Carbon monoxide is involved in melatonin-enhanced drought resistance in tomato seedlings by enhancing chlorophyll synthesis pathway.

BACKGROUND: Drought is thought to be a major abiotic stress that dramatically limits tomato growth and production. As signal molecule, melatonin (MT) and carbon monoxide (CO) can enhance plant stress resistance. However, the effect and underlying mechanism of CO involving MT-mediated drought resistance in seedling growth remains unknown. In this study, tomato (Solanum lycopersicum L. 'Micro-Tom') seedlings were used to investigate the interaction and mechanism of MT and CO in response to drought stress. RESULTS: The growth of tomato seedlings was inhibited significantly under drought stress. Exogenous MT or CO mitigated the drought-induced impairment in a dose-dependent manner, with the greatest efficiency provided by 100 and 500 µM, respectively. But application of hemoglobin (Hb, a CO scavenger) restrained the positive effects of MT on the growth of tomato seedlings under drought stress. MT and CO treatment promoted chlorophyll a (Chl a) and chlorophyll a (Chl b) accumulations. Under drought stress, the intermediate products of chlorophyll biosynthesis such as protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-Proto IX), potochlorophyllide (Pchlide) and heme were increased by MT or CO, but uroporphyrinogen III (Uro III) content decreased in MT-treated or CO-treated tomato seedlings. Meanwhile, MT or CO up-regulated the expression of chlorophyll and heme synthetic-related genes SlUROD, SlPPOX, SlMGMT, SlFECH, SlPOR, SlChlS, and SlCAO. However, the effects of MT on chlorophyll biosynthesis were almost reversed by Hb. CONCLUSION: The results suggested that MT and CO can alleviate drought stress and facilitate the synthesis of Chl and heme in tomato seedlings. CO played an essential role in MT-enhanced drought resistance via facilitating chlorophyll biosynthesis pathway.

Antimalarial activity and sensitization of chrysosplenetin against artemisinin-resistant genotype Plasmodium berghei K173 potentially via dual-mechanism of maintaining host P-glycoprotein homeostasis mediated by NF-κB p52 or PXR/CAR signaling pathways and regulating heme/haemozoin metabolism.

This study investigated antimalarial efficacy and sensitization of chrysosplenetin against artemisinin-resistant Plasmodium berghei K173 and potential molecular mechanism. Our data indicated a risk of artemisinin resistance because a higher parasitaemia% and lower inhibition% under artemisinin treatment against resistant parasites than those in the sensitive groups were observed. Two non-antimalarial components, verapamil and chrysosplentin, being P-gp inhibitors, possessed a strong efficacy against resistant parasites but it was not the case for Bcrp inhibitor novobiocin. Artemisinin-chrysosplenetin combination improved artemisinin susceptibility of resistant P. berghei. Artemisinin activated intestinal P-gp and Abcb1/Abcg2 expressions and suppressed Bcrp whereas chrysosplenetin reversed them. Resistant parasite infection led to a decreased haemozoin in organs or an increased heme in peripheral bloods compared with the sensitives; however, that in Abcb1-deficient knockout (KO)-resistant mice reversely got increased or decreased versus wild type (WT)-resistant animals. Chrysosplenetin as well as rifampin (nuclear receptor agonist) increased the transcription levels of PXR/CAR while showed a versatile regulation on hepatic and enternal PXR/CAR in WT- or KO-sensitive or -resistant parasites. Oppositely, hepatic and enteric NF-κB p52 mRNA decreased conformably in WT but increased in KO-resistant mice. NF-κB pathway potentially involved in the mechanism of chrysosplenetin on inhibiting P-gp expressions while PXR/CAR play a more complicated role in this mechanism.

