The composition and sequence specificity of Pro-Ala-Lys-OH for the thrombolytic activities of P6A and related oligopeptides

The in vitro and in vivo thrombolytic activities of Ala-Arg-Pro-Ala-Lys-OH, its analogs and the related peptides were assayed. The results indicate that when (5)Lys of Ala-Arg-Pro-Ala-Lys-OH is changed into (5)Arg, and (3)Lys of Pro-Ala-Lys-OH is changed into (3)Arg the thrombolytic activities are collapsed; when Pro-Ala-Lys-OH is changed into Ala-Pro-Lys-OH, and Ala-Arg-Pro-Ala-Lys-OH is changed into Ala-Arg-Ala-Pro-Lys-OH the thrombolytic activities are also collapsed; when (5)Lys of Ala-Arg-Pro-Ala-Lys-OH is changed into (5)nLeu the thrombolytic activities are again collapsed. All of the results indicate that for the thrombolytic activities of Ala-Arg-Pro-Ala-Lys-OH and the related peptides Pro-Ala-Lys-OH exhibits either amino acid composition specificity or sequence specificity. The composition and sequence specificity of Pro-Ala-Lys-OH reflects its rule as the pharmacophore of P6A and the related peptides.     

Synthesis and thrombolytic activity of carboline-3-carboxylic acid modified metabolites of Ala-Arg-Pro-Ala-Lys

From the metabolism of H-Ala-Arg-Pro-Ala-Lys-OH, four metabolites, H-Pro-Ala-Lys-OH, H-Arg-Pro-Ala-Lys-OH, H-Ala-Arg-Pro-OH, and H-Ala-Arg-Pro-Ala-OH were identified. In order to find a new lead compound of thrombolytic peptide, 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid was introduced to the N- and C-terminal of the metabolites by use of the common coupling strategy. Under this condition, the pseudopeptides (5a-d and 7a-d) were obtained with a good yield. The thrombolytic activities of 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid containing oligopeptides were evaluated in vitro and in vivo. The result indicated that the thrombolytic activity of the pseudopeptide depended on the sequence and the modification pattern of the metabolites, and only when 3S-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid was introduced into the C-terminal of H-Pro-Ala-Lys-OH or H-Arg-Pro-Ala-Lys-OH, the desirable thrombolytic activity was retained and enhanced significantly.     

PAK: an essential motif for forming beta-turn structures and exhibiting the thrombolytic effect of P6A and its analogs.

Ala-Arg-Pro-Ala-Lys (ARPAK; also known as P6A) and 19 of its analogs were synthesized, and their thrombolytic activities were assessed in vitro and in vivo. The solution structures of 12 of the P6A analogs were determined using nuclear magnetic resonance (NMR) spectroscopy. The thrombolytic activity and conformational structure relationship was analyzed. We found that the Pro-Ala-Lys (PAK) sequence was essential for thrombolytic activity and was also responsible for the beta-turn structure found in the P6A analogs studied. The well defined beta turn may act as a binding head with the protruding lysine side-chain (positively charged) found at the target site for target recognition. Additionally, the N-terminal residue may be critical for thrombolytic activity, which for PAK-containing peptides, is likely achieved via a plasminogen-dependent pathway.     

Characterization of Aspergillus niger phosphoglucose isomerase. Use for quantitative determination of erythrose 4-phosphate.

Phosphoglucose isomerase (PGI) was purified from Aspergillus niger and the in vitro kinetic properties of the enzyme were related to its functioning in vivo. A new assay method was developed to study the forward reaction making use of mannitol 1-P dehydrogenase as the coupling enzyme. In this simple assay system mannitol 1-P dehydrogenase converts fructose 6-P and NADH to mannitol 1-P and NAD+, respectively. At pH 7.5 the Km for glucose 6-P was 0.48 mM, whereas the Km for fructose 6-P was 0.32 mM. The pentose phosphate pathway intermediates 6-phosphogluconate and erythrose 4-P (E4P) were competitive inhibitors of PGI with Ki values of approximately 0.2 mM and 1 microM respectively. In citric acid producing A. niger mycelium inhibition by 6-phosphogluconate is of minor physiological significance (10% inhibition). Since E4P could not be detected by an existing procedure, a novel assay was developed based on the strong inhibition of PGI by E4P. Although the new assay is very sensitive (detection limit 25 pmol), E4P could still not be detected in metabolite extracts indicating that a very low level of E4P is present in the cells. Using in vitro kinetics and concentrations of intracellular metabolites the in vivo activity of PGI was calculated and closely matched the steady state glycolytic flux observed during citric acid production.     

