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Biomedical subjects

R Newbold

Publications and source records attributed to R Newbold.

At least 19 recordsLinked to original sources
PubMed2005

Structure of yeast 5-aminolaevulinic acid dehydratase complexed with the inhibitor 5-hydroxylaevulinic acid.

The X-ray structure of the enzyme 5-aminolaevulinic acid dehydratase (ALAD) from yeast complexed with the competitive inhibitor 5-hydroxylaevulinic acid has been determined at a resolution of 1.9 A. The structure shows that the inhibitor is bound by a Schiff-base link to one of the invariant active-site lysine residues (Lys263). The inhibitor appears to bind in two well defined conformations and the interactions made by it suggest that it is a very close analogue of the substrate 5-aminolaevulinic acid (ALA).

Aminolevulinic Acid↗
PubMed2002

Effect of dietary administration of genistein, nonylphenol or ethinyl estradiol on hepatic testosterone metabolism, cytochrome P-450 enzymes, and estrogen receptor alpha expression.

The objective of this study was to examine effects of estrogenic agents of varying potencies (genistein, p-nonylphenol, and ethinyl estradiol) on hepatic testosterone metabolism, cytochrome P-450 (CYP450) enzymes, and ERalpha expression. These endpoints were examined as potential biomarkers of, and contributors to, endocrine disruptive activity. Exposure occurred during critical developmental periods, from gestational day 7 through weaning via the mothers' diet. Thereafter, rats were exposed via their diet to the compounds until puberty (postnatal day 50). Testosterone hydroxylase and 5alpha-reductase activities, CYP2C and CYP3A levels were determined. In general, the compounds were more active in male rats than female rats. The only effect observed in female rats was at the 250 ppm genistein dose, in which an approximately 40% increase in 5alpha-reductase activity was observed. In male rats, genistein treatment had mixed effects on testosterone metabolism. The 1250 ppm dose decreased both CYP2C and CYP3A protein levels. Nonylphenol had the most profound effects on testosterone metabolism and CYP450 expression in male rats, with effects occurring at doses as low as 25 ppm. An increase in 5alpha-reductase activity and a decrease in the formation of 16alpha-OH-, 2alpha-OH-testosterone metabolites, CYP2C and CYP3A protein were observed. EE2 decreased the formation of several testosterone metabolites and CYP2C protein. All compounds had some effect on hepatic ERalpha expression, although a consistent effect was not observed. This study demonstrates that the test compounds can influence hepatic testosterone hydroxylase activity and CYP450 expression, as well as ERalpha expression, although these activities cannot be directly related to estrogenic activity.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2002

Telomerase activity and Bcl-2 expression in human breast cancer.

AIMS: Telomerase is a ribonucleoprotein that synthesizes telomeres and plays an important role in cellular immortalization. Bcl-2 gene encodes for a mitochondrial protein thought to prevent apoptosis of normal cells. We previously reported telomerase activity in 74% of human invasive breast cancers and detected a significant association between telomerase activity and prognostic parameters such as nodal status, tumour size and cellular proliferation. We hypothesized that telomerase reactivation in human breast cancer was associated with increased immunohistochemical expression of Bcl-2. METHODS: Bcl-2 immunohistochemical expression was determined in 25 infiltrating breast carcinomas with known telomerase activity (17 telomerase-positive and 8 telomerase-negative). The percentage of strongly and moderately stained tumour cells for Bcl-2 was determined by a breast pathologist who was blinded to telomerase data. Fisher's exact test was used to examine the association between telomerase activity and Bcl-2 expression. RESULTS: The median percentage of strongly stained tumour cells was 50% for telomerase-positive tumours (range, 0--100%) and 45% for telomerase-negative tumours (range, 0--100%). Twelve (70%) of 17 telomerase-positive tumours expressed strong or moderate Bcl-2 staining in >50% of tumour cells compared with six (75%) of eight telomerase-negative tumours (P=1.0). CONCLUSION: Telomerase reactivation seems to be independent of Bcl-2 protein expression in human breast cancer.

Adult↗
PubMed2001

The x-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors.

The structures of 5-aminolaevulinic acid dehydratase (ALAD) complexed with substrate (5-aminolaevulinic acid) and three inhibitors: laevulinic acid, succinylacetone and 4-keto-5-aminolaevulinic acid, have been solved at high resolution. The ligands all bind by forming a covalent link with Lys263 at the active site. The structures define the interactions made by one of the two substrate moieties that bind to the enzyme during catalysis. All of the inhibitors induce a significant ordering of the flap covering the active site. Succinylacetone appears to be unique by inducing a number of conformational changes in loops covering the active site, which may be important for understanding the co-operative properties of ALAD enzymes. Succinylacetone is produced in large amounts by patients suffering from the hereditary disease type I tyrosinaemia and its potent inhibition of ALAD also has implications for the pathology of this disease. The most intriguing result is that obtained with 4-keto-5-amino-hexanoic acid, which seems to form a stable carbinolamine intermediate with Lys263. It appears that we have defined the structure of an intermediate of Schiff base formation that the substrate forms upon binding to the P-site of the enzyme.

