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Biomedical subjects

J L Mitchell

Publications and source records attributed to J L Mitchell.

At least 37 records · Page 2Linked to original sources
PubMed1999

Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.

ADAM Proteins↗
PubMed1999

Use of cough and cold preparations during breastfeeding.

Adverse reactions in infants from maternal drug ingestion depend largely on the amount of milk consumed by the infant, timing of breastfeeding in relation to dosing, dose of the medication, dosing interval, and duration of therapy. When taking medications, breastfeeding mothers should be instructed to take their medication after breastfeeding, at the lowest effective dose and for the shortest duration. Overall, there are few data from human studies on the use of antihistamines, decongestants, and cough products during breastfeeding. Studies of pseudoephedrine, triprolidine, and loratadine in humans conclude that low levels of each drug would reach a breastfed infant. Since triprolidine and pseudoephedrine are also considered compatible with breastfeeding by the AAP, these 2 drugs should be the first-line choices. Codeine is considered compatible with breastfeeding by the AAP, and would be an acceptable choice for short-term use as a cough suppressant. It is important to note that many of the liquid cough and cold products contain alcohol. In addition, many of the combination products are a mixture of an antihistamine and a decongestant and may also contain aspirin, acetaminophen, ibuprofen, or caffeine. It is preferable for nursing mothers to only take medications that are necessary and to avoid such combination products. The AAP considers alcohol, acetaminophen, ibuprofen, and caffeine compatible with breastfeeding. Aspirin has been associated with significant negative effects on some nursing infants, and the AAP recommends giving aspirin to nursing mothers with caution. Mothers taking cough and cold products should watch for adverse events in their breastfed infants. Infants may experience paradoxical central nervous stimulation from antihistamines and irritability and insomnia from decongestants.

Antitussive Agents↗
PubMed1998

Mammalian cell polyamine homeostasis is altered by the radioprotector WR1065.

Mammalian cells become more susceptible to radiation-induced death and mutagenesis when restricted in their production of the natural polyamines putrescine, spermidine and spermine. The effects of polyamine deprivation are reversed by N-(2-mercaptoethyl)-1, 3-diaminopropane (WR1065), a simple aminothiol that has been extensively studied for its radioprotectant properties. Because this compound and its oxidized derivative WR33278 bear some resemblance to the polyamines, it was hypothesized that radioprotection by WR1065 or its metabolites is derived, at least in part, from their ability to supplement the natural polyamines. To evaluate the ability of these aminothiol compounds to emulate polyamine function in intact cells, rat liver hepatoma (HTC) cells were treated with radioprotective doses of WR1065; the ability of this compound to affect various aspects of normal polyamine metabolism was monitored. Although cellular WR1065 was maintained at levels exceeding those of the polyamines, this aminothiol did not have any polyamine-like effect on the initial polyamine biosynthetic enzyme, ornithine decarboxylase, or on polyamine degradative reactions. On the contrary, treatment with relatively low levels of WR1065 resulted in an unexpected increase in putrescine and spermidine synthesis. WR1065 treatment enhanced the stability, and consequently the activity, of ornithine decarboxylase. This stabilization seems to result from a WR1065-induced delay in the synthesis of antizyme, a critical regulatory protein required in the feedback modulation of polyamine synthesis and transport. The increase in cellular spermidine induced by WR1065 might explain its antimutagenic properties, but is probably not a factor in protection against cell killing by radiation. This is the first evidence that compounds can be designed to control polyamine levels by targeting the activity of the regulatory protein antizyme.

Animals↗
PubMed1998

Osmotic stress induces variation in cellular levels of ornithine decarboxylase-antizyme.