Paradoxical effect of sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethylbenz(a)anthracene

Effect of the induction of drug metabolizing enzymes by Sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethyl-benz(a)anthracene (DMBA) was investigated. A significant suppression of DMBA-induced micronucleated reticulocytes was observed in C57BL/6 mice treated with Sudan III intraperitoneally for 3 or 5 days before injection of the DMBA. However, the preincubation of DMBA with hepatic microsomes from Sudan III-treated rats caused a marked increase in the in vitro mutagenicity in the Ames assay, paradoxically. Sudan III was found to induce CYP 1A1, 7-ethoxycoumarin O-deethylase activity as well as both UDP-glucuronyl transferase and glutathione S-transferase activities. The increase of mutagenicity of DMBA observed in the Ames assay using hepatic microsomes from Sudan III-treated rats was inhibited by the addition of uridine 5′-diphosphoglucuronic add or reduced glutathione with cytosol. Mutagenic metabolites of DMBA formed by CYP1A1 appeared to be effectively detoxified by these phase II enzymes. The results of this study suggest that Sudan III-induced prevention of in vivo mutagenesis is due to the induction of both CYP 1A1 and detoxifying phase II enzymes. The induced CYP1A1 may accelerate formation of active metabolic intermediates, but phase II enzymes are also induced and detoxify these intermediates to inactive metabolites. This would reduce residence time of the carcinogen in the body and the time of exposure to active metabolites for target organs.     

Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

The role of suppressor macrophages (S-M phi) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2b/k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of M phi from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the M phi isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic M phi obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in M phi isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-M phi expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.     

In vivo and in vitro lysis of mouse cancer cells by antimetastatic effectors in normal plasma

Summary Results of parallel in vivo (IV. challenge) and in vitro (trypan blue staining) tests suggest that normal molecular factors in mouse blood can rapidly lyse malignant cells that enter the circulation. An inverse relationship was seen between the ability of malignant cells to form metastases after IV. injection and their susceptibility to lysis in vivo and in vitro. A mammary carcinoma lost its susceptibility to lysis and gained an ability to form metastases with conversion to the ascites form. The factor(s) active against tumor cells in vitro was were most evident in whole plasma, less so in serum form. The factors active against tumor cells in vitro globulin fraction of normal serum. Whole serum from tumor hosts had lower activity than whole normal serum, but had gained some activity in the immunoglobulin fraction. Treatment of normal animals with immunogens reduced the antitumor activity of whole serum. Whole-body irradiation, and, to a lesser degree, cyclophosphamide treatment, increased the activity.     

Suppression of low dose streptozotocin induced diabetes in mice by administration of a nitric oxide synthase inhibitor.

Nitric oxide has recently been identified as the primary toxic effector molecule in the lysis of islet cells by inflammatory macrophages. We show here that N-nitro-L-arginine-methylester (NAME), an inhibitor of endothelial and macrophage NO synthase partially suppresses diabetes development in the low dose streptozotocin induced diabetes model in C57BL/6J mice. Mean blood glucose levels were lower in the group receiving NAME throughout the observation period of 30d (p less than 0.05-0.001). Similar concentrations of NAME as expected in vivo were tested in vitro in macrophage-islet cell cocultures and were found to partially suppress NO production and islet cell lysis. We conclude that NO synthase activity is a pathogenetic factor in diabetes development.     

