Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate

Background: The enzyme coproporphyrinogen oxidase (copro\u27gen oxidase) converts coproporphyrinogen-Ill (GIII) to protoporphyrinogen-IX via an intermediary monovinyl porphyrinogen. The A ring isomer coproporphyrinogen-IV (C-IV) has previously been shown to be a substrate for copro\u27gen oxidase derived from avian erythrocytes. In contrast to the authentic substrate (GIII) where only a small amount of the monovinyl intermediate is detected, C-IV gives rise to a monovinyl intermediate that accumulates before being converted to an isomer of protoporphyrinogen-IX. No kinetic studies have been carried out using the purified human copro\u27gen oxidase to evaluate its ability to process both the authentic substrate as well as analogs. Material/Methods: Therefore, purified, cloned human copro\u27gen oxidase was incubated with GIII or C-IV at 37 degrees C with various substrate concentrations (from 0.005 mu M to 3.5 mu M). The Km (an indication of molecular recognition) and Kcat (turnover number) values were determined. Results: The Km value for total product formation was about the same with either C-III or C-IV indicating the same molecular recognition. However, the catalytic efficiency (Kcat/Km) of the enzyme for total product formation was not more than two fold higher using GIII relative to C-IV. Conclusions: Since the Km values are about the same for either substrate and the total Kcat/Km values are within two fold of each other, this could correlate with the increase of severity of porphyrias with monovinyl accumulation. The ability of the increased levels of C-IV to compete with the authentic substrate has important implications for clinical porphyrias

Departure from multiplicative interaction for catechol-O-methyltransferase genotype and active/passive exposure to tobacco smoke among women with breast cancer

BACKGROUND: Women with homozygous polymorphic alleles of catechol-O-methyltransferase (COMT-LL) metabolize 2-hydroxylated estradiol, a suspected anticarcinogenic metabolite of estrogen, at a four-fold lower rate than women with no polymorphic alleles (COMT-HH) or heterozygous women (COMT-HL). We hypothesized that COMT-LL women exposed actively or passively to tobacco smoke would have higher exposure to 2-hydroxylated estradiol than never-active/never passive exposed women, and should therefore have a lower risk of breast cancer than women exposed to tobacco smoke or with higher COMT activity. METHODS: We used a case-only design to evaluate departure from multiplicative interaction between COMT genotype and smoking status. We identified 502 cases of invasive incident breast cancer and characterized COMT genotype. Information on tobacco use and other potential breast cancer risk factors were obtained by structured interviews. RESULTS: We observed moderate departure from multiplicative interaction for COMT-HL genotype and history of ever-active smoking (adjusted odds ratio [aOR] = 1.6, 95% confidence interval [CI]: 0.7, 3.8) and more pronounced departure for women who smoked 40 or more years (aOR = 2.3, 95% CI: 0.8, 7.0). We observed considerable departure from multiplicative interaction for COMT-HL genotype and history of ever-passive smoking (aOR = 2.0, 95% CI: 0.8, 5.2) or for having lived with a smoker after age 20 (aOR = 2.8, 95% CI: 0.8, 10). CONCLUSION: With greater control over potential misclassification errors and a large case-only population, we found evidence to support an interaction between COMT genotype and tobacco smoke exposure in breast cancer etiology

Use of Di- and Tripropionate substrate analogs to probe the active site of human recombinant coproporphyrinogen oxidase

Background: Defects in the enzyme coproporphyrinogen oxidase result in accumulation of porphyrins which may affect the severity of a subset of porphyrias. Thus evaluation of this enzyme for substrate selectivity is of value. Kinetic evaluations of recombinant human coproporphyrinogen oxidase have been undertaken using six di- and tripropionate analogs of the natural substrate coproporphyrinogen-III. These Substrate analogs were modified by having alkyl groups in place of one or both of the ring 13- or 17-propionate moieties. Material/Methods: Cloned human enzyme was incubated with analogs under apparent first order conditions and with various substrate concentrations. The kinetic values, K-m and V-max, were determined. Results: Relative to the authentic substrate, the K-m values for the 13-ethyl, dimethyl and diethyl porphyrinogens were very comparable whereas the K-m values were much higher using dipropyl and dibutyl porphyrinogen and much lower for the 17-ethyl analog. For the dipropionate analogs, the V-max values were an apparent function of the carbon length of the substituent. on the C and D rings, with longer carbon length severely reducing product formation by some 4-5 orders of magnitude. Also, the two isomeric tripropionates that were tested indicated that it was more detrimental to have an ethyl group at the 13-position for both binding and catalysis. Conclusions: This work extends our understanding of porphyrin ring substituent effects reported by Cooper et al. (2005). The substituents on both the C and D rings have significant effects on both the substrate binding and catalysis by this important enzyme

