A theoretical study on the structure-activity relationships of metabolites of folates as antioxidants and its implications for rational design of antioxidants

To elucidate the structure-activity relationships of metabolites of folates as antioxidants, the O-H bond dissociation enthalpies (BDEs) and ionization potentials (IPs) for these compounds were calculated by density functional theory (DFT) on B3LYP/6-31+G(,3pd) level. Accordingly, the antioxidant activity difference for metabolites of folates can be elucidated by O-H BDE and IP values and can be further explained in terms of electronic effect and intramolecular hydrogen bond effect of substituents. Furthermore, the potential of the active center of metabolites of folates, 4-hydroxypyrimidine (4-HP), as lead antioxidant, was evaluated by comparing the BDEs and IPs of 4-HP with those of 5-hydroxypyrimidine (5-HP). It was revealed that 4-HP and 5-HP held identical IPs, but the O-H BDE of the former was 22.84 kcal/mol higher than that of the latter, which meant 4-HP was inert in H-atom donation. Nevertheless, the O-H BDE of 4-HP was very sensitive to the substituents, which made NH2-derivatives of 4-HP very active as antioxidants. Therefore, 4-HP is also a potential lead antioxidant and deserves attention in rational design of antioxidants.     

Betacyanin-Decolorizing Enzymes from Phytolacca americana

Betacyanin-decolorizing enzymes (BDEs) in Phytolacca americanawere partially purified and characterized. Acidic enzymes, BDE₁and BDE₂, and basic enzymes, BDE₃ and BDE₄, were partially purifiedby column chromatography on DEAE-Sepharose CL-6B and CM-SepharoseCL-6B. These enzymes degraded betanin to its aglycon (betanidin)and then to a product with a peak of absorbance at 485 nm. Theoptimum pH values were 5.0 for BDE₁, 3.5 for BDE₂, and 5.5 forBDE₃ and BDE₄. BDE activity was detected in extracts of theleaf, stem, seed, and root. The highest specific activity ofBDE₂ was found in the red epidermis of the stem. The activityof BDE₂ was inhibited by NH₄NO₃, KNO₃, KC1, and NaCl, but theactivities of the other BDEs were not inhibited. These resultssuggest that BDEs other than BDE₂ control the degradation ofbetacyanin in the intracellular space. (Received March 27, 1989; Accepted December 8, 1989)     

Probing the antioxidant potential of phloretin and phlorizin through a computational investigation

The structures and energetics of two dihydrochalcones (phloretin and its glycoside phlorizin) were examined with density functional theory, using the B3LYP, M06-2X, and LC-ω PBE functionals with both the 6-311G(d,p) and 6-311 + G(d,p) basis sets. Properties connected to antioxidant activity, i.e., bond dissociation enthalpies (BDEs) for OH groups and ionization potentials (IPs), were computed in a variety of environments including the gas-phase, n-hexane, ethanol, methanol, and water. The smallest BDEs among the four OH groups for phloretin (three for phlorizin) were determined (using B3LYP/6-311 + G(d,p) in water) to be 79.36 kcal/mol for phloretin and 79.98 kcal/mol for phlorizin while the IPs (at the same level of theory) were obtained as 139.48 and 138.98 kcal/mol, respectively. By comparing with known antioxidants, these values for the BDEs indicate both phloretin and phlorizin show promise for antioxidant activity. In addition, the presence of the sugar moiety has a moderate (0-6 kcal/mol depending on functional) effect on the BDEs for all OH groups. Interestingly, the BDEs suggest that (depending on the functional chosen) the sugar moiety can lead to an increase, decrease, or no change in the antioxidant activity. Therefore, further experimental tests are encouraged to understand the substituent effect on the BDEs for phloretin and to help determine the most appropriate functional to probe BDEs for dihydrochalcones.     