Aminolevulinic Acid↗
PubMed2001

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors.

The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.

Catalytic Domain↗
PubMed2000

The association between telomerase activity and hormone receptor status and p53 expression in breast cancer.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that seems to play an important role in cellular immortality and carcinogenesis. p53 mutations account for approximately 50% of human cancers and represent the most frequent genetic lesion in breast cancer. AIMS: This study aims to examine the association between telomerase reactivation and hormonal receptor status and p53 expression in invasive breast cancer. METHODS: Using a polymerase chain reaction-based assay, telomerase activity was determined in 47 invasive breast carcinomas and 21 adjacent non-cancerous breast tissue specimens (stored at -80 degrees C) prospectively collected from 47 women undergoing elective surgical treatment in our centre. The histopathological features of the tumour were determined by experienced breast pathologists using light microscopy and haematoxylin and eosin staining. Oestrogen receptor (ER), progesterone receptor (PR) and p53 expressions were determined using immunohistochemistry techniques. RESULTS: Telomerase activity was detected in 34 (72%) of 47 breast carcinomas and in none of the adjacent non-cancerous breast specimens. There was a significant association between telomerase reactivation, tumour size and nodal status. Telomerase positive tumours were more likely to be poorly differentiated (65% versus 46%), but this association failed to reach statistical significance. There was no significant difference in ER expression (68% versus 85%). PR expression (62% versus 62%) and p53 expression (19% versus 27%) between telomerase positive and telomerase negative cancers. CONCLUSION: Telomerase reactivation is associated with important prognostic factors such as tumour size and nodal status in invasive breast cancer and seems to be independent of hormonal receptor status and p53 expression.

Biopsy, Needle↗
PubMed1999

The Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with laevulinic acid.

The X-ray structure of the complex formed between yeast 5-aminolaevulinic acid dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15 A resolution. The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263. It is known that this lysine forms a Schiff base link with substrate bound at the enzyme's so-called P-site. The carboxyl group of laevulinic acid makes hydrogen bonds with the side-chain-OH groups of Tyr329 and Ser290, as well as with the main-chain >NH group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site. Our analysis strongly suggests that the same interactions will be made by P-side substrate and also indicates that the substrate that binds at the enzyme's A-site will interact with the enzyme's zinc ion bound by three cysteines (133, 135, and 143). Inhibitor binding caused a substantial ordering of the active site flap (residues 217-235), which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis.

Binding Sites↗
PubMed1999

New approaches for estimating risk from exposure to diethylstilbestrol.

A subgroup from a National Institute of Environmental Health Sciences, workshop concerned with characterizing the effects of endocrine disruptors on human health at environmental exposure levels considered the question, If diethylstilbestrol (DES) were introduced into the market for human use today and likely to result in low-dose exposure of the human fetus, what would be required to assess risk? On the basis of an analysis of the quality of data on human DES exposure, the critical times and doses for inducing genital tract malformations and cancer must be determined. This would be facilitated through analysis of the ontogeny of estrogen receptor expression in the developing human genital tract. Models of low-dose estrogenic effects will have to be developed for human and rodent genital tract development. Mouse models offer many advantages over other potential animal models because of the wealth of the earlier literature, the availability of sensitive end points, the availability of mutant lines, and the possibility of generating genetically engineered model systems. Through multidisciplinary approaches, it should be possible to elucidate the cellular and molecular mechanisms of endocrine disruption elicited by estrogens during development and facilitate an assessment of risk to humans.

Animals↗
PubMed1999

Potent and selective human beta(3)-adrenergic receptor antagonists.