Macrophage activation induced by a synthetic antioxidant

The effect of a synthetic antioxidant 2-tretbutyl-3-hydroxypyridine (TBHP) on the function of murine peritoneal macrophages (MP) has been studied. A direct contact of TBHP with MP in vitro increased the activity of a key enzyme of glucose monophosphate graft--glucose-6-phosphate dehydrogenase and the proportion of flattened MP. as compared to the control. Upon intraperitoneal MP injection the number of MP's in the abdominal cavity of mice increased. They differed from control MP's in enhanced flattening and phagocytosis. In mice with preinduced defect of abdominal clearance TBHP contributed to the recovery of the normal level of antibacterial protection. In all the in vitro and in vivo tests studying its activating effect on MP, the synthetic antioxidant was not inferior to the standard MP activator--bacterial lipopolysaccharide.     

In vivo and in vitro activation of pulmonary macrophages by IFN-gamma for enhanced killing of Paracoccidioides brasiliensis or Blastomyces dermatitidis

The fungicidal capacity of murine pulmonary macrophages (PuM) activated in vitro with IFN or lymphokines or in vivo with IFN was studied. PuM treated overnight with IFN (1000 U/ml), Con A-stimulated spleen cell culture supernatants, or lymph node cells plus Con A significantly killed yeast cells of the Gar w isolate of Paracoccidioides brasiliensis 45.5 +/- 2.1%, 72.0 +/- 4.2%, and 51.5 +/- 0.7% respectively. Two other isolates of P. brasiliensis (Ru and LA) were also killed (45 and 34%) by PuM activated by lymph node cells plus Con A. Control PuM had lesser but significant capacity for killing of P. brasiliensis isolates, ranging from 15 to 22%. Killing of P. brasiliensis by PuM activated by Con A-stimulated spleen cell culture supernatants could not be significantly inhibited by superoxide dismutase, catalase, or azide. When mice were treated in vivo with 4 X 10(5) IFN U i.p. and PuM isolated 24 h later, the PuM had significantly enhanced ability to kill P. brasiliensis (47.0 +/- 6.3%) compared with PuM from control mice (25.0 +/- 4.2%). PuM thus activated also showed enhanced killing (43%) of a second isolate compared with control PuM (22%). PuM from IFN-treated mice were able to significantly kill Blastomyces dermatitidis (37.5 +/- 0.7%) compared with control PuM (4.5 +/- 6.3%). These results show that PuM can be activated in vitro and in vivo by IFN for enhanced fungicidal activity against two pulmonary fungal pathogens and suggests that immunologic production of IFN could be an important factor in host defenses against these diseases.     

Effect of chronic IL-6 infusion on acute pressor responses to vasoconstrictors in mice

Interleukin (IL)-6 has been implicated as a contributing factor in the pathogenesis of hypertension, although the mechanisms involved are unclear. Studies conducted in vitro suggest that IL-6 may have a direct effect on vascular tone and may modulate constrictor responses to agonists. Whether this effect can be observed in vivo is unknown. Therefore, mice were treated with either IL-6 (16 ng/h sc) or vehicle for 14 days, and the acute blood pressure and heart rate responses to endothelin (ET)-1, angiotensin II (ANG II), and phenylephrine (PE) were assessed under isoflurane anesthesia. Blood pressure responses to ET-1 were identical in vehicle- and IL-6-infused mice, both in the presence and the absence of ganglion blockade with chlorisondamine. The fall in heart rate during ET-1 responses was significantly attenuated in IL-6-infused mice with autonomic reflexes intact (vehicle vs. IL-6, P < 0.05 at 1 and 3 nmol/kg of ET-1), but this difference was not observed after ganglionic blockade. Both blood pressure and heart rate responses to ANG II were indistinguishable between IL-6- and vehicle-infused mice, as were responses to PE except for a significant increase in the blood pressure response and decrease in the heart rate response in IL-6-infused mice observed only at the highest dose of PE (300 microg/kg; P < 0.05). These findings show that, despite what might be predicted from studies conducted in vitro, chronic exposure to elevated plasma IL-6 concentrations in itself does not predispose the mouse to enhanced responsiveness to vasoconstrictors in vivo.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Chronic intermittent hypoxia impairs baroreflex control of heart rate but enhances heart rate responses to vagal efferent stimulation in anesthetized mice