Heme protects Pseudomonas aeruginosa and Staphylococcus aureus from calprotectin-induced iron starvation.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that cause severe infections in immunocompromised individuals and patients with cystic fibrosis. Both P. aeruginosa and S. aureus require iron to infect the mammalian host. To obtain iron, these pathogens may rely on siderophore-mediated ferric iron uptake, ferrous iron uptake, or heme uptake at different points during infection. The preferred iron source depends on environmental conditions, including the presence of iron-sequestering host-defense proteins. Here, we investigate how the presence of heme, a highly relevant iron source during infection, affects bacterial responses to iron withholding by the innate immune protein calprotectin (CP). Prior work has shown that P. aeruginosa is starved of iron in the presence of CP. We report that P. aeruginosa upregulates expression of heme uptake machinery in response to CP. Furthermore, we show that heme protects P. aeruginosa from CP-mediated inhibition of iron uptake and iron-starvation responses. We extend our study to a second bacterial pathogen, S. aureus, and demonstrate that CP also inhibits iron uptake and induces iron-starvation responses by this pathogen. Similarly to P. aeruginosa, we show that heme protects S. aureus from CP-mediated inhibition of iron uptake and iron-starvation responses. These findings expand our understanding of microbial responses to iron sequestration by CP and highlight the importance of heme utilization for bacterial adaptation to host iron-withholding strategies.

Heme is involved in the exogenous ALA-promoted growth and antioxidant defense system of cucumber seedlings under salt stress.

A biosynthetic precursor of tetrapyrrol, 5-aminolevulinic acid (ALA), is widely used in agricultural production, as an exogenous regulatory substance that effectively regulates plant growth. Previous studies have shown that heme and chlorophyll accumulate in plants under salt stress, when treated with exogenous ALA. In this study, we explored the regulatory role of heme in plants, by spraying 25 mg L-1 ALA onto the leaves of cucumber seedlings treated with heme synthesis inhibitor (2,2'-dipyridyl, DPD) and heme scavenger (hemopexin, Hx), under 50 mmol L-1 NaCl stress. The results showed that NaCl alone and DPD + Hx treatments to cucumber seedlings subjected to salt stress adversely affected their growth, by decreasing biomass accumulation, root activity, and root morphology. In addition, these treatments induced an increase in membrane lipid oxidation, as well as enhancement of anti-oxidase activities, proline content, and glutamate betaine. However, exogenous ALA application increased the plant growth and root architecture indices under NaCl stress, owing to a lack of heme in the seedlings. In addition, cucumber seedlings treated with DPD and Hx showed inhibition of growth under salt stress, but exogenous ALA effectively improved cucumber seedling growth as well as the physiological characteristics; moreover, the regulation of ALA in plants was weakened when heme synthesis was inhibited. Heme biosynthesis and metabolism genes, HEMH and HO1, which are involved in the ALA metabolic pathway, were upregulated under salinity conditions, when ferrochelatase activity was inhibited. Application of exogenous ALA increased the heme content in the leaves. Thus, exogenous ALA may supplement the substrates for heme synthesis. These results indicated that heme plays a vital role in the response of plants to salinity stress. In conclusion, heme is involved in ALA-mediated alleviation of damage caused to cucumber seedlings and acts as a positive regulator of plant adaption.

Heme induces rapid endothelial barrier dysfunction via the MKK3/p38MAPK axis.

Several studies demonstrate that hemolysis and free heme in circulation cause endothelial barrier dysfunction and are associated with severe pathological conditions such as acute respiratory distress syndrome, acute chest syndrome, and sepsis. However, the precise molecular mechanisms involved in the pathology of heme-induced barrier disruption remain to be elucidated. In this study, we investigated the role of free heme in the endothelial barrier integrity and mechanisms of heme-mediated intracellular signaling of human lung microvascular endothelial cells (HLMVECs). Heme, in a dose-dependent manner, induced a rapid drop in the endothelial barrier integrity of HLMVECs. An investigation into barrier proteins revealed that heme primarily affected the tight junction proteins zona occludens-1, claudin-1, and claudin-5, which were significantly reduced after heme exposure. The p38MAPK/HSP27 pathway, involved in the regulation of endothelial cytoskeleton remodeling, was also significantly altered after heme treatment, both in HLMVECs and mice. By using a knockout (KO) mouse for MKK3, a key regulator of the p38MAPK pathway, we showed that this KO effectively decreased heme-induced endothelial barrier dysfunction. Taken together, our results indicate that targeting the p38MAPK pathway may represent a crucial treatment strategy in alleviating hemolytic diseases.

Effect of hemoglobin on Nile tilapia (Oreochromis niloticus) kidney (NTK) cell line damage.

