Mechanism of uptake and retention of F-18 BMS-747 158-02 in cardiomyocytes: a novel PET myocardial imaging agent

Background BMS-747158-02 is a novel fluorine 18-labeled pyridazinone derivative designed for cardiac imaging. The uptake and retention mechanisms of F-18 BMS-747158-02 in cardiac myocytes were studied in vitro, and the biodistribution of F-18 BMS-747158-02 was studied in vivo in mice. Methods and Results Fluorine 19 BMS-747158-01 inhibited mitochondrial complex I (MC-I) in bovine heart submitochondrial particles with an IC₅₀ of 16.6±3 nmol/L that was comparable to the reference inhibitors of MC-1, rotenone, pyridaben, and deguelin (IC₅₀ of 18.2±6.7 nmol/L, 19.8±2.6 nmol/L, and 23.1±1.5 nmol/L, respectively). F-18 BMS-747158-02 had high uptake in monolayers of neonatal rat cardiomyocytes (10.3%±0.7% of incubated drug at 60 minutes) that was inhibited by 200 nmol/L of rotenone (91%±2%) and deguelin (89%±3%). In contrast, an inactive pyridaben analog, P-0 (IC₅₀ value>4 μmol/L in MC-1 assay), did not inhibit the binding of F-18 BMS-747158-02 in cardiomyocytes. Uptake and washout kinetics for F-18 BMS-747158-02 in rat cardiomyocytes indicated that the time to half-maximal (t_1/2) uptake was very rapid (approximately 35 seconds), and washout t_1/2 for efflux of F-18 BMS-747158-02 was greater than 120 minutes. In vivo biodistribution studies in mice showed that F-18 BMS-747158-02 had substatial myocardial uptake (9.5%±0.5% of injected dose per gram) at 60 minutes and heart-to-lung and heart-to-liver ratios of 14.1±2.5 and 8.3±0.5, respectively. Positron emission tomography imaging in the mouse allowed clear cardiac visualization and demonstrated sustained myocardial uptake through 55 minutes. Conclusions F-18 BMS-747158-02 is a novel positron emission tomography cardiac tracer targeting MC-I in cardiomyocytes with rapid uptake and slow washout. These characteristics allow fast and sustained accumulation in the heart.     

Anopheles culicifacies: siblings species composition and its relationship to malaria incidence

Entomological and epidemiological surveys in May, August and November 1985 and March 1986 were conducted in villages in Bulandshahr, a western district in Uttar Pradesh and in three eastern districts, Jaunpur, Ballia and Saran. In Bulandshahr, Anopheles culicifacies sibling species A and B were found, with a predominance of species A. Both Plasmodium vivax and P. falciparum were present and the malaria incidence remained high (SPR, 6-50%) indicating an active transmission. In contrast, in three eastern districts predominance of species B with an occasional occurrence of species A was observed. Malaria cases were almost absent in Ballia and Saran and in Jaunpur 10.3% slide positivity rate was observed in May but in later surveys cases were considerably lower (SPR, 0.5-2.9%) indicating the absence of indigenous transmission. In the eastern districts, malaria parasites are regularly brought in from endemic areas by the migrant labor population. Although An. culicifacies s.l. occurs in both the areas, the difference in malaria incidence appears to be due to the difference in the composition of the sibling species which is, the predominant presence of species A in the western district and its absence in eastern districts. This indicates that species A is responsible for active malaria transmission while species B is not.     

Inhibition of spontaneous testicular Leydig cell tumor development in F-344 rats by dehydroepiandrosterone.

The incidence of spontaneous Leydig cell tumors of testis is very high in old F-344 rats. We have examined the effect of dehydroepiandrosterone (DHEA), a steroid hormone with antimitotic and anticarcinogenic properties, on spontaneous Leydig cell tumorigenesis. Fifteen-week-old male F-344 rats were fed a diet containing DHEA (0.45% w/w) for 84 weeks. At the termination of experiment none of the 13 rats had Leydig cell hyperplasia or Leydig cell tumors. All the eight control rats of comparable age had Leydig cell tumors. These findings suggest that DHEA is a potent inhibitor of spontaneous Leydig cell tumors of testis in aged rats.     

