The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: Potential role in temperature-dependent sex determination and testis development

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-α and PdgfR-β in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-α mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-β mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-α and PdgfR-β in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle.     

Reconstruction of hepatic organoid by hepatic stem cells

Recent advances in culture methods, stem cell research, and tissue engineering provide clues for making tissues in vitro that are functionally and structurally similar to hepatic tissues. To reconstruct hepatic organoids, two approaches to establish the methods have been proposed: the use of cells and the combination of cells and a scaffold (called tissue engineering). Recently, the coculture of hepatic cells (mature hepatocytes, small hepatocytes, hepatoblasts) and hepatic nonparenchymal cells has been reported to form hepatic organoids that possess differentiated hepatic functions. On the other hand, hepatocytes in a roller bottle were shown to form specific structures, consisting of biliary epithelial cells, connective tissue, mature hepatocytes, and endothelial cells. In this review, the studies of hepatic tissue formation in vitro will be summarized.     

Perinatal Tobacco Smoke Exposure Increases Vascular Oxidative Stress and Mitochondrial Damage in Non-Human Primates

Epidemiological studies suggest that events occurring during fetal and early childhood development influence disease susceptibility. Similarly, molecular studies in mice have shown that in utero exposure to cardiovascular disease (CVD) risk factors such as environmental tobacco smoke (ETS) increased adult atherogenic susceptibility and mitochondrial damage; however, the molecular effects of similar exposures in primates are not yet known. To determine whether perinatal ETS exposure increased mitochondrial damage, dysfunction and oxidant stress in primates, archived tissues from the non-human primate model Macaca mulatta (M. mulatta) were utilized. M. mulatta were exposed to low levels of ETS (1 mg/m³ total suspended particulates) from gestation (day 40) to early childhood (1 year), and aortic tissues were assessed for oxidized proteins (protein carbonyls), antioxidant activity (SOD), mitochondrial function (cytochrome oxidase), and mitochondrial damage (mitochondrial DNA damage). Results revealed that perinatal ETS exposure resulted in significantly increased oxidative stress, mitochondrial dysfunction and damage which were accompanied by significantly decreased mitochondrial antioxidant capacity and mitochondrial copy number in vascular tissue. Increased mitochondrial damage was also detected in buffy coat tissues in exposed M. mulatta. These studies suggest that perinatal tobacco smoke exposure increases vascular oxidative stress and mitochondrial damage in primates, potentially increasing adult disease susceptibility.     

Specificity of a porcine 127-kd nucleotide pyrophosphohydrolase for articular tissues

Objective. To determine the tissue specificity of a porcine 127-kd nucleotide pyrophosphohydrolase (NTPPHase) found in vesicles derived from hyaline articular cartilage (ACV). Methods. Homogenates of porcine brain, lung, liver, kidney, urinary bladder, pancreas, spleen, skin, vena cava, marrow, bone (cells), tendon (Achilles), ligament (anterior cruciate), elastic cartilage, meniscus, and hyaline cartilage were analyzed for NTPPHase activity (thymidine monophosphate paranitrophenyl ester substrate) and by Western blot using polyclonal antibodies against 127-kd NTPPHase and against recombinant PC-1, another ecto-NTPPHase. Results. All tissues contained NTPPHase activity; the highest specific activity was found in hyaline articular cartilage, the lowest in brain. ACV-associated 127-kd NTPPHase was expressed in cartilage, ligament, and tendon. PC-1 was also expressed in those tissues and in skin, kidney, bone cells, and (probably) in liver and muscle. Conclusion. The 127-kd NTPPHase appears to be highly specific for articular tissues.     

