Evolutionarily consistent families in SCOP: sequence, structure and function

Background

HutZ is the sole heme storage protein identified in the pathogenic bacterium Vibrio cholerae and is required for optimal heme utilization. However, no heme oxygenase activity has been observed with this protein. Thus far, HutZ??s structure and heme-binding mechanism are unknown.

Results

We report the first crystal structure of HutZ in a homodimer determined at 2.0 ? resolution. The HutZ structure adopted a typical split-barrel fold. Through a docking study and site-directed mutagenesis, a heme-binding model for the HutZ dimer is proposed. Very interestingly, structural superimposition of HutZ and its homologous protein HugZ, a heme oxygenase from Helicobacter pylori, exhibited a structural mismatch of one amino acid residue in ??6 of HutZ, although residues involved in this region are highly conserved in both proteins. Derived homologous models of different single point variants with model evaluations suggested that Pro¹⁴⁰ of HutZ, corresponding to Phe²¹⁵ of HugZ, might have been the main contributor to the structural mismatch. This mismatch initiates more divergent structural characteristics towards their C-terminal regions, which are essential features for the heme-binding of HugZ as a heme oxygenase.

Conclusions

HutZ??s deficiency in heme oxygenase activity might derive from its residue shift relative to the heme oxygenase HugZ. This residue shift also emphasized a limitation of the traditional template selection criterion for homology modeling.     

Effects of hematin and carbon monoxide on the salinity stress responses of Cassia obtusifolia L. seeds and seedlings

Aims

The purpose of the present study was to investigate the mechanism of carbon monoxide (CO) and hematin in alleviating the inhibition of Cassia obtusifolia seeds and seedlings. NaCl (100?mM) was used to mimic salinity stress in a series of experiments.

Methods

Varying combinations of CO in a saturated aqueous solution and hematin (1.0?μM) were added to seeds and seedlings under salinity stress. Seed germination indices and seedling parameters were investigated.

Results

Seed germination and seedling growth were significantly inhibited under salinity stress. NaCl-induced inhibitory effects on seed germination and seedling growth were ameliorated by hematin or the CO aqueous solution. Addition of 1.0?μM hematin or 5?% CO-saturated aqueous solution to seeds and seedlings significantly alleviated damage to the plant cells under salinity stress. Hematin and the CO aqueous solution enhanced chlorophyll concentration, total soluble sugars, free proline, and soluble protein, and improved photosystem II (PSII) photochemical efficiency levels, PSII actual photochemical efficiency, and the photochemical quench coefficient. In contrast, the non-photochemical quenching coefficient decreased. Hematin and the CO aqueous solution also enhanced the activities of superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, and glutathione reductase, thus alleviating oxidative damage, as indicated by decreases in hiobarbituric acid reactive substances, hydrogen peroxide concentration, relative conductivity, and lipoxygenase activity. Heme oxygenase (HO) activity was increased by hematin treatment. Hematin may contribute to endogenous HO-derived CO, since the addition of zinc protoporphyrin IX or hemoglobin reversed the protective effects conferred by hematin specified above.

Conclusions

Based on the experimental results, we conclude that hematin and CO induce advantageous effects on the attenuation of salt-stress inhibition of C. obtusifolia seeds and seedlings and alleviate oxidative damage by conferring beneficial cytoprotection and activating anti-oxidant enzymes.     

Heme oxygenase is involved in H2O2-induced lateral root formation in apocynin-treated rice

Key message

Apocynin is a natural organic compound structurally related to vanillin. We demonstrated that hydrogen peroxide and heme oxygenase participated in apocynin-induced lateral root formation in rice.