Bacteria or viral outbreaks can cause tilapia hemorrhage, ensuring considerable volume of hemoglobin (Hb) into the tissue. However, the hemoglobin toxicity on tissue and high doses also effect on tissue this phenomena is still under consideration. Therefore, current study exploited Nile tilapia kidney (NTK) cells to deeply expose the toxic effect of Hb on NTK cells. Toxicity of Hb on NTK cells was determined in terms of cells growth, expression of iron metabolism and inflammation-related genes, consequently examined antioxidant-related enzymes genes expression, intracellular iron and reactive oxygen species (ROS) contents, and apoptosis-related genes expression. The results showed that Hb and heme significantly inhibited NTK cells growth and up-regulated iron metabolism-related genes expression in different degrees. The Hb and heme activated the expression of pro-inflammatory cytokines (TNF-α, tumor necrosis factor-α; IL-1ß, interleukin 1ß; IL-6, interleukin 6), the anti-inflammatory factor (IL-10, interleukin 10) and the chemotactic factors (IL-4, interleukin 4; IL-8, interleukin 8) through NF-κB pathway, meanwhile activated the expression of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). Moreover, the Hb significantly increased intracellular iron and ROS contents while the expression of apoptosis-related genes was significantly activated by both Hb and heme. Current investigation suggested that high oxidative activity of Hb could activate iron metabolism- and inflammation-related genes expression, and increase intracellular iron and ROS levels, lead to up-regulated the expression of apoptosis genes in NTK cells.

A Comprehensive in vitro and in silico Assessment on Inhibition of CYP51B and Ergosterol Biosynthesis by Eugenol in Rhizopus oryzae.

Mucormycosis, also known as Zygomycosis, is a disease caused by invasive fungi, predominantly Rhizopus species belonging to the Order of Mucorales. Seeing from the chemistry perspective, heterocyclic compounds with an "azole" moiety are widely employed as antifungal agent for minimising the effect of mucormycosis as a prescribed treatment. These azoles serve as non-competitive inhibitors of fungal CYP51B by predominantly binding to its heme moiety, rendering its inhibition. However, long-term usage and abuse of azoles as antifungal medicines has resulted in drug resistance among certain fungal pathogens. Hence, there is an unmet need to find alternative therapeutic compounds. In present study, we used various in vitro tests to investigate the antifungal activity of eugenol against R. oryzae/R. arrhizus, including ergosterol quantification to test inhibition of ergosterol production mediated antifungal action. The minimum inhibitory concentration (MIC) value obtained for eugenol was 512 µg/ml with reduced ergosterol concentration of 77.11 ± 3.25% at MIC/2 concentration. Further, the molecular interactions of eugenol with fungal CYP51B were meticulously studied making use of proteomics in silico study including molecular docking and molecular dynamics simulations that showed eugenol to be strongly interacting with heme in an identical fashion to that shown by azole drugs (in this case, clotrimazole was evaluated). This is the first of a kind study showing the simulation study of eugenol with CYP51B of fungi. This inhibition results in ergosterol synthesis and is also studied and compared with keeping clotrimazole as a reference.

Repeated Episodes of Ischemia/Reperfusion Induce Heme-Oxygenase-1 (HO-1) and Anti-Inflammatory Responses and Protects against Chronic Kidney Disease.

Preconditioning episodes of ischemia/reperfusion (IR) induce protection against acute kidney injury (AKI), however their long-term effect still unknown. We evaluated AKI to chronic kidney disease (CKD) transition, after three-mild or three-severe episodes of IR. AKI was induced by single bilateral IR (1IR), or three episodes of IR separated by 10-day intervals (3IR) of mild (20 min) or severe (45 min) ischemia. Sham-operated rats served as controls. During 9-months, the 1IR group (20 or 45 min) developed CKD evidenced by progressive proteinuria and renal fibrosis. In contrast, the long-term adverse effects of AKI were markedly ameliorated in the 3IR group. The acute response in 3IR, contrasted with the 1IR group, that was characterized by an increment in heme oxygenase-1 (HO-1) and an anti-inflammatory response mediated by a NFkB-p65 phosphorylation and IL-6 decrease, together with an increase in TGF-ß, and IL-10 expression, as well as in M2-macrophages. In addition, three episodes of IR downregulated endoplasmic reticulum (ER) stress markers expression, CHOP and BiP. Thus, repeated episodes of IR with 10-day intervals induced long-term renal protection accompanied with HO-1 overexpression and M2-macrophages increase.