Although the functional presence of beta(3)-adrenergic receptors (beta(3)-AR) in rodents is well established, its significance in human adipose tissue has been controversial. One of the issues confounding the experimental data has been the lack of potent and selective human beta(3)-AR ligands analogous to the rodent-specific agonist BRL37344. Recently, we described a new class of aryloxypropanolamine beta(3)-AR agonists that potently and selectively activate lipolysis in rhesus isolated adipocytes and stimulate the metabolic rate in rhesus monkeys in vivo. In this article, we describe novel and selective beta(3)-AR antagonists with high affinity for the human receptor. L-748,328 and L-748,337 bind the human cloned beta(3)-AR expressed in Chinese hamster ovary (CHO) cells with an affinity of 3.7 +/- 1.4 and 4.0 +/- 0.4 nM, respectively. They display an affinity of 467 +/- 89 and 390 +/- 154 nM for the human beta(1)-AR. Their selectivity for human beta(3)-AR versus beta(2)-AR is greater than 20-fold (99 +/- 43 nM) and 45-fold (204 +/- 75 nM), respectively. These compounds are competitive antagonists capable of inhibiting the functional activation of agonists in a dose-dependent manner in cells expressing human cloned beta(3)-AR. Moreover, both L-748,328 and L-748,337 inhibit the lipolytic response elicited by the beta(3)-AR agonist L-742,791 in isolated nonhuman primate adipocytes. The aryloxypropanolamine benzenesulfonamide ligands illustrated here and elsewhere demonstrate high-affinity human beta(3)-AR binding. In addition, we describe specific 3'-phenoxy substitutions that transform these compounds from potent agonists into selective antagonists.

Adipocytes↗
PubMed1999

Using administrative data to compare the relative effectiveness of amlodipine vs nifedipine CC.

OBJECTIVE: To describe an approach for using claims data to compare the effectiveness of 2 similar drugs used for similar indications within a health maintenance organization. STUDY DESIGN: A database study comparing the effectiveness of amlodipine and nifedipine CC in the initial treatment of hypertension. PATIENTS AND METHODS: The claims records of Pennsylvania Medicaid patients between 18 and 64 years of age with continuous eligibility in 1994 were studied. Pharmacy, hospital, and outpatient claims data were merged, and adult patients receiving the target drugs for the specified indication were identified. The effectiveness of the 2 agents used were compared based on the concept that a change in dispensed medication suggested either an adverse event or lack of effectiveness. Adherence rates, adverse events, and pharmacy and nonpharmacy costs associated with the 2 agents were also compared. RESULTS: Patients receiving amlodipine and nifedipine CC as initial treatment for hypertension had similar demographic characteristics and numbers of comorbid conditions. More patients started on nifedipine CC switched to another calcium channel blocker (15.8% for nifedipine CC vs 10.3% for amlodipine). More patients started on amlodipine switched to another class of antihypertensive agent (13.2% for amlodipine vs 7.3% for nifedipine CC). Patients in both groups received adjunctive antihypertensive drugs at a similar frequency (35% for nifedipine CC vs 42%, for amlodipine). Rates of adherence were similar. In adherent patients, there was no difference in rates of reported adverse events. The nonpharmacy costs were similar between groups. Patients in the amlodipine group also had a trend toward higher overall pharmacy charges (all medications) and higher charges for antihypertensive medications other than the study drugs ($302 vs $188, P = .054). CONCLUSIONS: Claims data are often the best available evidence for comparing the effectiveness of pharmaceuticals in real clinical practice. While these comparisons have inherent limitations, the accuracy of the assessment can be maximized by limiting the assessment to agents with the same specific indications. Other important elements include comparison of crossover rates to other pharmaceuticals in the same class; rates of addition of other pharmaceuticals in the same class, adherence, adverse events, and overall healthcare charges.

Adult↗
PubMed1996

Mouse decay-accelerating factor: selective and tissue-specific induction by estrogen of the gene encoding the glycosylphosphatidylinositol-anchored form.

Neonatal exposure of mice to estrogen (diethylstilbestrol) results in a high incidence (90%) of uterine tumor later in life. In an effort to screen for estrogen-regulated genes in the uterus of the neonatal mouse, we have isolated a murine homologue of the human decay-accelerating factor (DAF), a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein and a member of the regulators of complement activation family of proteins that function to prevent autologous complement-mediated tissue damage. The induced mouse DAF cDNA has a 64% sequence identity with the human counterpart at the nucleotide level and a 50% identity in the deduced amino acid sequence. It consists of 390 amino acids and contains four short consensus repeats of internal homology characteristic of human DAF. It also contains a hydrophobic C-terminal that most likely serves as a signal for GPI anchor attachment. Sequence comparison with the recently reported mouse DAF cDNAs confirmed that the estrogen-inducible gene corresponds to the mouse GPI DAF gene. The induction of mouse DAF by estrogen is tissue specific and can be mimicked by the antiestrogen tamoxifen. Furthermore, the regulation of uterine DAF expression by estrogen is limited to the GPI DAF gene. The transmembrane DAF gene is not expressed in the mouse uterus, either with or without estrogen stimulation. These results suggest that the two mouse DAF genes are differentially regulated, and that the GPI-anchored DAF may play important roles in estrogen responses and other physiologic or pathophysiologic processes of the female reproductive system.

