Differential inhibition of rat and mouse microsome heme oxygenase by derivatives of imidazole and benzimidazole.

Metalloporphyrin heme oxygenase (HO) inhibitors have made an important contribution to elucidating the role of HO in physiological processes. Nevertheless, their off-target effects have drawn substantial criticism, which prompted us to develop non-porphyrin, azole-based inhibitors of HO. These second-generation HO inhibitors were evaluated using spleen and brain microsomes from rats as native sources of HO-1 and HO-2, respectively. Recently, the use of azole-based inhibitors of HO has been extended to other mammalian species and, as a consequence, it will be important to characterize the inhibitors in these species. The goal of this study was to compare the inhibitory profile of imidazole- and benzimidazole-based inhibitors of HO in a breast-cancer-implanted mouse to that of an untreated rat. For spleen and brain microsomes from both species, HO protein expression was determined by Western blotting and concentration-response curves for imidazole- and benzimidazole-derivative inhibition of HO activity were determined using a headspace gas-chromatographic assay. It was found that the effects on HO activity by imidazole and benzimidazole derivatives were different between the 2 species and were not explained by differences in HO expression. Thus, the HO inhibitory profile should be determined for azole derivatives before they are used in mammalian species other than rats.

Mitoxantrone and Etoposide for the Treatment of Acute Myeloid Leukemia Patients in First Relapse.

Relapsed acute myeloid leukemia (AML) represents a major therapeutic challenge. Achieving complete remission (CR) with salvage chemotherapy is the first goal of therapy for relapsed AML. However, there is no standard salvage chemotherapy. The current study evaluated outcomes and prognostic factors for achievement of CR in 91 AML patients in first relapse who were treated with the mitoxantrone-etoposide combination regimen. The overall response rate (CR and CRi) was 25%. Factors that were associated with a lower rate of CR included older age, shorter duration of first CR, low hemoglobin, and low platelet count. The median overall survival for all patients was 7.4 months. The survival of patients who achieved CR and underwent allogeneic hematopoietic cell transplantation (allo-HCT) was higher than those who achieved CR and did not undergo allo-HCT (35.3 months vs. 16.8 months, p = 0.057). The median duration of relapse-free survival was 12.7 months in the patients achieving CR. Older age at the time of AML relapse was associated with worse overall survival. The all-cause 4-week mortality rate was 4%, and the all-cause 8-week mortality rate was 13%. The findings of this study underscore the need for newer therapies, especially those that will improve the ability for patients with relapsed AML to achieve CR and to allow them to receive additional therapies.

Are icons sense data?

We argue that Hoffman, Singh, and Prakash (Psychon Bull Rev, this issue) have not made the case that "the language of space-time and physical objects is the wrong language for describing the true structure of the objective world." Further, we contend that, contrary to what Hoffman et al. claim, the perceptual icons posited by interface theory seem best taken to be sense data.

Structural analysis and identification of PhuS as a heme-degrading enzyme from Pseudomonas aeruginosa.

Bacterial pathogens require iron for proliferation and pathogenesis. Pseudomonas aeruginosa is a prevalent Gram-negative opportunistic human pathogen that takes advantage of immunocompromised hosts and encodes a number of proteins for uptake and utilization of iron. Here we report the crystal structures of PhuS, previously known as the cytoplasmic heme-trafficking protein from P. aeruginosa, in both the apo- and the holo-forms. In comparison to its homologue ChuS from Escherichia coli O157:H7, the heme orientation is rotated 180° across the α-γ axis, which may account for some of the unique functional properties of PhuS. In contrast to previous findings, heme binding does not result in an overall conformational change of PhuS. We employed spectroscopic analysis and CO measurement by gas chromatography to analyze heme degradation, demonstrating that PhuS is capable of degrading heme using ascorbic acid or cytochrome P450 reductase-NADPH as an electron donor and produces five times more CO than ChuS. Addition of catalase slows down but does not stop PhuS-catalyzed heme degradation. Through spectroscopic and mass spectrometry analysis, we identified the enzymatic product of heme degradation to be verdoheme. These data taken together suggest that PhuS is a potent heme-degrading enzyme, in addition to its proposed heme-trafficking function.

Phase I trial of carboplatin and etoposide in combination with panobinostat in patients with lung cancer.