GAPDH delivers heme to soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a key component of NO-cGMP signaling in mammals. Although heme must bind in the sGC ß1 subunit (sGCß) for sGC to function, how heme is delivered to sGCß remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGCß involves GAPDH. We utilized an sGCß reporter construct, tetra-Cys sGCß, whose heme insertion can be followed by fluorescence quenching in live cells, assessed how lowering cell GAPDH expression impacts heme delivery, and examined whether expressing WT GAPDH or a GAPDH variant defective in heme binding recovers heme delivery. We also studied interaction between GAPDH and sGCß in cells and their complex formation and potential heme transfer using purified proteins. We found that heme delivery to apo-sGCß correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme, that apo-sGCß associates with GAPDH in cells and dissociates when heme binds sGCß, and that the purified GAPDH-heme complex binds to apo-sGCß and transfers its heme to sGCß. On the basis of these results, we propose a model where GAPDH obtains mitochondrial heme and then forms a complex with apo-sGCß to accomplish heme delivery to sGCß. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.

Heme induces significant neutrophil adhesion in vitro via an NFκB and reactive oxygen species-dependent pathway.

Intravascular hemolysis, a major manifestation of sickle cell disease (SCD) and other diseases, incurs the release of hemoglobin and heme from red blood cells, in turn triggering inflammatory processes. This study investigated the in vitro effects of heme, a major inflammatory DAMP, on the adhesive properties of isolated human neutrophils. Heme (20 and 50 µM) significantly increased the adhesion of neutrophils to fibronectin and to recombinant ICAM-1, under static conditions, even more efficiently than the potent pro-inflammatory cytokine, tumor necrosis factor-α (TNF); a microfluidic assay confirmed that heme stimulated neutrophil adhesion under conditions of shear stress. Heme-induced neutrophil adhesion was associated with the increased activities, but not expressions, of the Mac-1 and LFA-1 integrin subunits, CD11b and CD11a, on the cell surface. Notably, heme (50 µM) significantly induced NFκB translocation in neutrophils, and inhibition of NFκB activity with the BAY11-7082 molecule abolished heme-induced cell adhesion to fibronectin and significantly decreased CD11a activity. Flow cytometric analysis demonstrated major reactive oxygen species (ROS) generation in neutrophils following heme stimulation that could be inhibited by the antioxidant, α-tocopherol, and by BAY11-7082. Furthermore, co-incubation with α-tocopherol abrogated both heme-stimulated neutrophil adhesion and CD11a/CD11b activation. Thus, our data indicate that heme, at clinically relevant concentrations, is a potent activator of neutrophil adhesion, increasing the ligand affinity of the ß2 integrins via a mechanism that may be partially mediated by an NFkB-dependent pathway and the generation of ROS. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in SCD vaso-occlusion and other vascular inflammatory processes, our findings provide further evidence that cell-free heme is a major therapeutic target in the hemolytic diseases.

Heme ameliorates dextran sodium sulfate-induced colitis through providing intestinal macrophages with noninflammatory profiles.

The local environment is crucial for shaping the identities of tissue-resident macrophages (Mϕs). When hemorrhage occurs in damaged tissues, hemoglobin induces differentiation of anti-inflammatory Mϕs with reparative function. Mucosal bleeding is one of the pathological features of inflammatory bowel diseases. However, the heme-mediated mechanism modulating activation of intestinal innate immune cells remains poorly understood. Here, we show that heme regulates gut homeostasis through induction of Spi-C in intestinal CX3CR1high Mϕs. Intestinal CX3CR1high Mϕs highly expressed Spi-C in a heme-dependent manner, and myeloid lineage-specific Spic-deficient (Lyz2-cre; Spicflox/flox ) mice showed severe intestinal inflammation with an increased number of Th17 cells during dextran sodium sulfate-induced colitis. Spi-C down-regulated the expression of a subset of Toll-like receptor (TLR)-inducible genes in intestinal CX3CR1high Mϕs to prevent colitis. LPS-induced production of IL-6 and IL-1α, but not IL-10 and TNF-α, by large intestinal Mϕs from Lyz2-cre; Spicflox/flox mice was markedly enhanced. The interaction of Spi-C with IRF5 was linked to disruption of the IRF5-NF-κB p65 complex formation, thereby abrogating recruitment of IRF5 and NF-κB p65 to the Il6 and Il1a promoters. Collectively, these results demonstrate that heme-mediated Spi-C is a key molecule for the noninflammatory signature of intestinal Mϕs by suppressing the induction of a subset of TLR-inducible genes through binding to IRF5.