Abstract

Apocynin, also known as acetovanillone, is a natural organic compound structurally related to vanillin. Information concerning the effect of apocynin on plants is limited. In this study, we examined the effect of apocynin on lateral root (LR) formation in rice. Treatment with apocynin induced LR formation and increased H₂O₂ production, but had no effect on nitric oxide production. Diphenyleneiodonium chloride, an inhibitor of H₂O₂ generating NADPH oxidase, was effective in reducing apocynin-induced H₂O₂ production and LR formation. Apocynin treatment also increased superoxide dismutase activity and decreased catalase activity. H₂O₂ application was able to increase the number of LRs. Moreover, H₂O₂ production caused by H₂O₂ and apocynin was localized in the root area corresponding to the LR emergence. Treatment with H₂O₂ and apocynin also increased heme oxygenase (HO) activity and induced OsHO1 mRNA expression. Lateral root formation and HO activity induced by H₂O₂ and apocynin were reduced by Zn protoporphyrin IX (the specific inhibitor of HO). Our data suggest that both H₂O₂ and HO are required for apocynin-induced LR formation in rice.     

Nitric oxide — a retrograde messenger for carbon monoxide signaling in ischemic heart

To examine the intracellular signaling mechanism of NO in ischemic myocardium, isolated working rat hearts were made ischemic for 30 min followed by 30 min of reperfusion. A separate group of hearts were pre-perfused with 3 mM L-arginine in the presence or absence of 650 M of protoporphyrin, a heme oxygenase inhibitor for 10 min prior to ischemia. The release of NO was monitored using an on-line amperometric sensor placed into the right atrium. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. Induction for the expression of heme oxygenase was studied by Northern hybridization. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [³H] myoinositol and [¹⁴C] arachidonic acid. Biopsies were processed to determine the isotopic incorporation into various phosphoinositols as well as phosphatidic acid and diacylglycerol. cGMP was assayed by radioimmunoassay and SOD content was determined by enzymatic analysis. The release of NO was diminished following ischemia and reperfusion and was augmented by L-arginine. L-arginine reduced ischemic/reperfusion injury as evidenced by the enhanced myocardial functional recovery. Protoporphyrin modulated the effects of L-arginine. cGMP, which was remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The NO-mediated augmentation of cGMP was reduced by protoporphyrin suggesting that part of the effects may be mediated by CO generated through the heme oxygenase pathway. Reperfusion of ischemic myocardium resulted in significant accumulation of radiolabeled inositol phosphate, inositol bisphosphate, and inositol triphosphate. Isotopic incorporation of [³H] inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly during reperfusion. Reperfusion of the ischemic heart prelabeled with [¹⁴C] arachidonic acid resulted in modest increases in [¹⁴C] diacylglycerol and [¹⁴C] phosphatidic acid. Pretreatment of the heart with L-arginine significantly reversed this enhanced phosphodiesteratic breakdown during ischemia and early reperfusion. However, at the end of the reperfision the inhibitory effect of L-arginine on the phosphodiesterases seems to be reduced. In L-arginine treated hearts, SOD activity was progressively decreased with the duration of reperfusion time. The results suggests for the first time that NO plays a significant role in transmembrane signaling in the ischemic myocardium. This signaling appears to be on- and off- nature, and linked with SOD content of the tissue. The signaling is transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. Our results also suggest that NO activates heme oxygenase which further stimulates the production of cGMP presumably by CO signaling. Thus, NO not only potentiates cGMP mediated intracellular signaling, it also functions as a retrograde messenger for CO signaling in heart.     

A simulation study of the reaction of human heart to biphasic electrical shocks

Background

This article presents a study, which examines the effects of biphasic electrical shocks on human ventricular tissue. The effects of this type of shock are not yet fully understood. Animal experiments showed the superiority of biphasic shocks over monophasic ones in defibrillation. A mathematical computer simulation can increase the knowledge of human heart behavior.

Methods

The research presented in this article was done with different models representing a three-dimensional wedge of ventricular myocardium. The electrophysiology was described with Priebe-Beuckelmann model. The realistic fiber twist, which is specific to human myocardium was included. Planar electrodes were placed at the ends of the longest side of the virtual cardiac wedge, in a bath medium. They were sources of electrical shocks, which varied in magnitude from 0.1 to 5 V. In a second arrangement ring electrodes were placed directly on myocardium for getting a better view on secondary electrical sources. The electrical reaction of the tissue was generated with a bidomain model.