The polyamines, and especially putrescine, play an integral role in the physiological response of cells to varying extracellular osmotic conditions. Ornithine decarboxylase (ODC) synthesis and stability, as well as the activity of the polyamine transporter, had all been reported to be very sensitive to media osmolarity in different cells and tissues, yet the mechanism of this complex, co-ordinated response was not known. In this study we have determined that all these aspects of osmotic-shock response may be mediated by the common regulatory protein, ODC-antizyme. HTC cells were induced for antizyme and then exposed to media of reduced osmotic strength. Both antizyme activity and protein decreased rapidly, under these conditions, to new steady-state levels that depended upon the degree of reduction in media tonicity. This antizyme reduction was found to be due to a rapid increase in antizyme degradation, with a half-life decrease from 75 min down to 45 min occurring immediately upon exchanging media. In complementary experiments, increased media tonicity induced elevated antizyme levels and stability. The sensitivity of antizyme turnover to osmotic conditions was also observed in DH23b cells, which contain elevated levels of more stable antizyme. Interestingly, the two main antizyme proteins, AZ-1 and AZ-2 (presumably products from the first and second translational start sites), differed in their responses to these changing osmotic conditions. Just as feedback regulation of antizyme synthesis provides an effective mechanism for maintaining stable polyamine levels, these studies suggest that alteration in the rate of antizyme degradation may be the mechanism whereby cells adjust steady-state polyamine levels in response to stimulation or stress.

Animals↗
PubMed1998

The special prenatal clinic: one approach to women and substance abuse.

We aimed to determine if the trend in substance use in this central core of a large inner-city area confirmed an increase in marijuana use among young women < 22 years of age as childbearing years were beginning or if marijuana use and harder drug use were greatest among women beyond the teens and the early second decade of life and to describe one prenatal care intervention. Statistical reports for the Special Prenatal Clinic, a Harlem clinic for women with a current or past history of substance use, were reviewed from 1985 (the clinic's inception) through March 1996 to determine the trends, patterns, and types of drug use. Findings chronicled the beginning and rise in crack cocaine use, especially after 1986, confirmed an increase in marijuana use by adolescents since 1992, and suggest that the risk for use of hard drugs increases with age, predominantly in the childbearing years. Intervention by the Special Prenatal Clinic aided case-finding, allowed for comprehensive care, offered women opportunities for behavior change, and increased their chances of keeping their babies after delivery.

Adolescent↗
PubMed1998

IgG subclasses in the serum and sputum from patients with bronchiectasis.

BACKGROUND: IgG subclass deficiency is often associated with recurrent pulmonary infections. The prevalence of deficiency in a large well characterised group of patients with bronchiectasis has not previously been established. METHODS: Serum IgG subclass concentrations in 89 patients with bronchiectasis were compared with those obtained from a group of 82 age and sex matched normal healthy controls. Sputum IgG subclass concentrations were also assessed in 44 of the patients. Albumin was measured as a marker of protein transudation from plasma to determine the degree of local IgG subclass production. RESULTS: The serum concentrations of IgG1, IgG2 and IgG3 were increased in the patients compared with the control group whereas IgG4 concentrations were not. There was an overall incidence of deficiency of 1% for subclasses 1-3 and 5% for subclass 4 in patients with bronchiectasis based on observed concentrations being below the lower limit of the control group range. The concentrations of IgG subclasses in sputum were partly dependent upon the degree of inflammation as assessed by the macroscopic appearance of purulence. A comparison of the ratio of sputum:serum subclass concentration and sputum:serum albumin, however, revealed that all of the subclasses were present at greater concentrations than could be accounted for by transudation alone. CONCLUSIONS: A new normal control range for serum IgG subclasses has been established and from this range it was found that IgG subclass deficiency in a group of unselected patients with bronchiectasis was comparatively rare. A significant degree of local IgG production was also suggested in the lungs of these patients.

Adult↗
PubMed1996

Feedback repression of ornithine decarboxylase synthesis mediated by antizyme.

The induction of antizyme by spermidine and the resulting enhancement of ornithine decarboxylase (ODC) degradation have been well studied; however, little is known about the mechanism whereby elevated spermidine levels decrease synthesis of the polyamine biosynthetic enzyme. To evaluate the relative contribution of inhibited synthesis, as distinct from enhanced degradation of ODC, spermidine levels were manipulated in a variant cell line that overproduces a stable form of ODC. Spermidine did not selectively inhibit ODC synthesis in these variant cells, supporting the concept that spermidine diminishes ODC synthesis in normal cells owing to enhanced degradation of the protein in the presence of elevated antizyme levels. This model was further investigated in vitro by use of rabbit reticulocyte lysate, which catalyses simultaneous ODC mRNA translation and antizyme-stimulated degradation of ODC protein. Antizyme strongly repressed the incorporation of labelled amino acids into normal rat ODC. Unexpectedly it also diminished the apparent translation of ODC mRNA species coding for enzyme forms that are not destabilized by the post-translational addition of antizyme. The effect of antizyme on ODC translation was not observed in wheatgerm extract, in which there is no antizyme-induced degradation. Further, deletion of a short segment of antizyme necessary for the destabilization of ODC (amino acid residues 113-118) resulted in a form that bound ODC but did not diminish its apparent translation. These results suggest that the co-translational addition of antizyme to ODC results in a complex that is different from, and innately less stable than, that formed when antizyme is added post-translationally.