Heme inhibits the activity of a c-di-GMP phosphodiesterase in Vibrio cholerae.

Heme, a complex of iron and protoporphyrin IX, plays an essential role in numerous biological processes including oxygen transport, oxygen storage, and electron transfer. The role of heme as a prosthetic group in bacterial hemoprotein gas sensors, which utilize heme as a cofactor for the binding of diatomic gas molecules, has been well studied. Less well known is the role of protein sensors of heme. In this report, we characterize the heme binding properties of a phosphodiesterase, CdpA, from Vibrio cholerae. We demonstrate that the N-terminal domain of CdpA is a NosP domain capable of heme binding, which consequently inhibits the c-di-GMP hydrolysis activity of the C-terminal phosphodiesterase domain. Further evidence for CdpA as a heme responsive sensor is supported by a relatively fast rate of heme dissociation. This study provides insight into an emerging class of heme-responsive sensor proteins.

Actovegin® reduces PMA-induced inflammation on human cells.

PURPOSE: The effect of Actovegin® was investigated on PMA- and LPS-induced human peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs (1 × 106 cells/ml) from five blood donors (2 f, 3 m; 45-55 years) were grown in medium and exposed to Actovegin® in the presence or absence of PMA or LPS. Supernatants were collected to assess the concentration of cytokines (TNF-α, IL-1beta, IL-6 and IL-10). The reactive oxygen species (ROS) were assessed by a ROS-GloTM H2O2 assay. RESULTS: Stimulation of cells by PMA or LPS (without Actovegin®) significantly increased the secretion of IL-1beta, IL-6, IL-10 and TNF-α from PBMCs, compared to controls. Pre-treatment of cells with Actovegin® (1, 5, 25, 125 µg/ml) plus PMA significantly decreased the secretion of IL-1beta from PBMCs, compared to controls (PMA without Actovegin®). In contrast, addition of Actovegin® (1, 5, 25, 125 and 250 µg/ml) plus LPS did not alter the IL-1beta production, compared to controls (LPS without Actovegin®). TNF-α, IL-6 and IL-10 do not contribute to the reduction of inflammatory reactions with Actovegin®. CONCLUSIONS: Actovegin® can reduce the PMA-induced IL-1beta release and the ROS production from PBMCs. These findings may help to explain the clinically known positive effects of Actovegin® on athletic injuries with inflammatory responses (e.g., muscle injuries, tendinopathies).

Update on the Role of Actovegin in Musculoskeletal Medicine: A Review of the Past 10 Years.

BACKGROUND: Actovegin is a biological drug with a controversial history of use in the treatment of sports injuries during the past 60 years. Particular concerns have been raised about its ergogenic potential to enhance performance, but some of these have been based on little more than anecdote. OBJECTIVES: In this article, we review the most recent scientific evidence to determine the clinical efficacy, safety profile, and legal status of Actovegin. METHODS: We considered all studies directly commenting on experience with Actovegin use as the primary intervention within the past 10 years. Outcomes included mechanisms of action, clinical efficacy in enhancing muscle repair, any report of safety issues, and any evidence for ergogenic effect. RESULTS: Our database search returned 212 articles, abstracts were screened, and after inclusion/exclusion criteria were applied, 25 articles were considered: Publications included 11 primary research articles (7 in vitro studies and 4 clinical trials), 8 review articles, 5 editorials, and a single case report. CONCLUSIONS: Current literature is still yet to define the active compound(s) of Actovegin, but suggests that it shows antioxidant and antiapoptotic properties, and may also upregulate macrophage responses central to muscle repair. Clinical efficacy was supported by one new original research article, and the use of Actovegin to treat muscle injuries remains safe and supported. Two articles argued the ergogenic effect of Actovegin, but in vitro findings did not to translate to the outcomes of a clinical trial. An adequate and meaningful scientific approach remains difficult in a field where there is immense pressure to deliver cutting-edge therapies.