Results

The reaction of the tissue to the electrical shock was specific to the initial imposed characteristics. Depolarization appeared in the first 5 ms in different locations. A further study of the cardiac tissue behavior revealed, which features influence the response of the considered muscle. It was shown that the time needed by the tissue to be totally depolarized is much shorter when a biphasic shock is applied. Each simulation ended only after complete repolarization was achieved. This created the possibility of gathering information from all states corresponding to one cycle of the cardiac rhythm.

Conclusions

The differences between the reaction of the homogeneous tissue and a tissue, which contains cleavage planes, reveals important aspects of superiority of biphasic pulses. ...     

Cryptococcus neoformans induces IL-8 secretion and CXCL1 expression by human bronchial epithelial cells

Background

Smoking is the most important cause for the development of COPD. Since not all smokers develop COPD, it is obvious that other factors must be involved in disease development. We hypothesize that heme oxygenase-1 (HO-1), a protective enzyme against oxidative stress and inflammation, is insufficiently upregulated in COPD. The effects of HO-1 modulation on cigarette smoke induced inflammation and emphysema were tested in a smoking mouse model.

Methods

Mice were either exposed or sham exposed to cigarette smoke exposure for 20 weeks. Cobalt protoporphyrin or tin protoporphyrin was injected during this period to induce or inhibit HO-1 activity, respectively. Afterwards, emphysema development, levels of inflammatory cells and cytokines, and the presence of B-cell infiltrates in lung tissue were analyzed.

Results

Smoke exposure induced emphysema and increased the numbers of inflammatory cells and numbers of B-cell infiltrates, as well as the levels of inflammatory cytokines in lung tissue. HO-1 modulation had no effects on smoke induced emphysema development, or the increases in neutrophils and macrophages and inflammatory cytokines. Interestingly, HO-1 induction prevented the development of smoke induced B-cell infiltrates and increased the levels of CD4⁺CD25⁺ T cells and Foxp3 positive cells in the lungs. Additionally, the CD4⁺CD25⁺ T cells correlated positively with the number of Foxp3 positive cells in lung tissue, indicating that these cells were regulatory T cells.

Conclusion

These results support the concept that HO-1 expression influences regulatory T cells and indicates that this mechanism is involved in the suppression of smoke induced B-cell infiltrates. The translation of this interaction to human COPD should now be pursued.     

Carbon monoxide reduces neuropathic pain and spinal microglial activation by inhibiting nitric oxide synthesis in mice

Background

Carbon monoxide (CO) synthesized by heme oxygenase 1 (HO-1) exerts antinociceptive effects during inflammation but its role during neuropathic pain remains unknown. Our objective is to investigate the exact contribution of CO derived from HO-1 in the modulation of neuropathic pain and the mechanisms implicated.

Methodology/Principal Findings

We evaluated the antiallodynic and antihyperalgesic effects of CO following sciatic nerve injury in wild type (WT) or inducible nitric oxide synthase knockout (NOS2-KO) mice using two carbon monoxide-releasing molecules (CORM-2 and CORM-3) and an HO-1 inducer (cobalt protoporphyrin IX, CoPP) daily administered from days 10 to 20 after injury. The effects of CORM-2 and CoPP on the expression of HO-1, heme oxygenase 2 (HO-2), neuronal nitric oxide synthase (NOS1) and NOS2 as well as a microglial marker (CD11b/c) were also assessed at day 20 after surgery in WT and NOS2-KO mice. In WT mice, the main neuropathic pain symptoms induced by nerve injury were significantly reduced in a time-dependent manner by treatment with CO-RMs or CoPP. Both CORM-2 and CoPP treatments increased HO-1 expression in WT mice, but only CoPP stimulated HO-1 in NOS2-KO animals. The increased expression of HO-2 induced by nerve injury in WT, but not in NOS2-KO mice, remains unaltered by CORM-2 or CoPP treatments. In contrast, the over-expression of CD11b/c, NOS1 and NOS2 induced by nerve injury in WT, but not in NOS2-KO mice, were significantly decreased by both CORM-2 and CoPP treatments. These data indicate that CO alleviates neuropathic pain through the reduction of spinal microglial activation and NOS1/NOS2 over-expression.

Conclusions/Significance

This study reports that an interaction between the CO and nitric oxide (NO) systems is taking place following sciatic nerve injury and reveals that increasing the exogenous (CO-RMs) or endogenous (CoPP) production of CO may represent a novel strategy for the treatment of neuropathic pain.     