Interaction of chemical carcinogens with macromolecules

Summary The nature of certain critical cellular reactions is discussed in terms of both mutagenic and carcinogenic effects. Emphasis is placed on the ability of the ultimate carcinogen, normally formed in vivo by metabolism, to react with nucleic acids and, in particular, with nuclear DNA. The actions of N-nitroso compounds is examined in some detail and a possible correlation of the carcinogenic action of these compounds with their ability to react with oxygenatoms in nucleic acids is considered. The formation of a specific lesion, O ⁶-alkyguanine, in DNA and the capacity for its repair in different tissues is discussed with respect to tissue susceptibility to tumor induction. This discussion is extended to compare differences between species in the (tissue) specificity of action of particular N-nitroso compounds.Abbreviations AAF 2-acetylaminofluorene - BP benzo()pyrene - DEN N, N-diethylnitrosamine - DES diethyl sulphate - DMAB N,N-dimethyl-4-aminoazobenzene - DMBA 7,12-dimethylbenz()-anthracene - DMH 1,2-dimethylhydrazine - DMN N,N-dimethylnitrosamine - DMPT 3,3-dimethyl-1-phenyltriazene - DMS dimethyl sulphate - EMS ethyl methanesulphonate - ENU N-ethyl-N-nitrosourea - MMPT 3-methyl-1-phenyltriazene - MMS methyl methanesulphonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - MNUreth N-methyl-N-nitrosourethane     

Pharmacokinetics of quinacrine in the treatment of prion disease

Background  

Prion diseases are caused by the accumulation of an aberrantly folded isoform of the prion protein, designated PrP^Sc. In a cell-based assay, quinacrine inhibits the conversion of normal host prion protein (PrP^C) to PrP^Sc at a half-maximal concentration of 300 nM. While these data suggest that quinacrine may be beneficial in the treatment of prion disease, its penetration into brain tissue has not been extensively studied. If quinacrine penetrates brain tissue in concentrations exceeding that demonstrated for in vitro inhibition of PrP^Sc, it may be useful in the treatment of prion disease.     

The13C isotope and nuclear magnetic resonance: unique tools for the study of brain metabolism

As studies of brain metabolism grow in complexity, investigators turn increasingly to nuclear magnetic resonance spectroscopy combined with¹³C isotopic labeling. The unique ability to detect labeling non-destructively in specific carbon positions of individual compounds has opened the way to investigate brain metabolism in systems ranging from cellular preparations to the human brainin vivo. This review is written for investigators whose backgrounds do not include detailed knowledge of principles of nuclear magnetic resonance. Its purpose is to show the wide array of NMR techniques for¹³C detection that are available for application in different systems to study aspects of brain metabolism, such as metabolic compartmentation and measurements of the tricarboxylic acid cycle ratein vivo. Basic NMR concepts are explained, and, because each detection method possesses specific advantages to address the requirements of different experimental goals, basic explanations and examples are given for each technique. The review should provide readers with a basic understanding of the methods of¹³C detection by NMR and assess which of the methods are most applicable to the particular issues they may face in their own research.     

Evidence for persistent Chlamydia pneumoniae infection of human coronary atheromas

To date, structures representing developmental stages of Chlamydia pneumoniae, especially persistent forms of this intracellular bacteria, have not been described in human atherosclerotic tissues using specific antibody labeling and transmission electron microscopy.Staining of atherosclerotic tissue from five patients seeking heart transplantation with gold-labeled antibodies specific for up-regulated chlamydial heat shock proteins, GroEL and GroES, and visualisation via transmission electron microscopy revealed intracellular, atypical, round to oval structures of variable diameter. These structures resembled reticulate bodies of Chlamydia, were surrounded by membranes and were located within smooth muscle cells, macrophages or fibroblasts. By using double immunogold electron microscopy technique (GroEL and GroES in combination with chlamydial LPS/MOMP antibodies), we demonstrated these structures were of chlamydial origin.In the current study, we demonstrated the presence of aberrant bodies of C. pneumoniae in vivo in archival coronary atheromatous heart tissues by the immunogold electron microscopy technique.     