UNLABELLED: A phase I trial consisting of panobinostat (a HDAC inhibitor), carboplatin and etoposide was conducted in patients with lung cancer. PATIENTS AND METHODS: Patients received carboplatin AUC5 on day 1 and etoposide 100 mg/m(2) on days 1, 2 and 3, every 21 days. Concurrent oral panobinostat was given 3 times weekly on a 2-weeks-on and 1-week-off schedule during the 4-6 cycles of chemotherapy and then continued as maintenance therapy. RESULTS: Six evaluable patients were treated at the first dose level of panobinostat (10 mg). Dose-limiting toxicity occurred in two patients (33%) during the first cycle. One patient developed grade 4 thrombocytopenia and another grade 4 febrile neutropenia. Therefore, the study was suspended based on the pre-specified study design. No recommended phase II starting dose was established. CONCLUSION: The addition of panobinostat to carboplatin and etoposide was not tolerable at the lowest dose level tested in this trial. Further research and development into this combination is not recommended.

Fludarabine and cytarabine in patients with relapsed acute myeloid leukemia refractory to initial salvage therapy.

The most effective regimen for relapsed acute myeloid leukemia (AML) patients who do not achieve complete remission (CR) after a course of salvage therapy has not been established. We evaluated the efficacy and toxicity of fludarabine and cytarabine in patients with AML in first relapse who did not respond to a course of salvage chemotherapy with mitoxantrone and etoposide. CR was achieved in 39 % of treated patients, and in 47 % of patients with a favorable/intermediate-risk karyotype. The median overall survival was 4.75 months. The median survival for patients achieving CR with fludarabine-cytarabine was significantly higher than for those who did not respond to therapy (9.6 vs. 4.5 months, P = 0.04). Our data suggest that the fludarabine-cytarabine regimen merits further investigation in relapsed AML patients with favorable or intermediate-risk karyotype with persistent leukemia after a course of salvage therapy.

Prosthetic limb sockets from plant-based composite materials.

BACKGROUND: There is a considerable demand for lower limb prostheses globally due to vascular disease, war, conflict, land mines and natural disasters. Conventional composite materials used for prosthetic limb sockets include acrylic resins, glass and carbon fibres, which produce harmful gasses and dust in their manufacture. OBJECTIVES: To investigate the feasibility of using a renewable plant oil-based polycarbonate-polyurethane copolymer resin and plant fibre composite, instead of conventional materials, to improve safety and accessibility of prosthetic limb manufacture. STUDY DESIGN: Experimental, bench research. METHODS: Test pieces of the resin with a range of plant fibres (10.0% by volume) were prepared and tensile strengths were tested. Test sockets of both conventional composite materials and plant resin with plant fibres were constructed and tested to destruction. RESULTS: Combinations of plant resin and either banana or ramie fibres gave high tensile strengths. The conventional composite material socket and plant resin with ramie composite socket failed at a similar loading, exceeding the ISO 10328 standard. Both wall thickness and fibre-matrix adhesion played a significant role in socket strength. CONCLUSIONS: From this limited study we conclude that the plant resin and ramie fibre composite socket has the potential to replace the standard layup. Further mechanical and biocompatibility testing as well as a full economic analysis is required. CLINICAL RELEVANCE: Using readily sourced and renewable natural fibres and a low-volatile bio-resin has potential to reduce harm to those involved in the manufacture of artificial limb sockets, without compromising socket strength and benefitting clinicians working in poorer countries where safety equipment is scarce. Such composite materials will reduce environmental impact.

Selective activation of heme oxygenase-2 by menadione.

While substantial progress has been made in elucidating the roles of heme oxygenases-1 (HO-1) and -2 (HO-2) in mammals, our understanding of the functions of these enzymes in health and disease is still incomplete. A significant amount of our knowledge has been garnered through the use of nonselective inhibitors of HOs, and our laboratory has recently described more selective inhibitors for HO-1. In addition, our appreciation of HO-1 has benefitted from the availability of tools for increasing its activity through enzyme induction. By comparison, there is a paucity of information about HO-2 activation, with only a few reports appearing in the literature. This communication describes our observations of the up to 30-fold increase in the in-vitro activation of HO-2 by menadione. This activation was due to an increase in Vmax and was selective, in that menadione did not increase HO-1 activity.

Recombinant truncated and microsomal heme oxygenase-1 and -2: differential sensitivity to inhibitors.