Metabolism and disposition of N-(2-cyanoethyl)amphetamine (fenproporex) and amphetamine: study in the rat brain

N-(2-Cyanoethyl)amphetamine (fenproporex, CE-AM) is a clinically used anorexiant claimed to be devoid of the stimulant properties associated with amphetamine (AM). This claim was inconsistent with preliminary studies conducted in our laboratories which indicated that CE-AM is metabolically dealkylated to AM to a considerable extent in the rat. Concentration-time profiles of CE-AM and its metabolites AM and 4-hydroxyamphetamine (4-OH-AM) in the rat brain were constructed after administration of CE-AM. Analyses of CE-AM, AM, and 4-OH-AM were performed by gas chromatography with electron-capture detection using pentafluorobenzoyl chloride (under aqueous conditions) as the derivatizing reagent. The half-life (t1/2) and the maximum concentration (Cmax) of AM after administration of CE-AM were calculated to be 2.04 and 0.56 times the respective t1/2 and Cmax obtained after an equimolar dose of AM. Significant differences in the profiles of 4-OH-AM were also observed. The Cmax of 4-OH-AM in rat brain after administration of CE-AM was nearly 4 times higher and the tmax (time at which concentration is maximum) 4 times lower than the respective Cmax and tmax values of 4-OH-AM observed after an equimolar dose of AM.     

Effects of structure on alpha C-H bond enthalpies of amino acid residues: relevance to H transfers in enzyme mechanisms and in protein oxidation.

The bond dissociation enthalpies (BDE) of all of the amino acid residues, modeled by HC(O)NHCH(R)C(O)NH(2) (PH(res)), were determined at the B3LYP/6-31G//B3LYP/6-31G level, coupled with isodesmic reactions. The results for neutral side chains with phi, psi angles approximately 180 degrees, approximately 180 degrees in ascending order, to an expected accuracy of +/-10 kJ mol(-)(1), are Asn 326; cystine 330; Asp 332; Gln 334; Trp 337; Arg 340; Lys 340; Met 343; His 344; Phe 344; Tyr 344; Leu 344; Ala 345; Cys 346; Ser 349; Gly 350; Ile 351; Val 352; Glu 354; Thr 357; Pro-cis 358; Pro-trans 369. BDEs calculated at the ROMP2/6-31G//B3LYP/6-31G level exhibit the same trends but are approximately 7 kJ mol(-)(1) higher. All BDEs are smaller than those of typical secondary or tertiary C-H bonds due to the phenomenon of captodative stabilization. The stabilization is reduced by changes in the phi,psi angles. As a result the BDEs increase by about 10 kJ mol(-)(1) in beta-sheet and 40 kJ mol(-)(1) in alpha-helical environments, respectively. In effect the alpha C-H BDEs can be "tuned" from about 345 to 400 kJ mol(-)(1) by adjusting the local environment. Some very significant effects of this are seen in the current literature on H-transfer processes in enzyme mechanisms and in oxidative damage to proteins. These observations are discussed in terms of the findings of the present study.     

In vivo effects of interferon-gamma and indomethacin on murine alveolar macrophage activity

This study determined the effects of treatment with recombinant interferon-gamma (rIFN-gamma) and the cyclooxygenase inhibitor, indomethacin (INDO), on alveolar macrophage (AM) immune function in AKR/J mice. Bactericidal activity, interleukin 1 (IL1) synthesis and antigen presentation by AM were enhanced at 24 hr after a single intravenous injection with 5 X 10(4) U of rIFN-gamma. Concomitant treatment with 2 mg INDO/kg given subcutaneously did not further enhance the effects of a single injection of rIFN-gamma, even though the prostaglandin E2 (PGE2) concentrations in lung airways were reduced by 50%. These results suggest that the stimulatory effects of rIFN-gamma on AM are not altered by blocking potentially immunosuppressive cyclooxygenase metabolites such as PGE2 with INDO. Mice given three consecutive daily intravenous injections of 5 X 10(4) U of rIFN-gamma had suppressed AM bactericidal activity and IL1 synthesis, while PGE2 concentrations in the lungs were increased. Concomitant treatment with INDO prevented suppression of these AM functions and elevation of PGE2 concentrations in the lungs. Therefore, it appears that INDO can prevent suppression of AM activity induced by multiple injections of rIFN-gamma and this effect may be by blockage of PGE2 synthesis or other cyclooxygenase-derived products.     

