. Actinoidin A2 is active against Staphylococcus aureus and coagulase-negative Staphylococci although it is less potent than actinoidin A.

Animals↗