Heme-Induced Oxidation of Cysteine Groups of Myofilament Proteins Leads to Contractile Dysfunction of Permeabilized Human Skeletal Muscle Fibres.

Heme released from red blood cells targets a number of cell components including the cytoskeleton. The purpose of the present study was to determine the impact of free heme (20-300 µM) on human skeletal muscle fibres made available during orthopedic surgery. Isometric force production and oxidative protein modifications were monitored in permeabilized skeletal muscle fibre segments. A single heme exposure (20 µM) to muscle fibres decreased Ca2+-activated maximal (active) force (Fo) by about 50% and evoked an approximately 3-fold increase in Ca2+-independent (passive) force (Fpassive). Oxidation of sulfhydryl (SH) groups was detected in structural proteins (e.g., nebulin, α-actinin, meromyosin 2) and in contractile proteins (e.g., myosin heavy chain and myosin-binding protein C) as well as in titin in the presence of 300 µM heme. This SH oxidation was not reversed by dithiothreitol (50 mM). Sulfenic acid (SOH) formation was also detected in the structural proteins (nebulin, α-actinin, meromyosin). Heme effects on SH oxidation and SOH formation were prevented by hemopexin (Hpx) and α1-microglobulin (A1M). These data suggest that free heme has a significant impact on human skeletal muscle fibres, whereby oxidative alterations in structural and contractile proteins limit contractile function. This may explain and or contribute to the weakness and increase of skeletal muscle stiffness in chronic heart failure, rhabdomyolysis, and other hemolytic diseases. Therefore, therapeutic use of Hpx and A1M supplementation might be effective in preventing heme-induced skeletal muscle alterations.

Intramuscular Injection of Combined Calf Blood Compound (CFC) and Homeopathic Drug Tr14 Accelerates Muscle Regeneration In Vivo.

Skeletal muscle injuries in competitive sports cause lengthy absences of athletes from tournaments. This is of tremendous competitive and economic relevance for both the athletes and their respective clubs. Therapy for structural muscle lesions aims to promote regeneration and fast-track return-to-play. A common clinical treatment strategy for muscle injuries is the intramuscular injection of calf blood compound and the homeopathic drug, Tr14. Although the combination of these two agents was reported to reduce recovery time, the regulatory mechanism whereby this occurs remains unknown. In this in vivo study, we selected a rat model of mechanical muscle injury to investigate the effect of this combination therapy on muscle regeneration. Gene expression analysis and histological images revealed that this combined intramuscular injection for muscle lesions can enhance the expression of pro-myogenic genes and proteins and accelerate muscle regeneration. These findings are novel and depict the positive effects of calf blood compound and the homeopathic drug, Tr14, which are utilized in the field of Sports medicine.

Cardiac expression of HMOX1 and PGF in sickle cell mice and haem-treated wild type mice dominates organ expression profiles via Nrf2 (Nfe2l2).

Haemolysis is a major feature of sickle cell disease (SCD) that contributes to organ damage. It is well established that haem, a product of haemolysis, induces expression of the enzyme that degrades it, haem oxygenase-1 (HMOX1). We have also shown that haem induces expression of placental growth factor (PGF), but the organ specificity of these responses has not been well-defined. As expected, we found high level expression of Hmox1 and Pgf transcripts in the reticuloendothelial system organs of transgenic sickle cell mice, but surprisingly strong expression in the heart (P < 0·0001). This pattern was largely replicated in wild type mice by intravenous injection of exogenous haem. In the heart, haem induced unexpectedly strong mRNA responses for Hmox1 (18-fold), Pgf (4-fold), and the haem transporter Slc48a1 (also termed Hrg1; 2·4-fold). This was comparable to the liver, the principal known haem-detoxifying organ. The NFE2L2 (also termed NRF2) transcription factor mediated much of the haem induction of Hmox1 and Hrg1 in all organs, but less so for Pgf. Our results indicate that the heart expresses haem response pathway genes at surprisingly high basal levels and shares with the liver a similar transcriptional response to circulating haem. The role of the heart in haem response should be investigated further.