Ambulatory oxygen: why do COPD patients not use their portable systems as prescribed? A qualitative study

Background

In mechanically ventilated preterm infants with respiratory distress syndrome (RDS), exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has not been studied directly on alveolar type II cells (ATII cells), a key cell type responsible for alveolar function and repair.

Objective

The aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis.

Methods

Curosurf^® and Alveofact^® were applied to two ATII cell lines (human A549 and mouse iMATII cells) and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation.

Results

Curosurf^® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact^® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models.

Conclusion

This study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis.     

Comparative study of cardiac electrophysiological effects of atrial natriuretic peptide

The effects of atrial natriuretic peptide (ANP) on action potential characteristics were studied in various (human, rabbit, guinea-pig) atrial and guinea-pig right ventricular papillary muscles. ANP (1–100 nM) did not modify the resting membrane potential nor the maximum rate of depolarization phase (V_max). Up to 10 nM, ANP dose-dependently decreased the action potential amplitude both in guinea-pig atrial and ventricular muscles, but it did not affect this parameter in the other atrial preparations. ANP caused a dose-dependent, marked decrease of action potential duration (APD) in practically every cardiac preparation studied (exception of guinea-pig left atrium). The strongest effect on APD can be observed in human atrial and guinea-pig ventricular fibers. The K⁺ channel blocker 4-aminopyridine (1 mM) and the ATP-dependent K⁺ channel inhibitor glibenclamide (10Nl) prevented the effect of ANP on APD in both ventricular atrial preparations. ANP prevented the appearance of isoprenaline (0.5 M) induced slow AP in K⁺ depolarized myocardium. The present data suggest that ANP may inhibit the slow inward Ca²⁺ channel activity and facilitate the K⁺ channel activity.     

Role of heme oxygenase in heme-mediated inhibition of rat brain Na+−K+-ATPase: Protection by tin-protoporphyrin

Hemoglobin has been shown to inhibit brain Na⁺–K⁺-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na⁺–K⁺-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na⁺–K⁺-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na⁺–K⁺-ATPase. However, in the presence of heme oxygenase, heme inhibited Na⁺–K⁺-ATPase by 75%. Pretreatment of rats with SnCl₂, a known inducer of heme oxygenase, reduced the basal activity of the brain Na⁺–K⁺-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na⁺–K⁺-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage.     

Cardiac reflections and natural vibrations: Force-frequency relation recording system in the stress echo lab

Background

The inherent ability of ventricular myocardium to increase its force of contraction in response to an increase in contraction frequency is known as the cardiac force-frequency relation (FFR). This relation can be easily obtained in the stress echo lab, where the force is computed as the systolic pressure/end-systolic volume index ratio, and measured for increasing heart rates during stress. Ideally, the noninvasive, imaging independent, objective assessment of FFR would greatly enhance its practical appeal.

Objectives

1 – To evaluate the feasibility of the cardiac force measurement by a precordial cutaneous sensor. 2 – To build the curve of force variation as a function of the heart rate. 3 – To compare the standard stress echo results vs. this sensor operator-independent built FFR.

Methods

The transcutaneous force sensor was positioned in the precordial region in 88 consecutive patients referred for exercise, dipyridamole, or pacing stress. The force was measured as the myocardial vibrations amplitude in the isovolumic contraction period. FFR was computed as the curve of force variation as a function of heart rate. Standard echocardiographic FFR measurements were performed.

Results

A consistent FFR was obtained in all patients. Both the sensor built and the echo built FFR identifiy pts with normal or abnormal contractile reserve. The best cut-off value of the sensor built FFR was 15.5 g * 10^-3 (Sensitivity = 0.85, Specificity = 0.77). Sensor built FFR slope and shape mirror pressure/volume relation during stress. This approach is extendable to daily physiological exercise and could be potentially attractive in home monitoring systems.     

Developmental expression of a functional TASK-1 2P domain K+ channel in embryonic chick heart

Background

Background K⁺ channels are the principal determinants of the resting membrane potential (RMP) in cardiac myocytes and thus, influence the magnitude and time course of the action potential (AP).