Tannic acid-induced nucleolar changes in hepatocytes transplanted into syngeneic or xenogeneic host and in hepatocytes maintained in primary culture

In this study we have investigated the effect of a single dose of tannic acid, administered s.c., on the nucleolar ultrastructure of hepatocytes transplanted into a syngeneic or xenogeneic host in order to evaluate the validity of our hepatocyte transplantation system as an in vivo alternative to the use of whole animals to test for species and strain differences to the effects of hepatotoxins. Within 4-6 h following tannic acid injection, rat hepatocytes transplanted into the anterior chamber of eye and inguinal fat pads of rat and athymic nude mouse, showed changes of nucleolar components, with separation of ribonucleoprotein containing granules into discrete dark zones. These dark areas were surrounded by light areas consisting of granular and fibrillar components of the nucleolus. These changes were identical to tannic acid-induced nucleolar alterations in the homotopic liver. Hamster and rat hepatocytes xenotransplanted into athymic nude mice also displayed prominent nucleolar alterations in response to tannic acid. The similarity and extent of nucleolar alterations observed in transplanted hepatocytes and the in situ homotopic liver cells attest to the usefulness of the hepatocyte transplantation system for the evaluation of species differences in biological response to toxic/carcinogenic effects of xenobiotics.     

Role of Periductal and Ductular Epithelial Cells of the Adult Rat Pancreas in Pancreatic Hepatocyte Lineage: A Change in the Differentiation Commitment

Development of pancreatic hepatocytes in adult rats maintained on copper deficient diet containing 0.6% trien (CuDT) has been reported recently. To elucidate the histogenesis of hepatocytes a sequential study was undertaken using morphologic, histochemical, immunochemical, in situ hybridization, and Northern blot analysis. Male F-344 rats weighing 80 to 90 g were fed CuDT for 8 weeks and returned to normal rat chow. Beginning from 4 weeks of copper depletion, there was a progressive loss of acinar cells and by 8 weeks more than 90% of the acinar tissue was lost. During this period, there was an increase in the number of adipocytes in the interstitium, and in the number of interstitial and ductular cells. Morphologic observations were confirmed by immunoblot and Northern blot analysis, in which the amount of pancreatic proteins and their mRNAs decreased between 5 and 8 weeks. During this period, a progressive increase in the level of albumin mRNA was observed. In situ hybridization, performed at 7 weeks of copper deficiency, showed localization of albumin mRNA over interstitial and ductular cells. Pancreatic hepatocytes were identified immediately after the rats were returned to a normal diet and gradually increased in number. The hepatocytes occupied almost 60% of the pancreatic volume by 8 weeks. During the early recovery phase, hepatocytes were identified in ductules as well as in the interstitium. Based on these studies, it is concluded that both the ductular cells and interstitial cells, which resemble oval cells of liver, are capable of transforming into pancreatic hepatocytes and these cells may be considered stem-cell equivalent.     

Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae

Verticillium dahliae is an important soil-borne fungal pathogen that causes vascular wilt diseases in a large variety of important crop plants. Due to its persistence in the soil, control of Verticillium wilt relies heavily on soil fumigation. The global ban on methyl bromide, a highly effective soil fumigant, poses an urgent need to develop alternative control measures against Verticillium wilt; and these might be more forthcoming with a better understanding of the molecular and cellular mechanisms that underpin the pathogenicity of V. dahliae. In this study, we assessed the role in growth, development, and pathogenicity of VMK1, a gene encoding a mitogen-activated protein (MAP) kinase (hence, Verticillium MAP Kinase 1). Disruption of VMK1 via Agrobacterium tumefaciens-mediated transformation, in two V. dahliae isolates, one from lettuce and the other from tomato, resulted in severely reduced virulence in diverse host plants, suggesting that VMK1 is essential for pathogenicity and that the MAP kinase-mediated signaling pathway has a conserved role in fungal pathogenicity. The vmk1 mutants also exhibited reduced conidiation and microsclerotia formation, suggesting that the gene is important for multiple cellular processes. P.R. and R.G.B. equally contributed to the work     

Leukotriene B4 Receptor (BLT-1) Modulates Neutrophil Influx into the Peritoneum but Not the Lung and Liver during Surgically Induced Bacterial Peritonitis in Mice