Effects of human alveolar macrophages on the induction of lymphokine (IL 2)-activated killer cells

Normal human alveolar macrophages (AM) significantly and reproducibly suppress induction of IL 2-activated killer (LAK) cell activity against allogeneic Burkitt's lymphoma (Daudi) cells. Incubation of purified peripheral blood lymphocytes for 4 days with autologous AM and 1 U/ml of IL 2 resulted in AM-mediated suppression of LAK activity, whereas peripheral blood monocytes isolated freshly by centrifugal elutriation from the same donor potentiated induction of LAK activity by IL 2. The suppression of LAK cell induction by human AM was dependent on the density of AM added to the lymphocyte cultures. Recombinant IFN-gamma did not affect AM-mediated suppression of LAK cell induction by IL 2. Both AM and monocytes stimulated with lipopolysaccharide markedly suppressed LAK cell induction by IL 2. AM-mediated down-regulation was seen only when AM were added immediately after the start of incubation of lymphocytes with IL 2; AM potentiated LAK activity when added 1 day later. Similar AM-mediated suppression of LAK cell induction was observed with four lines of allogeneic lung cancer cells as targets for LAK activity. These results indicate that AM may be important in regulation of in situ induction of LAK activity in the lung.     

Superoxide- and 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activities of soyasaponin beta g related to gallic acid.

Soyasaponin beta p g at 1 mm had 8% scavenging activity for O2-, and 25 microM beta g scavenged 20.9% for the DPPH radical (IC50: 63.8 microM). In the soyasaponin beta g-gallic acid system, synerigistic effects were observed at a low level of gallic acid concentration. The spin density distribution calculated by the MNDO/AM1 method showed unpaired electron localization on the carbons at C-4 and C-6, and on the ketone group at C-4 of the DDMP moiety. Furthermore, for soyasaponin beta g, the MNDO/AM1 method gave an ionization potential of 8.38 eV, electron affinity of 1.16 eV and Mulliken electronegativity of 4.77 eV. Based on this evidence, the synergistic antiradical effects of the soyasaponin beta g-gallic acid system are assumed to involve two-electron reduction from gallic acid.     

Polyhydroxylated 4-thiaflavans as multipotent antioxidants: protective effect on oxidative DNA damage in vitro

Hydroxylated 4-thiaflavans, possessing the antioxidant features of catechol containing flavonoids and/or tocopherols, were evaluated as protective agents against oxidation damage induced in herring sperm DNA by cumene hydroperoxide (CumOOH) or by the glutathione/ferric ion (GSH/Fe(3+)) system. Our data indicate that the effective protection exerted by some of the tested compounds is overall higher than those provided by catechin and alpha-tocopherol, which might be attributed both to the scavenging properties and chelation of Fe(2+) ions.     

Immune responses during human Schistosoma mansoni. XVII. Recognition by monoclonal anti-idiotypic antibodies of several idiotopes on a monoclonal anti-soluble schistosomal egg antigen antibody and anti-soluble schistosomal egg antigen antibodies from patients with different clinical forms of infection

A monoclonal human anti-soluble schistosomal egg Ag(SEA) antibody (E5) that stimulates anti-Id T cells and is idiotypically represented in pools of immunoaffinity-purified human anti-SEA antibodies from chronic, generally asymptomatic, intestinal (INT) patients (AM1 and AM5) was used to raise several monoclonal anti-Id: 1C2, 1C6, 4A8, 4F9, and 2A7. Cross-inhibition between these anti-Id identified distinct idiotopes on E5. Anti-SEA preparations from schistosomiasis patients (AM1, AM5, and others) were tested for their inhibition of the E5/monoclonal anti-Id reactions, in competitive ELISA. In either the E5/4A8 or E5/1C6 ELISA system, anti-SEA from INT (AM1 or AM5) or hepatointestinal (HI) (AM7) patients were able to inhibit these reactions. However, anti-SEA antibodies from acute (AM9) or hepatosplenic (HS) (AM3 or AM8) patients did not express Id that were inhibitory in these systems. These results suggest that a relatively high proportion of INT and HI anti-SEA antibodies express a dominant cross-reactive idiotope (CRI) recognized by 1C6/4A8. This CRI is also easily detected in plasmas from individual INT patients. Anti-Id 1C2 reacted strongly with an Id in AM1, AM5, or AM7, but one which also occurred, to a lesser extent, in AM3, AM8, and AM9. Monoclonal anti-Id 4F9 and 2A7 reacted weakly with idiotopes expressed by antibodies from all patients, regardless of the clinical form of their infection. These observations indicate that anti-SEA antibodies from INT and HI, but not acute or HS patients express dominant, CRI that are identified by 1C6, 4A8, or 1C2 and are also expressed on the INT-derived anti-SEA mAb E5.     