Aging leads to increased levels of protein O-linked <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine in heart,aorta, brain and skeletal muscle in Brown-Norway rats

Changes in the levels of O-linked N-acetyl-glucosamine (O-GlcNAc) on nucleocytoplasmic protein have been associated with a number of age-related diseases such as Alzheimer’s and diabetes; however, there is relatively little information regarding the impact of age on tissue O-GlcNAc levels. Therefore, the goal of this study was to determine whether senescence was associated with alterations in O-GlcNAc in heart, aorta, brain and skeletal muscle and if so whether there were also changes in the expression of enzymes critical in regulating O-GlcNAc levels, namely, O-GlcNAc transferase (OGT), O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase (GFAT). Tissues were harvested from 5- and 24-month old Brown-Norway rats; UDP-GlcNAc, a precursor of O-GlcNAc was assessed by HPLC, O-GlcNAc and OGT levels were assessed by immunoblot analysis and GFAT1/2, OGT, O-GlcNAcase mRNA levels were determined by RT-PCR. In the 24-month old animals serum insulin and triglyceride levels were significantly increased compared to the 5-month old group; however, glucose levels were unchanged. Protein O-GlcNAc levels were significantly increased with age (30–107%) in all tissues examined; however, paradoxically the expression of OGT, which catalyzes O-GlcNAc formation, was decreased by ∼30% in the heart, aorta and brain. In the heart increased O-GlcNAc was associated with increased UDP-GlcNAc levels and elevated GFAT mRNA while in other tissues we found no difference in UDP-GlcNAc or GFAT mRNA levels. These results demonstrate that senescence is associated with increased O-GlcNAc levels in multiple tissues and support the notion that dysregulation of pathways leading to O-GlcNAc formation may play an important role in the development of age-related diseases.     

Expression of Basic Fibroblast Growth Factor mRNA and Protein in the Human Brain following Ischaemic Stroke

Our previous work has demonstrated that angiogenesis occurs in the damaged brain tissue of patients surviving acute ischaemic stroke and increased microvessel density in the penumbra is associated with longer patient survival. The brain is one of the richest sources of FGF-2 and several studies have noted its angiogenic and neuroprotective effects in the nervous system. These findings led us to investigate the expression and localisation of both FGF-2 mRNA and protein in brain tissue collected within 12 h of death from 10 patients who survived for between 24 h and 43 days after acute stroke caused by thrombosis or embolus. Western blot analysis demonstrated increased FGF-2 protein expression in both grey and white matter in the infarcted core and the penumbra region compared to the normal contralateral hemisphere of all 10 patients studied. Using indirect immunoperoxidase staining of paraffin embedded sections, we observed the presence of FGF-2 in neurones, astrocytes, macrophages and endothelial cells. In situ hybridisation was used to localise and quantify mRNA expression in ischaemic brain tissue of the same 10 patients. The expression of FGF-2 in the penumbra of all patients was significantly raised compared with infarcted tissue and normal-looking contralateral hemisphere. In addition, serum FGF-2 was significantly increased between 1 and 14 days (P < 0.001) in many patients with both ischaemic stroke (n = 28) and intra-cerebral haemorrhage (n = 16) compared with age-matched control subjects undergoing routine medical examinations (n = 20). We suggest that up-regulation of FGF-2 is one of the mechanisms that leads to angiogenesis and neuro-protection in the penumbra region after acute stroke in man.     

Serum and Tissue Beta-2 Microglobulin Levels in Patients with Helicobacter pylori Infection

Background and aim Beta-2 microglobulin (β₂-m) is a minor plasma protein, secreted from the plasma membranes as a result of the continuous regeneration of membrane proteins in the cell surface of all nucleated cells. The relationship between Helicobacter pylori and β₂-m has not been adequately established in studies. In this study, we aimed to compare the levels of serum and tissue β₂-m in patients with and without H. pylori infection, and to examine the relationship between levels of serum and tissue β₂-m. Material and methods About 30 patients with H. pylori gastritis and 22 healthy persons were enrolled in this study. Gastric biopsies were histologically analyzed and compared according to tissue and serum β₂-m levels. Results Serum β₂-m levels were comparable in H. pylori and control groups. There was no significant link between tissue H. pylori grade and serum β₂-m levels. Subendothelial β₂-m was detected in 19 (63.3%) cases with H. pylori and none of the control group with immunohistochemical staining (P < 0.001). There was no correlation between serum and tissue levels of β₂-m. Conclusion β₂-m accumulates in the majority of gastric tissues of patients with active chronic gastritis who were H. pylori (+), whereas no accumulation was found in H. pylori (−) control subjects.     