Chronic intermittent hypoxia (CIH) leads to increased sympathetic nerve activity and arterial hypertension. In this study, we tested the hypothesis that CIH impairs baroreflex (BR) control of heart rate (HR) in mice, and that decreased cardiac chronotropic responsiveness to vagal efferent activity contributes to such impairment. C57BL/6J mice were exposed to either room air (RA) or CIH (6-min alternations of 21% O(2) and 5.7% O(2), 12 h/day) for 90 days. After the treatment period, mice were anesthetized (Avertin) and arterial blood pressure (ABP) was measured from the femoral artery. Mean ABP (MABP) was significantly increased in mice exposed to CIH (98.7 +/- 2.5 vs. RA: 78.9 +/- 1.4 mmHg, P < 0.001). CIH increased HR significantly (584.7 +/- 8.9 beats/min; RA: 518.2 +/- 17.9 beats/min, P < 0.05). Sustained infusion of phenylephrine (PE) at different doses (0.1-0.4 microg/min) significantly increased MABP in both CIH and RA mice, but the ABP-mediated decreases in HR were significantly attenuated in mice exposed to CIH (P < 0.001). In contrast, decreases in HR in response to electrical stimulation of the left vagus nerve (30 microA, 2-ms pulses) were significantly enhanced in mice exposed to CIH compared with RA mice at low frequencies. We conclude that CIH elicits a sustained impairment of baroreflex control of HR in mice. The blunted BR-mediated bradycardia occurs despite enhanced cardiac chronotropic responsiveness to vagal efferent stimulation. This suggests that an afferent and/or a central defect is responsible for the baroreflex impairment following CIH.     

肺炎链球菌溶血素黏膜免疫抗定植保护作用的研究

目的原核表达△A146Ply蛋白,评价△A146Ply黏膜免疫对肺炎链球菌（Streptococcuspneumon—ioe,5．Pn）在宿主鼻咽部定植的保护作用。方法IPTG诱导、Ni—NTA树脂纯化获得纯化的△A146Ply蛋白,经黏膜免疫BALB／C小鼠,制备其特异性抗血清;进行体内抗定植实验,观察小鼠鼻咽部灌洗液和肺部残存的细菌数量,检测△A146Ply黏膜免疫对19F型肺炎链球菌在鼻咽部定植的保护作用。为验证该保护作用是否具有广谱性,培养血清型14型、3型、6型和2型S．pn,经鼻腔感染免疫后小鼠,评价△A146Ply蛋白黏膜免疫对多株肺炎链球菌定植的保护作用。进行体外抗黏附实验,检测△A146Ply蛋白及其抗血清是否对无荚膜的肺炎链球菌R6黏附A549细胞具有抑制作用。结果获得了纯度〉90％的目的蛋白;体内实验结果显示,△A146Ply黏膜免疫可以显著降低肺炎链球菌19F在宿主鼻咽部和肺部残存的细菌数量（P〈0．01）;14型和3型肺炎链球菌在免疫后小鼠鼻咽部及肺部定植的数量均显著下降（P〈0．05）,2型肺炎链球菌在免疫后小鼠肺部定植的数量显著下降（P〈0．05）,6B型肺炎链球菌在免疫组与对照组小鼠鼻咽部及肺部均无显著差异（P〉0．05）;△A146Ply特异性抗血清△A146Ply蛋白对R6黏附A549细胞的抑制效应呈剂量依赖性。结论△A146Ply蛋白经黏膜免疫BALB／C小鼠可以对多种血清型的肺炎链球菌在宿主鼻咽部及肺部的定植提供显著保护作用,为Ply作为肺炎链球菌疫苗候选蛋白的应用提供了实验依据。     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