Recombinant truncated forms of heme oxygenase-1 and -2 (HO-1 and HO-2) were compared with their crude microsomal counterparts from brain and spleen tissue of adult male rats with respect to their inhibition by azole-based, nonporphyrin HO inhibitors. The drugs tested were an imidazole-alcohol, an imidazole-dioxolane, and a triazole-ketone. Both the recombinant and crude forms of HO-2 were similarly inhibited by the 3 drugs. The crude microsomal spleen form of HO-1 was more susceptible to inhibition than was the truncated recombinant form. This difference is attributed to the extra amino acids in the full-length enzyme. These observations may be relevant in the design of drugs as inhibitors of HO and other membrane proteins.

The effects of azole-based heme oxygenase inhibitors on rat cytochromes P450 2E1 and 3A1/2 and human cytochromes P450 3A4 and 2D6.

Heme oxygenases (HOs) catalyze the degradation of heme to biliverdin, carbon monoxide (CO), and free iron. The two major isoforms, HO-1 (inducible) and HO-2 (constitutive), are involved in a variety of physiological functions, including inflammation, apoptosis, neuromodulation, and vascular regulation. Major tools used in exploring these actions have been metalloporphyrin analogs of heme that inhibit the HOs. However, these tools are limited by their lack of selectivity; they affect other heme-dependent enzymes, such as cytochromes P450 (P450s), soluble guanylyl cyclase (sGC), and nitric-oxide synthase (NOS). Our laboratory has successfully synthesized a number of nonporphyrin azole-based HO inhibitors (QC-xx) that had little or no effect on sGC and NOS activity. However, their effects on various P450 isoforms have yet to be fully elucidated. To determine the effects of the QC-xx inhibitors on P450 enzyme activity, microsomal preparations of two rat P450 isoforms (2E1 and 3A1/3A2) and two human P450 supersome isoforms (3A4 and 2D6) were incubated with varying concentrations of HO inhibitor, and the activity was determined by spectrophotometric or fluorometric analysis. Results indicated that some QC compounds demonstrated little to no inhibition of the P450s, whereas others did inhibit these P450 isoforms. Four structural regions of QC-xx were analyzed, leading to the identification of structures that confer a decreased effect on both rat and human P450 isoforms studied while maintaining an inhibitory effect on the HOs.

Extravascular lung water indexed to predicted body weight is a novel predictor of intensive care unit mortality in patients with acute lung injury.

OBJECTIVES: Acute lung injury and the acute respiratory distress syndrome are characterized by noncardiogenic pulmonary edema, which can be assessed by measurement of extravascular lung water. Traditionally, extravascular lung water has been indexed to actual body weight (mL/kg). Because lung size is dependent on height rather than weight, we hypothesized indexing to predicted body weight may be a better predictor of mortality in acute lung injury/acute respiratory distress syndrome. DESIGN: Prospective observational cohort study. SETTING: A tertiary referral intensive care unit. PATIENTS: Patients were recruited within 48 hrs of fulfilling the American European Consensus Conference definition of acute lung injury/acute respiratory distress syndrome. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Demographics, severity of illness scores, and respiratory parameters were collected. Extravascular lung water was measured using the PiCCO system. This was indexed to actual and predicted body weight. Statistically significant predictors of mortality identified using single regressor logistic regression and additional variables known to be associated with outcome were entered into a multiple logistic regression analysis. Receiver operator characteristic curves were generated. Forty-four patients were recruited (septic 34%). Using single regressor logistic regression, six variables were statistically significantly related to mortality: Acute Physiology and Chronic Health Evaluation II, PaO2, PaO2/Fio2 ratio, oxygenation index, actual extravascular lung water, and predicted extravascular lung water. In multiple logistic regression analysis, predicted extravascular lung water but not actual extravascular lung water was a predictor of mortality with an odds ratio of 4.3 (95% confidence interval, 1.5-12.9) per sd. Although the area under the curve for predicted extravascular lung water (0.8; confidence interval, 0.65-0.94) was larger than for actual extravascular lung water (0.72; confidence interval, 0.53-0.91), this was not statistically significant (p = .12). A baseline predicted extravascular lung water value of 16 mL/kg predicted intensive care unit mortality with a sensitivity of 0.75 (confidence interval, 0.47-0.91) and specificity of 0.78 (confidence interval, 0.61-0.89). CONCLUSIONS: Early measurement of predicted extravascular lung water is a better predictor than actual extravascular lung water to identify patients at risk for death in acute lung injury/acute respiratory distress syndrome.