Stretch and CO2 modulate the inflammatory response of alveolar macrophages through independent changes in metabolic activity

Ventilatory-induced strain can exacerbate acute lung injury (ALI). Current ventilation strategies favour low tidal volumes and high end-expiratory volumes to 'rest' the lung, but can lead to an increase in CO2. Alveolar macrophages (AM) play a pivotal role in ALI through the release of inflammatory mediators. The effect of physical strain and CO2 on the release of pro-inflammatory mediators was examined in isolated rat AM. AM were cultured on IgG-coated silastic membranes with or without lipopolysaccharide (LPS) and 5% or 20% CO2 and subjected to a repetitive sinusoidal mechanical strain (30%, 60 cycles/min) for 4 h. Cell viability and metabolic activity were assessed. In both the presence and absence of LPS, physical strain increased metabolic activity by approximately 5%, while 20% CO2 decreased metabolic activity by approximately 40%. Twenty per cent CO2 decreased TNF-alpha secretion by approximately 45%, without affecting cell viability. Physical strain enhanced LPS-induced secretion of TNF-alpha by 1.5%, but not IL-6 or CINC-1. Hence, the effects of both CO2 and physical strain are mediated independently through changes in AM metabolic activity. Physical strain is not a major determinant of TNF-alpha, IL-6 or CINC-1 in AM. Our results confirm that high CO2 can lessen the TNF-alpha inflammatory response of AM.     

Superoxide- and 1,1-Diphenyl-2-picrylhydrazyl Radical-scavenging Activities of Soyasaponin β g Related to Gallic Acid

Soyasaponin β g at 1 mM had 8% scavenging activity for O₂ ^-, and 25 μM β scavenged 20.9% for the DPPH radical (IC₅₀: 63.8 μM). In the soyasaponin β g-gallic acid system, synerigistic effects were observed at a low level of gallic acid concentration. The spin density distribution calculated by the MNDO/AM1 method showed unpaired electron localization on the carbons at C-4 and C-6, and on the ketone group at C-4 of the DDMP moiety. Furthermore, for soyasaponin β g, the MNDO/AM1 method gave an ionization potential of 8.38 eV, electron affinity of 1.16 eV and Mulliken electronegativity of 4.77 eV. Based on this evidence, the synergistic antiradical effects of the soyasaponin β g-gallic acid system are assumed to involve two-electron reduction from gallic acid.     

Impaired functional activity of alveolar macrophages from GM-CSF-deficient mice

We hypothesized that pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically involved in determining the functional capabilities of alveolar macrophages (AM) for host defense. To test this hypothesis, cells were collected by lung lavage from GM-CSF mutant mice [GM(-/-)] and C57BL/6 wild-type mice. GM(-/-) mice yielded almost 4-fold more AM than wild-type mice. The percentage of cells positive for the beta(2)-integrins CD11a and CD11c was reduced significantly in GM(-/-) AM compared with wild-type cells, whereas expression of CD11b was similar in the two groups. The phagocytic activity of GM(-/-) AM for FITC-labeled microspheres was impaired significantly compared with that of wild-type AM both in vitro and in vivo (after intratracheal inoculation with FITC-labeled beads). Stimulated secretion of tumor necrosis factor-alpha (TNF-alpha) and leukotrienes by AM from the GM(-/-) mice was greatly reduced compared with wild-type AM, whereas secretion of monocyte chemoattractant protein-1 was increased. Transgenic expression of GM-CSF exclusively in the lungs of GM(-/-) mice resulted in AM with normal or supranormal expression of CD11a and CD11c, phagocytic activity, and TNF-alpha secretion. Thus, in the absence of GM-CSF, AM functional capabilities for host defense were significantly impaired but were restored by lung-specific expression of GM-CSF.     