Investigation of the catalytic and structural roles of conserved histidines of human coproporphyrinogen oxidase using site-directed mutagenesis

Background: The catalytic contribution of four conserved histidines of human coproporphyrinogen oxidase (CPO) has been investigated using site-directed mutagenesis to change histidine (H) into alanine (A). Material/Methods: The wild-type and mutant enzyme forms were analyzed for their ability to utilize coproporphyrinogen-III, mesoporphvrinogen-VI, and harderoporphyrinogen as substrates. Results: Wild-type CPO had specific activities of 4.9 +/- 0.9 nmole product/min/mg for coproporphyrinogen-III, 1.7 +/- 0.7 nmole ptoduct/min/mg for mesoporphyrinogen-VI, and 5.1 +/- 1.8 nmole product/min/mg for harderoporphyrinogen. The four mutant enzymes were catalytically competent With all three substrates, but to varying degrees. The most affected Mutant was the H158A enzyme which exhibited approximately 50-fold lower activity than wild-type recombinant CPO. Conclusions: Thus, His 158 of human CPO may have a role ill the active site, but none of the conserved histidine residues of human coproporphyrinogen oxidase is essential for catalytic activity although changes in histidines have been implicated in the disease state hereditary coproporphyria

Use of selective serotonin reuptake inhibitors and risk of re-operation due to post-surgical bleeding in breast cancer patients: a Danish population-based cohort study

<p>Abstract</p> <p>Background</p> <p>Selective serotonin reuptake inhibitors (SSRI) decrease platelet-function, which suggests that SSRI use may increase the risk of post-surgical bleeding. Few studies have investigated this potential association.</p> <p>Methods</p> <p>We conducted a population-based study of the risk of re-operation due to post-surgical bleeding within two weeks of primary surgery among Danish women with primary breast cancer. Patients were categorised according to their use of SSRI: never users, current users (SSRI prescription within 30 days of initial breast cancer surgery), and former users (SSRI prescription more than 30 days before initial breast cancer surgery). We calculated the risk of re-operation due to post-surgical bleeding within 14 days of initial surgery, and the relative risk (RR) of re-operation comparing SSRI users with never users of SSRI adjusting for potential confounders.</p> <p>Results</p> <p>389 of 14,464 women (2.7%) were re-operated. 1592 (11%) had a history of SSRI use. Risk of re-operation was 2.6% among never users, 7.0% among current SSRI users, and 2.7% among former users. Current users thus had an increased risk of re-operation due to post-operative bleeding (adjusted relative risk = 2.3; 95% confidence interval (CI) = 1.4, 3.9) compared with never users. There was no increased risk of re-operation associated with former use of SSRI (RR = 0.93, 95% CI = 0.66, 1.3).</p> <p>Conclusions</p> <p>Current use of SSRI is associated with an increased risk of re-operation due to bleeding after surgery for breast cancer.</p

Triple-negative breast cancers are increased in black women regardless of age or body mass index