Management of patients treated with chemoradiotherapy for head and neck cancer without prophylactic feeding tubes: the University of Pittsburgh experience.

OBJECTIVES/HYPOTHESIS: Mucositis and dysphagia are common complications of chemoradiotherapy (CRT) for head and neck cancer that may necessitate nutritional support with a gastrostomy tube (G-tube). METHODS: We reviewed records of patients who underwent and completed CRT, which included at least one traditional chemotherapeutic, for previously untreated head and neck cancer. G-tubes were placed as needed. The timing and duration of G-tube placement and treatment-related complications and risk factors for long-term G-tube use were analyzed. RESULTS: A total of 91 consecutive patients who received CRT, 68 as primary and 23 as postoperative treatment, were studied. Radiation doses ranged from 59.4 to 74 Gy (median, 70 Gy). Seventy-nine percent of patients received platinum-based therapy during CRT. Severe mucositis occurred in 40% of patients. Forty percent of patients required G-tube placement (15 prior to CRT and 21 during CRT). Median duration of G-tube use was 5.8 months. Two patients who had a G-tube placed during CRT developed a G-tube-related complication. At 6 and 12 months, 15 (18%) and four (6%) patients who were disease free were using G-tubes, respectively. Patients with G-tubes placed prior to CRT or advanced T stage had longer G-tube dependence. CONCLUSIONS: With aggressive supportive care it is feasible to avoid G-tubes in the majority of patients undergoing CRT for head and neck cancer. G-tube placement prior to CRT due to pre-existing dysphagia and advanced T stage are associated with prolonged G-tube dependence.

Dialysis disequilibrium syndrome: a consideration in patients with hydrocephalus.

Dialysis disequilibrium syndrome is a rare neurological manifestation of intermittent hemodialysis. Urea removal occurs more slowly across the blood-brain barrier than from the plasma, generating an osmotic gradient that promotes water movement into the brain and cerebral edema. The authors report the development of dialysis disequilibrium syndrome in a patient with spina bifida and an adequately functioning shunt.

Chronic prenatal ethanol exposure and increased concentration of fatty acid ethyl esters in meconium of term fetal Guinea pig.

In humans, the occurrence of prenatal exposure to ethanol is difficult to validate objectively. Increased concentration of fatty acid ethyl esters (FAEE) in the meconium of the newborn may be a biomarker of prenatal ethanol exposure. The validity of this proposed biomarker was tested in pregnant guinea pigs that received chronic oral administration of 4 g ethanol/kg maternal body weight/day (n=8), isocaloric-sucrose/pair-feeding (n=8) or water (n=2) throughout gestation. At gestational day 65 (term, gestational day 66 to 69), each dam and her offspring were euthanized, and meconium was collected from the term fetal large intestine. Eight individual FAEE (lauric, myristic, palmitic, palmitoleic, stearic, oleic, linolenic and arachidonic AEE) were measured by gas chromatography--flame ionization detection and confirmed by gas chromatography--mass spectrometry. The chronic maternal ethanol regimen decreased fetal body weight and brain weight. There was virtually no measurable FAEE in the meconium for the water group (n=3 fetuses). For meconium of the ethanol offspring (n=25 fetuses) compared with the sucrose offspring (n=23 fetuses), the total FAEE concentration was 8-fold higher; and lauric, palmitic, stearic and oleic AEE concentrations were at least 5-fold higher for the ethanol group. The data indicate that fetal meconium FAEE constitute a biomarker of prenatal ethanol exposure for a maternal ethanol regimen that restricts fetal development, with an inverse relationship between meconium total FAEE concentration and both body weight and brain weight.

The source of heme for vascular heme oxygenase I: heme uptake in rat aorta.