Methods

RT-PCR and in situ hybridization are used to study the distribution of TASK-1 and whole-cell patch clamp technique is employed to determine the functional expression of TASK-1 in embryonic chick heart.

Results

Chicken TASK-1 was expressed in the early tubular heart, then substantially decreased in the ventricles by embryonic day 5 (ED5), but remained relatively high in ED5 and ED11 atria. Unlike TASK-1, TASK-3 was uniformly expressed in heart at all developmental stages. In situ hybridization studies further revealed that TASK-1 was expressed throughout myocardium at Hamilton-Hamburger stages 11 and 18 (S11 &; S18) heart. In ED11 heart, TASK-1 expression was more restricted to atria. Consistent with TASK-1 expression data, patch clamp studies indicated that there was little TASK-1 current, as measured by the difference currents between pH 8.4 and pH 7.4, in ED5 and ED11 ventricular myocytes. However, TASK-1 current was present in the early embryonic heart and ED11 atrial myocytes. TASK-1 currents were also identified as 3 μM anandamide-sensitive currents. 3 μM anandamide reduced TASK-1 currents by about 58% in ED11 atrial myocytes. Zn²⁺ (100 μM) which selectively inhibits TASK-3 channel at this concentration had no effect on TASK currents. In ED11 ventricle where TASK-1 expression was down-regulated, I_K1 was about 5 times greater than in ED11 atrial myocytes.

Conclusion

Functional TASK-1 channels are differentially expressed in the developing chick heart and TASK-1 channels contribute to background K⁺ conductance in the early tubular embryonic heart and in atria. TASK-1 channels act as a contributor to background K⁺ current to modulate the cardiac excitability in the embryonic heart that expresses little I_K1.     

血红素氧合酶-1/一氧化碳通路参与辛伐他汀抗高血压诱发的大鼠心肌肥厚

为了探讨他汀类药物抑制心肌肥厚的作用机制,本研究应用一氧化氮合酶抑制剂左旋硝基精氨酸[N-nitro-L-arginine, L-NNA,15 mg／(kg·d)]制备大鼠高血压心肌肥厚模型,并分别给予不同剂量辛伐他汀[5或30 mg／(kg·d)进行干预。6周后测大鼠左心室功能、左心室重量指数(left ventricular mass index,LVMI)、心肌脑钠素(brain natriuretic peptide,BNP)含量、心肌羟脯氨酸含量和心肌血红素氧合酶(heme oxygenase,HO)活性。在体外培养的新生大鼠心肌细胞中,观察辛伐他汀对血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)引起的心肌细胞肥大的抑制作用与细胞血红素氧合酶-1(HO-1)表达、HO活性及CO生成间的关系。结果表明,辛伐他汀干预明显减轻L-NNA处理大鼠的心肌肥厚(LVMI值、心肌BNP和羟脯氨酸含量均显著低于单纯L-NNA处理组),改善左心室舒张功能,而且心肌HO活性显著升高。在离体培养的原代乳鼠心肌细胞,辛伐他汀浓度依赖性地抑制Ang Ⅱ引起的细胞肥大(3H-亮氨酸掺入),并相应增加HO-1 mRNA表达、HO活性和CO生成量。应用HO抑制剂锌卟啉能有效抑制辛伐他汀抗Ang Ⅱ诱导的心肌肥大作用。结果提示:辛伐他汀上调HO-1／CO通路是其抗高血压诱发的心肌肥厚的机制之一。     

Ferrochelatase activity inAzospirillum brasilense with reference to the influence of metal cations

Summary Ferrochelatase in membrane preparations fromAzospirillum brasilense displayed an activity of 2.17 mol protoheme formed · h^–1 · mg protein^–1 which is 10-fold greater than previous reports for other bacteria. This ferrochelatase showed an apparentK _m of 20.9 M for Fe²⁺, a pH optimum of 6.0–6.5, and stimulation by oleic or stearic acids. Co²⁺, Cu²⁺ and Zn²⁺ inhibited the incorporation of Fe²⁺ into protoporphyrin IX while Ni² and Mg²⁺ had no effect on protoheme synthesis. Activity with Fe²⁺ and mesoporphyrin IX was less than with protoporphyrin IX but deuteroporphyrin IX produced the highest rate of protoheme synthesis. The membrane fraction containing ferrochelatase activity was found to insert Cu²⁺, Ni²⁺, Zn²⁺ and Co²⁺ enzymatically into protoporphyrin IX to produce metalloporphyrins. Cu²⁺ incorporation into protoporphyrin IX proceeded at a rate greater than with Fe²⁺ and theK _m for Cu²⁺ was 21.9 M.     

