PubMed Health⌕ Search

Biomedical subjects

S Glass

Publications and source records attributed to S Glass.

At least 19 recordsLinked to original sources
PubMed2000

SCF(beta-TRCP) and phosphorylation dependent ubiquitinationof I kappa B alpha catalyzed by Ubc3 and Ubc4.

NF kappa B is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NF kappa B is anchored in the cytoplasm via the inhibitory protein I kappa B alpha. Following exposure to diverse pro-inflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transduction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF beta-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of I kappa B alpha. Here we reconstitute phosphorylation dependent ubiquitination of I kappa B alpha using recombinant components. Our results suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1. We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCF beta-TRCP dependent manner and that both are capable of associating with the SCF beta-TRCP complex isolated from human cells. Finally, we show that Ubc4 is in excess to Ubc3 in THP.1 cells and 19 times more efficient in catalyzing the reaction, suggesting that Ubc4 is the preferentially used Ubc in this reaction in vivo. Our results also suggest that ubiquitin is transferred directly from the Ubc to phospho-I kappa B alpha in a SCF beta-TRCP dependent reaction. Oncogene (2000) 19, 3529 - 3536

Amino Acid Sequence↗
PubMed2000

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer.

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.

Dimethyl Sulfoxide↗
PubMed1997

Antisense targeting of E6AP elevates p53 in HPV-infected cells but not in normal cells.

Invasive cervical cancer is very highly correlated with the presence of high-risk human papillomavirus (HPV) types 16 and 18. Two viral proteins, E6 and E7, act in concert to subvert growth control of infected cells by inactivating the tumor suppressor proteins, p53 and Rb, respectively. E6 is thought to abrogate p53 function by stimulating its degradation via ubiquitin-mediated proteolysis in a reaction requiring E6AP (E6-Associated Protein). Here we evaluate the in vivo role of E6AP in p53 degradation in normal and HPV-infected cell types using antisense phosphorothioate oligodeoxynucleotides (S-ODNs). This study shows that reduction of E6AP in vivo in high-risk HPV-infected cells leads to an elevation of p53, confirming the function of E6AP predicted by in vitro experiments. Further, we demonstrate that reduction of E6AP in normal cells has no effect on p53 levels, indicative of an E6AP-indpendent mechanism for p53 degradation. These experiments show that inhibition of intermediate proteins in the ubiquitin-mediated proteolysis pathway (ubiquitin-conjugating enzymes or associated recognition proteins) can result in specific inhibition of substrate degradation. We propose that modulation of p53 levels by elimination of E6AP function may have therapeutic potential for cervical cancer.

Apoptosis↗
PubMed1997

Butorphanol (Stadol): a study in problems of current drug information and control.

Butorphanol (Stadol, Bristol-Meyers Squibb, Princeton, NJ) is a synthetically derived opiate. As a nasal spray, it was approved for release in 1991 and was subsequently promoted as a safe treatment for migraine. Since then, there have been numerous reports of problems with butorphanol similar to those of any narcotic, especially dependence-addiction and major psychological disturbances. These problems have been documented by the Food and Drug Administration, but the information can be obtained only through the Freedom of Information Act. The experience with butorphanol indicates the need for physicians to have additional sources of information about drugs than are presently available.

Administration, Inhalation↗
PubMed1995

Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

Amino Acid Sequence↗
PubMed1993

Risk factors for hepatitis C virus seropositivity in heterosexual couples.

OBJECTIVES: To determine the risk of heterosexual transmission of hepatitis C virus (HCV) and to identify other risk factors for HCV seropositivity in heterosexual couples. DESIGN: Retrospective cross-sectional study comparing HCV-seropositive and HCV-seronegative heterosexual men and women. SETTING: Couples recruited from the community and screened for participation in a study of the heterosexual transmission of human immunodeficiency virus. PARTICIPANTS: A total of 340 subjects, 170 men and 170 women in sexual partnerships, aged 18 through 61 years. MAIN OUTCOME MEASURE: Seropositivity for HCV antibodies. RESULTS: Overall, 31 (18%) of the 170 women and 56 (33%) of the 170 men were positive by a four-antigen HCV immunoblot. Injection drug use and hemophilia were strongly associated with HCV seropositivity. Sixty-four percent of injection drug users were positive (odds ratio [OR], 27.0; 95% confidence interval [CI], 13.4 to 56.1; P < .0001), as were all four hemophiliacs in the study. History of blood transfusion was significantly associated with HCV seropositivity (OR, 2.7; 95% CI, 1.1 to 7.0; P = .02). Positivity for HCV was not associated with measures of sexual behavior within couples or with numbers of other sexual partners, history of sexually transmitted diseases, or human immunodeficiency virus seropositivity. However, two of the 31 women without parenteral risk but with a long-term HCV-positive male partner were HCV seropositive compared with none of 81 women with an HCV-negative male partner (P = .07). CONCLUSIONS: These results provide little evidence of HCV sexual transmission but are consistent with infrequent sexual transmission. They corroborate the importance of injection drug use and transfusion of blood or blood products in transmitting HCV and underscore the importance of ascertaining parenteral exposures when examining sexual transmission of HCV.

Adult↗
PubMed1993

Time to change?

Explore the source record for details and available documents.

Clinical Protocols↗
PubMed1993

Prevention of heterosexual transmission of human immunodeficiency virus through couple counseling.