Animals↗
PubMed1996

Ornithine decarboxylase stability in HMOA and DH23b cells is not due to post-translational truncation of a C-terminal recognition site.

The normally labile ornithine decarboxylase (ODC) becomes unusually stable when Cys-441 is replaced with Trp in the variant cell lines HMOA and DH23b. This stable ODC is also observed to have higher mobility on SDS/PAGE. Because previous studies have shown that ODC stability can be achieved when as few as five amino acid residues are removed from its C-terminus, it was suggested that the amino acid substitution in the variant ODC might alter its conformation sufficiently to promote a similar proteolytic loss of a C-terminal degradation signal, resulting in a stable yet active ODC. To examine this mechanism, amino acids in the C-terminal regions of both wild-type and stable (Trp-441) ODC proteins were released, by means of carboxypeptidase-Y digestion, and identified by HPLC. The C-terminal ends were found to be the same, and are as predicted from the cDNA sequence. This study proves that stability of the Trp-441 form of ODC is not simply due to proteolytic removal of a C-terminal proteasome-targeting sequence, thereby implying that the stabilization of this mutant ODC form must result directly from a conformational change associated with the loss of Cys-441.

Amino Acid Sequence↗
PubMed1996

Overproduction of stable ornithine decarboxylase and antizyme in the difluoromethylornithine-resistant cell line DH23b.

DH23b cells, a variant of the HTC line selected for their resistance to difluoromethylornithine, exhibit defective feedback regulation of ornithine decarboxylase (ODC) stability and polyamine transport, and accumulate ODC protein to > 1000 times normal concentrations. The components of the polyamine feedback regulation system have been examined in an attempt to understand these unusual responses. Southern-blot analysis revealed an amplification (approx. 10-fold) in ODC DNA sequence without any concomitant increase in antizyme. Moreover, the amplified ODC sequence contains a single base substitution that results in the conversion of Cys-441 into Trp. This modification has previously been shown to cause ODC stability in HMOA cells. Although antizyme activity has not been noted in DH23b cells, Western-blot analysis revealed the accumulation of antizyme protein to > 50 times that induced in parental HTC cells. This increase is consistent with a 6-9-fold increase in the half-life of antizyme in these cells, a consequence of the inability of the mutant ODC-antizyme complex to be degraded by 26 S proteasome. Associated with the stabilization of antizyme in both DH23b and HMOA cells is the appearance of two additional forms of antizyme protein with apparent molecular masses of 22 and 18.5 kDa. It is suggested that these result from proteolytic removal of discrete fragments from the N-terminal end of antizyme, perhaps an indication of an initial step in rapid antizyme turnover.

Animals↗
PubMed1995

Involvement of the polyamine transport system in cellular uptake of the radioprotectants WR-1065 and WR-33278.

High levels of the aminothiol WR-1065 protect cells from ionizing radiation, while much lower levels of this compound or its disulfide, WR-33278, impart anti-mutagenic effects. In view of the structural similarity of these agents to the essential cellular polyamines putrescine, spermidine and spermine we investigated the possibility transport system. WR-33278 appears to be a very close analog of spermidine or spermine in that it not only inhibits spermidine incorporation, but is also transported at the same velocity as spermidine, with a Kt of approximately 0.8 microM compared with 0.4 microM for the polyamine. Further, repression of the activity of the polyamine transporter by antizyme or its elimination by selected mutation affected both transport of WR-33278 and spermidine equally. In contrast, WR-1065 is not a good substrate for the polyamine transporter and appears to enter cells predominantly by non-mediated passive diffusion. There appears to be no uptake of either WR-33278 or the polyamines by this non-mediated diffusion. Thus both the form of the aminothiol and the activity of the polyamine transport system need to be considered in evaluating the efficacy of low exogenous levels of this drug on mutagenesis or carcinogenesis.