Amino Acid Sequence↗
PubMed1996

Death rates among patients hospitalized with community-acquired pneumonia: a reexamination with data from three states.

OBJECTIVES: Death rates for community-acquired pneumonia based on relatively small-scale, published studies tend to exceed 15% to 20%. This study reexamined these estimates by using very large, population-based databases. METHODS: Death rates from 1993 associated with community-acquired pneumonia were reexamined with hospital discharge data from all of Washington, Illinois, and Florida. RESULTS: These death rates were substantially lower (7.0%, 8.1%, and 9.7%, respectively) than what appears in the literature. Significant risk factors for dying were being 65 years of age or older (odds ratio [OR] = 2.9), being positive for human immunodeficiency virus (OR = 2.9), and having a high severity of illness (OR = 7.1). CONCLUSION: Sampling bias associated with selection for hospital admissions explain the discrepancy between previous and this study's results.

AIDS-Related Opportunistic Infections↗
PubMed1995

Cellular and molecular effects of developmental exposure to diethylstilbestrol: implications for other environmental estrogens.

Concerns have been raised regarding the role of environmental and dietary estrogens as possible contributors to an increased incidence of various abnormalities in estrogen-target tissues of both sexes. These abnormalities include breast cancer, endometriosis, fibroids, and uterine adenocarcinoma in females, as well as alterations in sex differentiation, decreased sperm concentrations, benign prostatic hyperplasia, prostatic cancer, testicular cancer, and reproductive problems in males. Whether these concerns are valid remains to be determined; however, studies with the potent synthetic estrogen diethylstilbestrol (DES) suggest that exogenous estrogen exposure during critical stages of development can result in permanent cellular and molecular alterations in the exposed organism. These alterations manifest themselves in the female and male as structural, functional, or long-term pathological changes including neoplasia. Although DES has potent estrogenic activity, it may be used as a model compound to study the effects of weaker environmental estrogens, many of which may fit into the category of endocrine disruptors.

Animals↗
PubMed1994

Cytogenetic analysis of murine cell lines from diethylstilbestrol-induced uterine endometrial adenocarcinomas.

Treatment of female CD-1 mice with the synthetic estrogen diethylstilbestrol (DES) on days 1 through 5 after birth results in a 90% incidence of endometrial adenocarcinomas by 18 months of age Three cell lines were established from DES-induced uterine carcinomas and studied for specific chromosomal changes. Each cell line exhibited numerical decreases in chromosomes 9, 11, 13, and X as common abnormalities. Structural alterations involving chromosomes 3, 6, 11, and 19 occurred nonrandomly among the three cell lines. Every cell line showed a rearrangement in the long arm of chromosome 3 (3q+), a translocation between chromosomes 3 and 19 [t(3;19)], isochromosome of chromosome 11 [i(11)], and a marker chromosome (M2) either as common abnormalities or recurrent abnormalities. t(3;19), i(11), and M2 were observed also in the primary colonies from which the cell lines arose. The changes were not observed in a cell line derived from the uterus of one untreated control mouse, suggesting that these chromosomal alterations may have occurred during DES-induced neoplastic transformation. The chromosomal alterations found in the present study may prove useful in investigating the genetic changes involved in DES carcinogenesis.

Adenocarcinoma↗
PubMed1991

Regional differences in the prostate of the neonatally estrogenized mouse.

Neonatal estrogenization of the mouse with diethylstilbestrol resulted in time-of-exposure and dose-dependent inhibition of the growth of the prostatic lobes observed at the age of 2 mon. The critical time was the days 1-6 of postnatal life. In neonatally estrogenized (neoDES) mice, responses to 5 alpha-dihydrotestosterone in terms of nuclear 3H-thymidine labelling were altered concomitantly with the inhibition of growth and were in accordance with changes in the relative volumes of epithelium, glandular lumina, and interacinar stroma. Secondary estrogen treatment of neoDES mice with 17 beta-estradiol did not increase 3H-thymidine labelling in the prostate of control or neoDES mice. However, it induced squamous epithelial metaplasia in periurethral collecting ducts and proximal parts of coagulating glands of neoDES animals. In control mice only slight epithelial hyperplasia could be observed after similar treatment. Estrogen receptors, located immunocytochemically in nuclei of stromal cell, corresponded with the sites of increased estrogen sensitivity, observed as metaplastic transformation. When the neoDES animals aged, epithelial hyperplasia and dysplasia could be observed at distinct prostatic sites, ie, the periurethral collecting ducts and the coagulating glands and periurethral glands, and stromal inflammation become more extensive. Almost identical location of the epithelial changes and the altered estrogen response is suggestive of causal relationship.

Aging↗