During the last decade, heme oxygenase (HO) and carbon monoxide (CO) have garnered substantial research interest in terms of cell and organ regulation, especially as they bear on the central nervous system, organ transplantation, and the cardiovascular system. While the enzymatic mechanism, substrates, and products of HO are well known, it is not clear whether the cardiovascular system derives its supply of the heme substrate through de novo synthesis or uptake from the extracellular milieu. The objective of the present study was to test the latter possibility in rat aorta and to determine the influence of plasma proteins that bind heme in vivo, viz. hemopexin and albumin. Aortic tissue was exposed to [14C]heme in vitro, and the concentration and time dependence of heme uptake was assessed. The presence of hemopexin or albumin in the incubation medium dramatically decreased heme uptake by the aorta. Heme uptake by aortic tissue was not altered after induction of HO-1, which would be expected to increase tissue heme demand. In summary, the rat, isolated aorta was capable of obtaining heme from its external milieu, but this was obtunded in the presence of the plasma proteins hemopexin or albumin. For normal physiological situations, heme uptake may not be a usual source of substrate for vascular HO and hemoenzymes such as nitric oxide synthase, soluble guanylyl cyclase, and cyclooxygenase.

The source of heme for vascular heme oxygenase II: de novo heme biosynthesis in rat aorta.

Heme is an essential prosthetic group or substrate for many proteins, including hemoglobin, and hemo enzymes such as nitric oxide synthase, soluble guanylyl cyclase, and heme oxygenase (HO). HO is responsible for the breakdown of heme into equimolar amounts of biliverdin, iron, and carbon monoxide, the latter of which is thought to play a role in the regulation of vascular tone. It is not clear whether the source of heme for cardiovascular functions is derived from uptake from the extracellular milieu or synthesis. In this study, we tested the hypothesis that blood vessels obtain their supply of heme for HO through de novo synthesis. Adult male Sprague-Dawley rat aorta was incubated at 37 degrees C in Krebs' solution with 1 micro M [14C]delta-aminolevulinic acid (ALA). [14C]ALA uptake was linear for about 30 min and reached a plateau at approximately 100 min. The radioactivity was incorporated into porphyrins and heme as determined by esterification of 14C-labelled metabolites and thin-layer chromatography. The first and rate-limiting step of heme biosynthesis is catalyzed by ALA synthase (ALA-S), the activity of which was determined in rat aorta using a radiometric assay, approximately 250 nmol x (g wet mass)(-1) x h(-1). Inducing HO-1 in rat aorta with S-nitroso-N-acetylpenicillamine (500 micro M) did not increase ALA-S activity as compared with basal activity levels of the enzyme. It appears that there is a sufficient amount of heme available under basal ALA-S activity conditions to meet the increased demand for heme resulting from HO-1 induction. These observations indicate that the complete enzymatic pathway for de novo heme biosynthesis resides in rat aorta and furthermore indicate that de novo heme synthesis is capable of supplying a substantial portion of the heme substrate for HO in the aorta.

Heme oxygenase expression in selected regions of term human placenta.

Carbon monoxide (CO), formed during heme oxygenase (HO)-catalyzed oxidation of heme, has been proposed to play a complementary role with nitric oxide in the regulation of placental hemodynamics. The objective of this study was to elucidate HO enzymatic activity and HO-1 (inducible) and HO-2 (constitutive) protein content in the microsomal subcellular fraction of homogenate of selected regions of placenta from normotensive and mild pre-eclamptic pregnancies. HO enzymatic activity was measured under optimized conditions by gas chromatography using CO formation as an index of activity, and HO-1 and HO-2 protein content were determined by Western immunoblot analysis. Microsomal HO activity in each of the four placental regions was not different between normotensive and mild pre-eclamptic pregnancies. Microsomal HO-2 protein content was not different between normotensive and mild pre-eclamptic pregnancies, whereas there was increased expression of microsomal HO-1 protein in chorionic villi and fetal membranes from pre-eclamptic pregnancy compared with normotensive pregnancy. Microsomal HO enzymatic activity correlated with HO-2, but not HO-1, protein content.

Measurement of endogenous carbon monoxide formation in biological systems.

Endogenous carbon monoxide (CO) formation has been measured in different biological systems using a variety of analytical procedures. The methods include gas chromatography-reduction gas detection, gas chromatography-mass spectroscopic detection, laser sensor-infrared absorption, UV-visible spectrophotometric measurement of CO-hemoglobin or CO-myoglobin complex, and formation of (14)CO from (14)C-heme formed following [2-(14)C]glycine administration. CO formation ranged from a low of 0.029 nmol/mg of protein/h in chorionic villi of term human placenta to a high of 0.28 nmol/mg of protein/h in rat olfactory receptor neurons in culture and rat liver perfusate.