Molecular confirmation of Xiphinema italiae Meyl, 1953 (Nematoda: Longidoridae) from the Slovak Republic

Summary  Identification of the nematode Xiphinema italiae relies mainly on time-consuming morphological and morphometrical studies. A polymerase chain reaction protocol has been used for the reliable and specific identification of X. italiae. Moreover, four independently evolving molecular markers (cox1- cytochrome c oxidase subunit 1; ITS2-second internal transcribed spacer; 18S gene and D2/D3 expansion segments of 28S gene) were amplified and sequenced in both directions.     

Biochemistry and bioenergetics of glutaryl-CoA dehydrogenase deficiency

Summary Glutaryl-CoA dehydrogenase (GCDH) is a central enzyme in the catabolic pathway of l-tryptophan, l-lysine, and l-hydroxylysine which catalyses the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA and CO₂. Glutaryl-CoA dehydrogenase deficiency (GDD) is an autosomal recessive disease characterized by the accumulation of glutaric and 3-hydroxyglutaric acids in tissues and body fluids. Untreated patients commonly present with severe striatal degeneration during encephalopathic crises. Previous studies have highlighted primary excitotoxicity as a trigger of striatal degeneration. The aim of this PhD study was to investigate in detail tissue-specific bioenergetic and biochemical parameters of GDD in vitro, post mortem, and in Gcdh ^−/− mice. The major bioenergetic finding was uncompetitive inhibition of α-ketoglutarate dehydrogenase complex by glutaryl-CoA. It is suggested that a synergism of primary and secondary excitotoxic effects in concert with age-related physiological changes in the developing brain underlie acute and chronic neurodegenerative changes in GDD patients. The major biochemical findings were highly elevated cerebral concentrations of glutaric and 3-hydroxyglutaric acid despite low permeability of the blood–brain barrier for these dicarboxylic acids. It can be postulated that glutaric and 3-hydroxyglutaric acids are synthesized de novo and subsequently trapped in the brain. In this light, neurological disease in GDD is not ‘transported’ to the brain in analogy with phenylketonuria or hepatic encephalopathy as suggested previously but is more likely to be induced by the intrinsic biochemical properties of the cerebral tissue and the blood–brain barrier. Competing interests: None declared References to electronic databases: Glutaryl-CoA dehydrogenase deficiency (GDD), OMIM #231670). Glutaryl-CoA dehydrogenase (GCDH) EC 1.3.99.7.     

Preproenkephalin hypermethylation in the pure pancreatic juice compared with p53 mutation in the diagnosis of pancreatic carcinoma

Background Aberrant methylation of CpG islands is a common mechanism for the dysregulation of tumor suppressor genes in a variety of human malignancies. Preproenkephalin ppENK) hypermethylation is recognized in 90% of pancreatic carcinoma (PCa) tissues, but not in normal pancreas. We analyzed ppENK hypermethylation in pure pancreatic juice (PPJ) in patients with PCa, intraductal papillary mucinous neoplasms (IPMN), and chronic pancreatitis (CP), and elucidated its usefulness as a marker in the diagnosis of PCa compared with p53 mutation. Methods PPJ was collected endoscopically from 28 patients with PCa, 15 patients with IPMN, and 20 patients with CP. DNA was extracted from the supernatant and the sediment of PPJ. Methylation-specific polymerase chain reaction was performed for hypermethylation analysis of ppENK. In addition, single-strand conformation polymorphism and direct sequencing were performed simultaneously to identify p53 mutations. Results The incidence of ppENK hypermethylation in the supernatant and/or the sediment of PPJ was 50% (14 of 28) in patients with PCa. In contrast, the incidence of ppENK hypermethylation was 26.7% (4 of 15) in patients with IPMN, and 5% (1 of 20) in patients with CP (P < 0.002). The incidence of p53 mutations in the PPJ was 42.9% (12 of 28) in patients with PCa and 0% (0 of 20) in patients with CP. Furthermore, the incidence of ppENK hypermethylation and/or p53 mutations in the PPJ was enhanced to 67.9% (19 of 28) in patients with PCa in the combination assay. Conclusions These results suggest that ppENK hypermethylation in PPJ is specific for cancer, and the combination assay with p53 enhances the genetic diagnosis of PCa.     