Leukotriene B₄ (LTB₄) is a rapidly synthesized, early neutrophil chemoattractant that signals via its cell surface receptor, BLT-1, to attract and activate neutrophils during peritonitis. BLT-1-deficient (BLT-1^−/−) mice were used to determine the effects of LTB₄ on neutrophil migration and activation, bacterial levels, and survival after cecal ligation and puncture (CLP). Male BLT-1^−/− or wild-type (WT) BALB/c mice underwent CLP. Tissues were harvested for determination of levels of bacteria, myeloperoxidase (MPO), LTB₄, macrophage inflammatory protein 2 (MIP-2), and neutrophil (polymorphonuclear leukocyte [PMN]) numbers at 4 and 18 h after CLP. PMN activation was determined by an assessment of phagocytosis ability and CD11b expression. Survival was also determined. BLT-1^−/− mice had decreased numbers of PMNs in the peritoneum at both 4 and 18 h after CLP but increased numbers of PMNs in the blood at 18 h compared with WT mice. Liver and lung MPO levels were significantly higher in BLT-1^−/− mice at both 4 and 18 h after CLP, with increased bacterial levels in the blood, the liver, and peritoneal fluid at 4 h. Bacterial levels remained higher in peritoneal fluid at 18 h, but blood and liver bacterial levels at 18 h were not different from levels at 4 h. PMN phagocytosis and CD11b levels were decreased in BLT-1^−/− mice. LTB₄ levels were similar between the groups before and after CLP, but MIP-2 levels were decreased both locally and systemically in BLT-1^−/− mice. Survival was significantly improved in BLT-1^−/− mice (71%) compared with WT mice (14%) at 48 h post-CLP. Thus, LTB₄ modulates neutrophil migration into the mouse peritoneum, but not the lung or liver, after CLP. Despite higher bacterial and PMN levels at remote sites, there was increased survival in BLT-1^−/− mice compared to WT mice. Decreased PMN activation may result in less remote organ dysfunction and improved survival.     

Alterations of pancreatic hepatocytes in rats exposed to carcinogens.

In this study, acute and chronic responses of pancreatic hepatocytes induced in F-344 rats by copper depletion-repletion protocol to certain hepatocarcinogens were examined. Administration of a single dose of tannic acid (subcutaneous), aflatoxin B1 (gavage), or lasiocarpine (intraperitoneally) caused characteristic nucleolar segregation in parenchymal cells of liver as well as in pancreatic hepatocytes. Chronic dietary administration of 2-acetylaminofluorene (0.025%) for 12 to 32 weeks led to the development of glutathione S-transferase-P-positive pancreatic hepatocytes in the pancreas. In addition, oval cell proliferation was observed in close association with pancreatic hepatocytes, but not in other areas of pancreas containing residual acinar cells. Oval cells in the pancreas and in the liver that developed in rats after chronic 2-acetylaminofluorene treatment and pancreatic duct cells stained positively with rat liver oval cell marker OV-6 antibodies by immunoperoxidase. These findings indicate that pancreatic hepatocytes respond to carcinogens in a fashion similar to parenchymal cells of liver.     

Macrophages but not B cells from aged mice are defective in stimulating autoreactive T cells in vitro

In the present study the effect of aging on the capacity of Ia+ cells to stimulate autoreactive T cells in the syngeneic mixed lymphocyte reaction (SMLR) was investigated. Using young CD4+ T cells as responders, it was observed that unseparated whole spleen cells from aged mice had normal stimulatory activity comparable to that of young spleen cells. Interestingly, however, when purified splenic adherent cells (SAC) enriched for macrophages or splenic B cells were used as stimulators, aged SAC but not aged B cells were found to be defective in stimulating autoreactive T cells. This defect in aged SAC was not due to decreased expression of Ia antigens since the percentage of Ia+ SAC and density of Ia antigen expression was similar in both young and old mice. Also, the B cells from aged mice expressed normal levels of Ia antigens. Aged SAC, when mixed with young SAC could also actively suppress the normal SMLR. However, this suppression was not due to increased prostaglandin production but was found to be associated with interleukin-1 (IL-1) regulation, inasmuch as addition of exogenous IL-1 could completely reconstitute the defective stimulatory activity of aged SAC and also abolished the suppressor activity of the SAC. Aged mice also demonstrated an intrinsic defect in the CD4+ T cells responding in the SMLR. Together, our studies on the SMLR demonstrate an age-related defect in responder autoreactive T cells and in stimulator splenic macrophages but not in the stimulatory activity of B cells.