Sex differences in vascular reactivity in mesenteric arteries from a mouse model of acute intermittent porphyria.

BACKGROUND AND AIMS: Acute intermittent porphyria (AIP) results from a partial deficiency of porphobilinogen deaminase (PBGD). Symptomatic AIP patients, most of whom are women, experience acute attacks characterized by severe abdominal pain and abrupt increases in blood pressure. Here, we characterized the reactivity of mesenteric arteries from male and female AIP mice with ~30% of normal PBGD activity and wild type C57BL/6 mice. METHODS: An acute porphyric attack was induced in AIP mice by treatment with phenobarbital. Vascular responses to K+, phenylephrine (PE), acetylcholine (ACh), and hemin were determined (Wire Multi Myograph). RESULTS: Maximal contraction to PE was increased in arteries from male and female AIP mice (p < .05) during an induced attack of acute porphyria. Female AIP arteries had increased sensitivity to PE (p < .05) even after nitric oxide (NO) blockade with Nω-nitro-L-arginine methyl ester (L-NAME) (p < .05). Maximal relaxation to ACh was similar in males and females with lower sensitivity in female AIP arteries (p < .05). Hemin induced greater relaxation in AIP arteries in both males and females (p < .05). SUMMARY/CONCLUSIONS: Sex differences in this AIP mouse model include a pro-contractile response in females. These alterations may contribute to the increased blood pressure during an acute attack and provide a novel mechanism of action whereby heme ameliorates the attacks.

Calf Blood Compound (CFC) and Homeopathic Drug Induce Differentiation of Primary Human Skeletal Muscle Cells.

The use of injections to treat structural muscle injuries is controversially discussed. In our controlled in vitro study, we investigated the biological impact of Actovegin and Traumeel alone and in combination on primary human skeletal muscle cells. Cells were characterized by immunofluorescence staining for myogenic factor 5 (Myf5) and MyoD, and cultured with or without Actovegin and / or Traumeel. The effects of these agents were assayed by cell viability and gene expression of the specific markers MyoD, Myf5, neural adhesion molecule (NCAM), and CD31. Myotube formation was determined by myosin staining. Neither Actovegin nor Traumeel showed toxic effects or influenced cell viability significantly. High volumes of Actovegin down-regulated gene expression of NCAM after 3 days but had no effect on MyoD, Myf5, and CD31 gene expression. High volumes of Traumeel inhibited MyoD gene expression after 3 days, whereas after 7 days MyoD expression was significantly up-regulated. The combination of both agents did not significantly influence cell viability or gene expression. This is the first study demonstrating that Actovegin and Traumeel potentially modulate human skeletal muscle cells. The relevance of these in vitro findings has to be highlighted in further in vivo studies.

Outer Membrane Vesicle Proteome of Porphyromonas gingivalis Is Differentially Modulated Relative to the Outer Membrane in Response to Heme Availability.

Porphyromonas gingivalis is an anaerobic, Gram-negative oral pathogen associated with chronic periodontitis. P. gingivalis has an obligate requirement for heme, which it obtains from the host. Heme availability has been linked to disease initiation and progression. In this study we used continuous culture of the bacterium to determine the effect of heme limitation and excess on the P. gingivalis proteome. Four biological replicates of whole cell lysate (WCL) and outer membrane vesicle (OMV) samples were digested with trypsin and analyzed by tandem mass spectrometry and MaxQuant label-free quantification. In total, 1211 proteins were quantified, with 108 and 49 proteins significantly changing in abundance more than 1.5-fold ( p < 0.05) in the WCLs and OMVs, respectively. The proteins most upregulated in response to heme limitation were those involved in binding and transporting heme, whereas the four proteins most upregulated under the heme-excess condition constitute a putative heme efflux system. In general, the protein abundance ratios obtained for OMVs and WCLs agreed, indicating that changes to the OM protein composition are passed onto OMVs; however, 16 proteins were preferentially packaged into OMVs under one condition more than the other. In particular, moonlighting cytoplasmic proteins were preferentially associated with OMVs under heme excess.