Role of T lymphocytes in secretory response to an enteric nematode parasite

Athymic (nude) rats have been used to assess the role of thymus-dependent T cells in the control of the intestinal response following infection with the enteric parasite,Nippostrongylus brasiliensis. Tissues from infected rats were excised on days 4, 7, 10, and 21 postinfection (p-i) for physiological and morphological studies; uninfected (day 0) rats served as controls. In response to the worm burden, jejunal tissues displayed a secretory response, indicated by an elevated baseline short-circuit current (I _sc ) on days 7 and 10 p-i, and were more responsive to histamine than control tissues. Despite this enhanced secretory response, 35% of the worm burden was still present on day 21 p-i (compared with expulsion of >95% by day 14 p-i in normal rats). Mast cell activation and hyperplasia, increased goblet cell (implying increased mucus synthesis) and intraepithelial leukocyte numbers, and abnormalities inI _sc responses after electrical stimulation of enteric nerves were identified following infection. These events in nude rats were attenuated or delayed in onset as compared with conventional immunocompetent rats. Our results support the postulate that thymus-dependent T cells regulate the timing and/or nature of the mucosal response to enteric parasitic infections. However, ion secretion was not altered in the absence of T cells and, therefore, is more likely to be a consequence of mast cell activation.This work was conducted with financial support from The Canadian Medical Research Council and the National Institutes of Health (NS 29536).     

Amelioratory Effects of Zinc Supplementation on <Emphasis Type="Italic">Salmonella</Emphasis>-induced Hepatic Damage in the Murine Model

Zinc (Zn) has been reported to influence the susceptibility of the host to a diverse range of infectious pathogens, including viruses, bacteria, fungi and protozoa. We report here an evaluation of the effects of Zn supplementation on Salmonella enterica serovar Typhimurium (S. typhimurium)-induced hepatic injury in the murine model. Zinc levels in the plasma and liver tissues were measured by atomic absorption spectroscopy. The effect of Zn supplementation was evaluated by assessing the bacterial load and levels of lipid peroxidation (LPO), antioxidants and monokines present in the hepatic tissue as well as by histopathological studies. Zinc supplementation reduced the bacterial load in the liver and reversed hepatic microscopic abnormalities. It also decreased the levels of LPO but increased the levels of reduced glutathione (GSH) as well as the activities of superoxide-dismutase (SOD) and catalase in the livers of infected mice supplemented with Zn compared to the livers of infected mice not supplemented with Zn. Zinc supplementation was also able to modulate the levels of monokines such as tumour necrosis factor alpha (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6). Our results indicate a role for Zn in downregulating oxidative stress and upregulating antioxidant defense enzymes through the action of monokines, suggesting that supplementation with Zn has a protective function in Salmonella-induced liver injury.     

Intracellular localization of lipoplexed siRNA in vascular endothelial cells of different mouse tissues

Liposomally formulated siRNA can be used for RNAi applications in vivo. Intravenous bolus administration of lipoplexed siRNA has been shown to reduce gene expression in the vascular endothelium. Here, we applied immunofluorescence staining for different endothelial markers (PECAM-1, CD34, laminin) on paraffin sections to compare the respective expression pattern with the intracellular localization of intravenously administered, fluorescently labeled siRNA (siRNA-Cy3-lipoplex). By confocal microscopy, lipoplexed siRNA-Cy3 was detected inside vascular endothelial cells in vivo, which where identified with co-staining of endothelial markers. Consequently, the finding of intracellular siRNA uptake by vascular endothelial cells correlated with RNAi based specific protein reduction in situ as revealed by PECAM-1 specific immunofluorescence staining in lung tissue sections. Therefore, by using a cell biological approach these in situ data emphasize the functional uptake of liposomal siRNA molecules in vascular endothelial cells of different mouse tissues as indicated in our previous molecular study.