INTRODUCTION. We investigated clinical and pathologic features of breast cancers (BC) in an unselected series of patients diagnosed in a tertiary care hospital serving a diverse population. We focused on triple-negative (Tneg) tumours (oestrogen receptor (ER), progesterone receptor (PR) and HER2 negative), which are associated with poor prognosis. METHODS. We identified female patients with invasive BC diagnosed between 1998 and 2006, with data available on tumor grade, stage, ER, PR and HER2 status, and patient age, body mass index (BMI) and self-identified racial/ethnic group. We determined associations between patient and tumour characteristics using contingency tables and multivariate logistic regression. RESULTS. 415 cases were identified. Patients were racially and ethnically diverse (born in 44 countries, 36% white, 43% black, 10% Hispanic and 11% other). 47% were obese (BMI > 30 kg/m2). 72% of tumours were ER+ and/or PR+, 20% were Tneg and 13% were HER2+. The odds of having a Tneg tumour were 3-fold higher (95% CI 1.6, 5.5; p = 0.0001) in black compared with white women. Tneg tumours were equally common in black women diagnosed before and after age 50 (31% vs 29%; p = NS), and who were obese and non-obese (29% vs 31%; p = NS). Considering all patients, as BMI increased, the proportion of Tneg tumours decreased (p = 0.08). CONCLUSIONS. Black women of diverse background have 3-fold more Tneg tumours than non-black women, regardless of age and BMI. Other factors must determine tumour subtype. The higher prevalence of Tneg tumours in black women in all age and weight categories likely contributes to black women's unfavorable breast cancer prognosis.LaPann Fund; Research Enhancement Fun

Prescriptions for selective cyclooxygenase-2 inhibitors, non-selective non-steroidal anti-inflammatory drugs, and risk of breast cancer in a population-based case-control study

INTRODUCTION. Non-steroidal anti-inflammatory drugs (NSAIDs) prevent the growth of mammary tumours in animal models. Two population-based case-control studies suggest a reduced risk of breast cancer associated with selective cyclooxygenase-2 (sCox-2) inhibitor use, but data regarding the association between breast cancer occurrence and use of non-selective NSAIDs are conflicting. METHODS. We conducted a population-based case-control study using Danish healthcare databases to examine if use of NSAIDs, including sCox-2 inhibitors, was associated with a reduced risk of breast cancer. We included 8,195 incident breast cancer cases diagnosed in 1991 through 2006 and 81,950 population controls. RESULTS. Overall, we found no reduced breast cancer risk in ever users (>2 prescriptions) of sCox-2 inhibitors (odds ratio (OR) = 1.08, 95% confidence interval (95% CI) = 0.99, 1.18), aspirin (OR = 0.98, 95% CI = 0.90-1.07), or non-selective NSAIDs OR = 1.04, (95% CI = 0.98, 1.10)). Recent use (>2 prescriptions within two years of index date) of sCox-2 inhibitors, aspirin, or non-selective NSAIDs was likewise not associated with breast cancer risk (Ors = 1.06 (95% CI = 0.96, 1.18), 0.96 (95% CI = 0.87, 1.06) and 0.99 (95% CI = 0.85, 1.16), respectively). Risk estimates by duration (<10, 10 to 15, 15+ years) or intensity (low/medium/high) of NSAID use were also close to unity. Regardless of intensity, shorter or long-term NSAID use was not significantly associated with breast cancer risk. CONCLUSIONS. Overall, we found no compelling evidence of a reduced risk of breast cancer associated with use of sCox-2 inhibitors, aspirin, or non-selective NSAIDs.Karen Elise Jensen Foundatio

The Somatic Genomic Landscape of Glioblastoma

We describe the landscape of somatic genomic alterations based on multi-dimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer

Organometallic Chemistry within the Structured Environment Provided by the Macrocyclic Cores of Carbaporphyrins and Related Systems

The unique environment within the core of carbaporphyrinoid systems provides a platform to explore unusual organometallic chemistry. The ability of these structures to form stable organometallic derivatives was first demonstrated for N-confused porphyrins but many other carbaporphyrin-type systems were subsequently shown to exhibit similar or complementary properties. Metalation commonly occurs with catalytically active transition metal cations and the resulting derivatives exhibit widely different physical, chemical and spectroscopic properties and range from strongly aromatic to nonaromatic and antiaromatic species. Metalation may trigger unusual, highly selective, oxidation reactions. Alkyl group migration has been observed within the cavity of metalated carbaporphyrins, and in some cases ring contraction of the carbocyclic subunit takes place. Over the past thirty years, studies in this area have led to multiple synthetic routes to carbaporphyrinoid ligands and remarkable organometallic chemistry has been reported. An overview of this important area is presented