In the absence of an effective vaccine, behavior change remains the most effective means to prevent the spread of HIV. We examined behavior change over time and rates of HIV seroconversion in a cohort of HIV individuals and their heterosexual partners recruited since 1985. Participants were recruited from various HIV counseling and testing sources throughout California and were usually interviewed and tested in their own homes. Couple counseling and risk assessments were conducted at average intervals of six months. Data from 144 couples who were discordant for HIV serostatus are reported. Of the index cases, 78% were men. Most male index cases were bisexuals, and most female index cases were infected through heterosexual intercourse with a previous sexual partner. The mean duration of the relationship for the couple at intake was 5.6 years. Both condom use and sexual abstinence increased over time (p < 0.001 for both), and most behavior change occurred between intake and first follow-up visit. We observed no seroconversion after 193 couple-years of follow-up. Couple counseling in combination with social support appears to be an effective means to promote and sustain behavior change among HIV-infected individuals and their heterosexual partners.

Adolescent↗
PubMed1992

Mitochondrial myopathy of childhood associated with depletion of mitochondrial DNA.

We have studied five children with mitochondrial myopathy manifesting within or soon after the first year of life. Muscle biopsies showed ragged-red fibers and decreased respiratory chain activity. All five patients had a severe decrease (2 to 34% of normal) in the amount of muscle mitochondrial DNA (mtDNA). The depletion of mtDNA correlated with absence of mtDNA-encoded translation products and with loss of cytochrome c oxidase enzyme activity in individual muscle fibers. This mitochondrial myopathy of childhood illustrates one phenotypic expression of a novel pathogenetic mechanism in mitochondrial diseases, the specific depletion of mtDNA in affected tissues.

Blotting, Southern↗
PubMed1991

Biological monitoring of 2,4-dichlorophenoxyacetic acid-exposed workers in agriculture and forestry.

This is a report on the application of a radioimmunoassay (RIA) for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in biological monitoring of occupationally exposed sprayers. Urinalysis was conducted on two workers involved in spraying 2,4-D sodium salt solution with car mounted ground rigs in agriculture and on the pilot and the mixer-loader of a helicopter crew applying 2,4-D dimethylamine salt for brush control in forestry. All sprayers showed detectable quantities of 2,4-D in morning urine samples voided over 4 or 6 days in the post-spraying period. The highest 2,4-D urinary concentrations of about 2.5 ppm could be measured in a car driver on the 3 day after exposure. The 2,4-D level in urine was much lower in forestry workers, with 0.365 ppm for the mixer-loader and 0.052 ppm for the pilot on the 1 day after spraying. Urinary 2,4-D concentrations in the agricultural study were adjusted for endogenous creatinine and, when normalized for body weight, resulted in a total 2,4-D uptake of about 5.7 or 84.9 micrograms/kg body wt. for a single spraying operation. Thus, the calculated absorbed dose was less than the NOAEL (no observed adverse effect level) of 10 mg/kg body weight per day by a large margin of safety. Further, the calculated amounts excreted seemed to be sufficiently reliable to be used for assessing the risk of human exposure to 2,4-D. However, more studies are desirable for confirmation of the association of urinary 2,4-D and creatinine excretion. Clearance occurs with a 12-h to 22-h half-life.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1990

Rheumatology algorithms for primary care physicians.

Primary care physicians were trained on three rheumatology topics to assess the effectiveness of an educational strategy for continuing medical education. Algorithm training was shown to be at least as effective as that based on standard prose monographs. Both training groups improved their knowledge of patient management skills but there were no statistically significant differences between groups in the amount learned. When algorithms were used to design text materials, the designed texts required less study time than did the annotated clinical algorithms alone. That difference was significant for the shoulder pain materials (P less than 0.05) but not for the osteoporosis materials. The ratio of knowledge gained to study time was significantly higher for the algorithm group on the low back pain topic (P less than 0.05) but not for the other topics. Taped interview problems tests were studied as a method for assessing patient management skills related to problem-specific indicator conditions and were found to produce interrater reliability greater than 0.80 on five of the six tests.

Clinical Protocols↗
PubMed1989

Fecal isolation of Pseudomonas aeruginosa from patients with cystic fibrosis.

Fecal isolation of Pseudomonas aeruginosa was observed in 8 of 10 patients with cystic fibrosis who at the time of sampling also exhibited colonization of the respiratory tract. In contrast, P. aeruginosa cells were isolated at low frequency (9.1%) from the stools of 44 patients with cystic fibrosis with no previous history of chronic colonization. The results of this study suggest that the gastrointestinal tract is not a significant chronic reservoir of P. aeruginosa prior to pulmonary colonization.

Adolescent↗
PubMed1987

Serum concentrations of a granulocyte-derived calcium-binding protein in cystic fibrosis patients and heterozygotes.

Serum samples from cystic fibrosis homozygotes and heterozygotes contain elevated levels of a protein, known as cystic fibrosis antigen, which is synthesized primarily in granulocytes. We have produced monoclonal antibodies against cystic fibrosis antigen and developed a sensitive two-site immunoassay. Antigen levels were evaluated in serum samples from 50 cystic fibrosis homozygotes, 34 heterozygotes, 60 healthy controls and 25 disease controls. Simultaneous measurement was made of another granulocyte-derived serum protein, lactoferrin, and also of the acute-phase reactant, C-Reactive Protein. Attempts to use the concentrations of cystic fibrosis antigen in serum to distinguish cystic fibrosis patients from heterozygotes were unsuccessful, even when these concentrations were expressed as a ratio with lactoferrin or with C-Reactive Protein. However, examination of the ratio of cystic fibrosis antigen to lactoferrin in serum samples from cystic fibrosis heterozygotes suggests that there is some specific association between this antigen and the cystic fibrosis gene.

Antibodies, Monoclonal↗