Lysis of uninfected and virus-infected cells in vivo: a rejection mechanism in addition to that mediated by natural killer cells

To examine the lysis of virus-infected cells in vivo, uninfected and lymphocytic choriomeningitis virus (LCMV)-infected L-929 cells were labeled in vitro with [125I]-iododeoxyuridine and implanted intravenously into mice. Natural cytotoxicity against both uninfected and virus-infected cells was demonstrated in normal uninfected mice, but LCMV-infected cells were cleared from the lungs and whole bodies more rapidly than uninfected cells. Treatment of L-929 cells with defective interfering LCMV inhibited standard virus synthesis and protected the target cells from enhanced in vivo rejection. The in vivo rejection was apparently mediated by a cellular constituent of the host immune response and not simply a result of virus-induced cytopathic effects on the target cell, as hydrocortisone acetate and cyclophosphamide each reduced rejection of both target cell types and eliminated the enhanced rejection of LCMV-infected cells. The enhanced rejection of LCMV-infected cells was not restricted by histocompatibility antigens, indicating that classic T-cell recognition was not involved in the lysis, and since the enhanced rejection of LCMV-infected cells was mediated by mice treated with cobra venom factor, complement was also not involved in the lysis. Although moderate levels of interferon (102 U/ml) were present in the sera and although there was a modest activation of natural killer (NK) cells in the lungs of LCMV-infected cell recipients but not uninfected cell recipients, the enhanced rejection of virus-infected cells did not appear to be NK cell mediated. Normal mice and mice depleted of NK cell activity by in vivo treatment with antibody to asialo ganglio-n-tetraosylceramide ( AGM1 ) rejected uninfected and LCMV-infected L-929 cells similarly. This antibody markedly inhibited the rejection of NK-sensitive YAC-1 cells. In addition to the natural cytotoxicity directed against virus-infected cells, a second nonspecific rejection mechanism appeared in response to treatment protocols which induced interferon. Polyinosinic-polycytidylic acid and infection with LCMV augmented in vivo rejection of both uninfected and LCMV-infected L-929 cells but eliminated the differential rejection of the virus-infected cells. Infection with LCMV also augmented the in vivo rejection of the NK-sensitive target cell, YAC-1. In vivo treatments with anti- AGM1 sera only moderately inhibited the elevated rejection of uninfected and LCMV-infected L-929 cells, indicating that the enhanced rejection of these target cells was predominantly mediated by a mechanism other than that mediated by NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The genetic control of natural killer cell activity and its association with in vivo resistance against a moloney lymphoma isograft

Spleens of normal young mice of certain strains contain lymphocytes that can kill strain A-derived YAC-1 lymphoma cells in a⁵¹Cr release cytotoxic assay in vitro. We have previously classified mouse genotypes as high or low reactors, according to their responses in this test. In vivo resistance to small numbers of YAC ascites lymphoma cells is correlated with in vitro cytolytic activity. In vitro and in vivo tests were carried out on the same individual (A x C57BL)F₁ x A backcross mice. Natural in vitro killer cell activity appeared to be under polygenic control, including a strong H-2-linked factor. No linkage was found with five different isozyme loci, with theIg-l locus or with C5 serum activity. Also in vivo resistance showed strong linkage with theH-2 complex. In (A x CBA)F₁ x A backcross mice, a weak linkage was found with the coat color locusC. There was a correlation between in vitro killer activity and in vivo resistance in the same backcross mice. In vivo resistance was particularly strong in mice that combined theH-2 ^b -linked resistance factor with a high cytolytic activity in vitro.     