Synergistic activation of the human adrenomedullin gene promoter by Sp1 and AP-2alpha

Adrenomedullin (AM) is a potent vasodilator peptide, which is ubiquitously expressed and has various biological actions, such as proliferative action and anti-oxidative stress action. AM expression is induced by various stresses, such as hypoxia and inflammatory cytokines, and during cell differentiation. The human AM gene promoter region (-70/-29) contains binding sites for stimulatory protein 1 (Sp1) and activator protein-2alpha (AP-2alpha), and has been shown to be important for the AM gene expression during cell differentiation to macrophages or adipocytes. We here show that Sp1 and AP-2alpha synergistically activate the AM gene promoter. Co-transfection of the reporter plasmid containing the AM promoter region (-103/-29) with Sp1 and AP-2alpha expression plasmids showed that Sp1 and AP-2alpha synergistically increased the promoter activity in HeLa cells. Sp1 or AP-2alpha alone caused only small increases in the promoter activity. EMSA showed that Sp1 bound to the promoter region (-70/-29), whereas AP-2alpha bound to a more upstream promoter region (-103/-71). Thus, the synergistic activation of the human AM gene promoter by Sp1 and AP-2alpha may be mediated by the binding of Sp1 to the promoter region (-70/-29) and the interaction with AP-2alpha, which binds to the promoter region (-103/-71).     

Acute, same-day effects of antecedent exercise on counterregulatory responses to subsequent hypoglycemia in type 1 diabetes mellitus

Exercise-induced hypoglycemia can occur within hours after exercise in type 1 diabetes mellitus (T1DM) patients. This study tested the hypothesis that an acute exercise bout causes (within hours) blunted autonomic and metabolic responses to subsequent hypoglycemia in patients with T1DM. Twelve T1DM patients (3 W/9 M) were studied during a single-step, 2-h hyperinsulinemic (572 +/- 4 pmol/l) hypoglycemic (2.8 +/- 0.1 mmol/l) clamp 2 h after either a hyperinsulinemic euglycemic (AM EUG) or hypoglycemic clamp (AM HYPO) or after sitting in a chair with basal insulin infusion (AM CON) or 90 min of moderate-intensity exercise (50% Vo(2 max), AM EX). Both AM HYPO and AM EX significantly blunted epinephrine responses and muscle sympathetic nerve activity responses to subsequent hypoglycemia compared with both control groups. Endogenous glucose production was significantly lower and the exogenous glucose infusion rate needed to maintain the hypoglycemic level was significantly greater during subsequent hypoglycemia in AM EX vs. CON. Rate of glucose disposal (Rd) was significantly reduced following AM HYPO. In summary, within 2.5 h, both moderate-intensity AM EX and AM HYPO blunted key autonomic counterregulatory responses. Despite this, glucose Rd was reduced during afternoon hypoglycemia following morning hypoglycemia, indicating posthypoglycemic insulin resistance. After morning exercise, endogenous glucose production was blunted, but glucose Rd was maintained during afternoon hypoglycemia, thereby indicating reduced metabolic defenses against hypoglycemia. These data suggest that exercise-induced counterregulatory failure can occur very rapidly, increasing the risk for hypoglycemia in T1DM within hours.     

Adrenomedullin and its receptor components in adipose tissues: Differences between white and brown fats and the effects of adrenergic stimulation

Male Sprague-Dawley rats were subcutaneously injected with 2.5mg/kg phenylephrine or 2.5mg/kg isoproterenol or both (2.5mg/kg for each drug) for 4 days, twice a day. Samples of scapular brown adipose tissue (BAT) and epididymal white adipose tissue (WAT) were collected for the measurement of adrenomedullin (AM) levels and the gene expression of preproAM, calcitonin receptor like receptor (CRLR) and its activity modifying proteins (RAMPs) by radioimmunoassay and RT-PCR. These values were compared with those in the rats that received 0.9% saline. The gene expression of AM and AM receptor components in BAT are much less than that in epididymal WAT. In BAT there were an increase in AM peptide level after a combined treatment of alpha(1) and beta adrenoceptor agonists and increases in preproAM mRNA levels for rats treated with alpha(1) and beta receptor agonists alone or in combination. Both CRLR and RAMP2 mRNA levels of alphabeta group were increased significantly. In WAT, AM peptide level, RAMP1 and RAMP2 mRNA expression levels were augmented in the alpha group while CRLR mRNA level was enhanced in the beta group. The levels of AM, its receptor and RAMPs are much less in BAT than in WAT but adrenergic stimulation has a greater effect on the AM and its receptor components in BAT than those in WAT. AM stimulates lipolysis and increases the level of uncoupling protein-1 (UCP-1) in BAT. It may therefore enhance thermogenesis by increasing the availability of free fatty acids substrate as well as the UCP-1 level on the mitochondrial membrane.     