Antimutagenic Agents↗
PubMed1995

The isolation and characterization of non-typeable Haemophilus influenzae from the sputum of adult cystic fibrosis patients.

The role of non-typeable Haemophilus influenzae in cystic fibrosis (CF) remains unclear. We wanted, therefore, to determine the presence and characteristics of non-typeable H. influenzae in sputum samples from patients with CF. In order to do this, we have assessed sputum samples from 55 consecutive clinically stable patients seen routinely at an adult CF out-patient clinic. Quantitative bacterial culture was performed using a selective media containing cefsoludin, and isolates were characterized by biotyping and outer membrane protein profile analysis. In 17 (30%) of these samples, non-typeable H. influenzae was isolated and was present in similar viable numbers (mean 7.7 x 10(8) colony-forming units (cfu).mL-1; SEM 3.1) to Pseudomonas aeruginosa (mean 8 x 10(8) cfu.mL-1: SEM 2.4). All non-typeable H. influenzae isolates recovered were beta-lactamase negative and sensitive to a range of antibiotics. Several biotypes and outer membrane protein profiles were observed, with no apparent association between these two phenotypic characteristics. The study showed that large numbers of non-typeable H. influenzae are often present in sputum from adult patients with CF. Further longitudinal studies of outer-membrane protein profile analysis are required to determine the dynamics of non-typeable H. influenzae colonization in individual patients and the clinical significance.

Adolescent↗
PubMed1995

Salmonella typhi chorioamnionitis in a human immunodeficiency virus-infected pregnant woman. A case report.

Opportunistic prenatal infection is a recognized problem in human immunodeficiency virus (HIV)-infected women from inner city communities. We report a case of intrapartum Salmonella typhi infection and discuss the possible route of infection. An HIV-infected pregnant woman was admitted for fever and ruptured membranes. Maternal blood, cervical and uterine cultures, and placental surface and intramembranous space cultures grew S typhi. The patient responded to antibiotic therapy, with no relapse. The infant did not show signs or symptoms of Salmonella sepsis. Salmonella infection should be treated aggressively in HIV-infected pregnancies with chorioamnionitis and postpartum endometritis.

Adult↗
PubMed1995

In vivo study of indomethacin in bronchiectasis: effect on neutrophil function and lung secretion.

Bronchiectasis is associated with sputum containing high levels of the proteolytic enzyme elastase, which is thought to be involved in the pathogenesis of the disease. Agents which inhibit neutrophil function and interfere with neutrophil elastase release may have a beneficial effect on the development and progression of such diseases. We have studied the effects of the nonsteroidal anti-inflammatory agent indomethacin on neutrophil function in nine patients with clinically stable bronchiectasis. All patients remained clinically stable during the study. We observed a significant reduction in peripheral neutrophil chemotaxis to 10 nmol.L-1 N-formyl-methionyl-leucyl-phenylalanine (FMLP) from a mean of 19.86 (SEM 1.35) to 8.46 (0.68) cells.field-1 after 4 weeks of therapy. There was also a significant reduction in fibronectin degradation both by resting and FMLP-stimulated neutrophils, from a mean of 1.90 (0.19) micrograms x 3 x 10(5) cells at the start of therapy to 0.87 (0.08) micrograms after 4 weeks, and from 3.17 (0.35) micrograms to 1.48 (0.05) micrograms, respectively. There was no effect on spontaneous or stimulated superoxide anion generation by neutrophils. Despite the marked changes in peripheral neutrophil function, no adverse effect was observed on viable bacterial load in the bronchial secretions. In addition, there was no difference in sputum albumin, elastase or myeloperoxidase levels, and only minor changes in the chemotactic activity of the sputum. These results suggest that nonsteroidal anti-inflammatory agents have a major effect on peripheral neutrophil function but do not appear to have an adverse effect on bacterial colonization of the airways.

Adult↗
PubMed1994

Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells.

Antizyme, a spermidine-induced protein that binds and stimulates ornithine decarboxylase degradation, is now shown also to mediate the rapid feedback inhibition of polyamine uptake into mammalian cells. Using a cell line (HZ7) transfected with truncated antizyme cDNA, and mutant ornithine decarboxylase cell lines, we demonstrate that this newly discovered action of antizyme is distinct from its role in modulating polyamine biosynthesis.

Animals↗