Differential mutagenic activity of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) in Salmonella typhimurium strains in vitro and in vivo, in Drosophila, and in mice

IQ, a heterocyclic aromatic amine which is formed during the frying of meat, was prepared by chemical synthesis. Its genotoxic potential was studied in bacteria, Drosophila and in mice. A mutagenic effect of IQ (frameshift induction) was detected in Salmonella typhimurium in experiments without metabolic activation; this effect was several orders of magnitude lower than that observed in the presence of an activation system. Ames tests with liver-homogenate S9 fraction from PCB-induced mice and rats confirmed the high mutagenic potency of IQ metabolites (Kasai et al., 1980a). Comparative studies on diagnostic Salmonella strains revealed that the high frameshift-inducing activity is independent of the plasmid pkM101; it is, however, greatly reduced by an intact excision-repair system for DNA lesions. The mutagenic activity of the metabolite(s) formed in vitro by S9 mix has a half-life of ca. 14 min. In the fruit fly, Drosophila melanogaster, IQ induced when used at sublethal concentrations, X-chromosomal recessive lethal mutations in male germ cells in a dose-dependent manner. In mice, tests were performed to detect somatic mutations: chromosomal anomalies (micronuclei) in bone marrow, and gene mutations (affecting coat pigmentation) in mice exposed to IQ in utero. No genotoxic effects were observed in these assays. However, the formation of mutagenic metabolites in the liver of IQ-treated mice was unequivocally demonstrated in host-mediated assays using Salmonella as mutagen probes in mice. The data demonstrate genotoxic activity of IQ in prokaryotic and eukaryotic organisms. The possible reasons for the different response of mammalian systems in vivo and the Salmonella system are discussed.     

Lack of mannose-binding lectin-A enhances survival in a mouse model of acute septic peritonitis

The mannose-binding lectin (MBL) (also known as the mannose-binding protein) is a serum protein that plays a role as an "ante-antibody" in innate immunity. In man, MBL is encoded by a single gene, whereas in mice there are two homologous proteins, MBL-A and MBL-C. In order to evaluate the relative roles of these two forms of MBL, we created MBL-A null mice that were MBL-C sufficient. We found MBL-A null mice had enhanced survival in a septic peritonitis model compared to wild-type mice and complement 3 null mice at 24 h, 48 h and 10 d (P < 0.05). Reconstitution of these mice with human MBL reversed the phenotype. Surviving mice had significantly decreased TNF-alpha and IL-6 levels in the blood and peritoneal cavity (P < 0.01). In vitro studies indicate that bacteria opsonized with MBL-A-deficient serum induced significantly less cytokine by peritoneal macrophages compared to those with wild-type serum. Our results indicate that MBL-A is a modulator of inflammation in vivo and in vitro in the mouse and that the role of MBL may extend beyond its role as an opsonin.     

Reduced DNA oxidation in aged prostaglandin H synthase-1 knockout mice

Prostaglandin H synthase (PHS)-2 (COX-2) is implicated in the neurodegeneration of Alzheimer and Parkinson diseases. Multiple mechanisms may be involved, including PHS-catalyzed bioactivation of neurotransmitters, precursors, and metabolites to neurotoxic free radical intermediates. Herein, in vitro studies with the purified PHS-1 (COX-1) isoform and in vivo studies of aging PHS-1 knockout mice were used to evaluate the potential neurodegenerative role of PHS-1-catalyzed bioactivation of endogenous neurotransmitters to free radical intermediates that enhance reactive oxygen species formation and oxidative DNA damage. The brains of 2-year-old wild-type (+/+) PHS-1 normal and heterozygous (+/-) and homozygous (-/-) PHS-1 knockout mice were analyzed for 8-oxo-2'-deoxyguanosine formation, characterized by high-performance liquid chromatography with electrochemical detection and by immunohistochemistry. Compared to aging PHS-1(+/+) normal mice, aging PHS-1(-/-) knockout mice had less oxidative DNA damage in the cortex, hippocampus, cerebellum, and brain stem. This PHS-1-dependent oxidative damage was not observed in young mice. In vitro incubation of purified PHS-1 and 2'-deoxyguanosine with dopamine, L-DOPA, and epinephrine, but not glutamate or norepinephrine, enhanced oxidative DNA damage. These results suggest that PHS-1-dependent accumulation of oxidatively damaged macromolecules including DNA may contribute to the mechanisms and risk factors of aging-related neurodegeneration.