Adrenomedullin in adipocyte differentiation of human mesenchymal stem cells

Expression of adrenomedullin (AM), a potent vasodilator peptide, was studied during adipocyte differentiation of human mesenchymal stem cells (hMSCs). Immunoreactive AM levels in the medium were increased at day 4 and 8 of the adipocyte differentiation. Northern blot analysis showed increased expression of AM mRNA in hMSCs-derived adipocytes at day 4, 8, 12, and 18. Transient transfection assay showed that the promoter activity was higher in hMSCs-derived adipocytes than in hMSCs, when cells were transfected with plasmids containing a cis-acting region (-70/-29) of the human AM gene. Electrophoretic mobility shift assay showed that specific bands bound to the region (-70/-29) in hMSCs-derived adipocytes but not in hMSCs, and were abolished by the stimulatory protein 1 (Sp1) antibody. The present study has shown that AM expression is up-regulated during adipocyte differentiation of hMSCs probably via the interaction between Sp1 or Sp1-related factor(s) and the AM promoter region (-70/-29).     

Circulating hormone adrenomedullin and its binding protein protect neural cells from hypoxia-induced apoptosis

Background

Brain ischemia is the underlying cause of neuron death during stroke and brain trauma. Neural cells exposed to ischemia can undergo apoptosis. Adrenomedullin (AM) in combination with its enhancing binding protein, AMBP-1, has been shown to reduce tissue damage in inflammation.

Methods

To evaluate a beneficial effect of AM/AMBP-1 administration in brain ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human neuroblastoma SH-SY5Y cells.

Results

After exposure to 1% O₂ for 20 h, neural cells were injured with decreased ATP levels and increased LDH release. Pre-administration of AM/AMBP-1 significantly reduced hypoxia-induced cell injury. Moreover, AM/AMBP-1 treatment reduced the number of TUNEL-positive cells and activation of caspase-3, compared to cells exposed to hypoxia alone. AM/AMBP-1 prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after hypoxia exposure. Correspondingly, an elevation of cAMP levels by forskolin protected neural cells from hypoxia-induced injury. Inhibition of PKA by KT5720 abolished the protective effect of AM/AMBP-1 on hypoxia-induced apoptosis.

Conclusions

AM/AMBP-1 elevates cAMP levels, followed by activating PKA, to protect neural cells from the injury caused by hypoxia.

General significance

AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from brain ischemia.     

Effects of macrophage inducible nitric oxide synthase in murine septic lung injury

Inducible nitric oxide synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion (via intratracheal instillation of clodronate liposomes) and subsequent AM reconstitution (via intratracheal instillation of donor iNOS+/+ or iNOS-/- AM). Sepsis was induced by cecal ligation and perforation, and ALI was assessed at 4 h: protein leak by the Evans blue (EB) dye method, neutrophil infiltration via myeloperoxidase (MPO) activity, and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 +/- 0.1 vs. 1.4 +/- 0.1 microg EB.g lung(-1).min(-1); P < 0.05) and MPO activity (37 +/- 4 vs. 67 +/- 8 U/g lung; P < 0.05) compared with that shown in non-AM-depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9 +/- 0.3 microg EB.g lung(-1).min(-1)) but not with iNOS-/- donor AM. In iNOS-/- mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS-/- mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus septic pulmonary protein leak is absolutely dependent on the presence of functional AM and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.     

Physiological and biochemical analysis of the transformants of aerobic methylobacteria expressing the dcm A gene of dichloromethane dehydrogenase

The transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr-/pME8220 and DM4-2cr-/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl- ions, CH2O, and HCOOH, nor to the impairment of glutathione synthesis or to the maintenance of intracellular pH. The (exo-) Klenow fragment-mediated incorporation of [alpha-32P]dATP into the DNA of the transformants DM4-2cr-/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH2Cl2 than when they were incubated with CH3OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH2Cl2 showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH2Cl2 degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.