Insulin resistance in Chinese patients with type 2 diabetes is associated with C-reactive protein independent of abdominal obesity

Background

Patients with diabetes mellitus (DM) have high risk of heart failure. Whether some of the risk is directly linked to metabolic derangements in the myocardium or whether the risk is primarily caused by coronary artery disease (CAD) and hypertension is incompletely understood. Echocardiographic tissue Doppler imaging was therefore performed in DM patients without significant CAD to examine whether DM per se influenced cardiac function.

Methods

Patients with a left ventricular (LV) ejection fraction (EF) > 35% and without significant CAD, prior myocardial infarction, cardiac pacemaker, atrial fibrillation, or significant valve disease were identified from a tertiary invasive center register. DM patients were matched with controls on age, gender and presence of hypertension.

Results

In total 31 patients with diabetes and 31 controls were included. Mean age was 58 ± 12 years, mean LVEF was 51 ± 7%, and 48% were women. No significant differences were found in LVEF, left atrial end systolic volume, or left ventricular dimensions. The global longitudinal strain was significantly reduced in patients with DM (15.9 ± 2.9 vs. 17.7 ± 2.9, p = 0.03), as were peak longitudinal systolic (S') and early diastolic (E') velocities (5.7 ± 1.1 vs. 6.4 ± 1.1 cm/s, p = 0.02 and 6.1 ± 1.7 vs. 7.7 ± 2.0 cm/s, p = 0.002). In multivariable regression analyses, DM remained significantly associated with impairments of S' and E', respectively.

Conclusion

In patients without significant CAD, DM is associated with an impaired systolic longitudinal LV function and global diastolic dysfunction. These abnormalities are likely to be markers of adverse prognosis.     

Kinetics and Specificity of Feline Leukemia Virus Subgroup C Receptor (FLVCR) Export Function and Its Dependence on Hemopexin

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [⁵⁵Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K_d < 1 pm) compared with albumin (K_d = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7–25 μm) equivalent to that of the iron transport protein transferrin (22–31 μm), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.     

The diagnostic role of glycosaminoglycans in pleural effusions: A pilot study

Background

While modulation of the serotonin transporter (5HTT) has shown to be a risk factor for pulmonary arterial hypertension for almost 40 years, there is a lack of in vivo data about the broad molecular effects of pulmonary inhibition of 5HTT. Previous studies have suggested effects on inflammation, proliferation, and vasoconstriction. The goal of this study was to determine which of these were supported by alterations in gene expression in serotonin transporter knockout mice.

Methods

Eight week old normoxic mice with a 5-HTT knock-out (5HTT-/-) and their heterozygote(5HTT+/-) or wild-type(5HTT+/+) littermates had right ventricular systolic pressure(RVSP) assessed, lungs collected for RNA, pooled, and used in duplicate in Affymetrix array analysis. Representative genes were confirmed by quantitative RT-PCR and western blot.

Results

RVSP was normal in all groups. Only 124 genes were reliably changed between 5HTT-/- and 5HTT+/+ mice. More than half of these were either involved in inflammatory response or muscle function and organization; in addition, some matrix, heme oxygenase, developmental, and energy metabolism genes showed altered expression. Quantitative RT-PCR for examples from each major group confirmed changes seen by array, with an intermediate level in 5HTT +/- mice.

Conclusion

These results for the first time show the in vivo effects of 5HTT knockout in lungs, and show that many of the downstream mechanisms suggested by cell culture and ex vivo experiments are also operational in vivo. This suggests that the effect of 5HTT on pulmonary vascular function arises from its impact on several systems, including vasoreactivity, proliferation, and immune function.