Novel aspects of murine B cell lymphomas.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J M Ward
Publications and source records attributed to J M Ward.
Thyroid-specific enhancer-binding protein/thyroid transcription factor 1 is not required for the initial specification of the thyroid and lung primordia.
Targeted disruption of the homeobox gene T/ebp (Ttf1) in mice results in ablation of the thyroid and pituitary, and severe deformities in development of the lung and hypothalamus. T/ebp is expressed in the thyroid, lung, and ventral forebrain during normal embryogenesis. Examination of thyroid development in T/ebp homozygous null mutant embryos revealed that the thyroid rudiment is initially formed but is eliminated through apoptosis. Absence of T/EBP expression in the lung primordium does not activate apoptosis since a lung tissue, albeit dysmorphic, is nevertheless formed in T/ebp-/- embryos. These results demonstrate that T/EBP is not required for the initial specification of thyroid or lung primordia, but is absolutely essential for the development and morphogenesis of these organs.
Animals↗
The mitochondrial permeability transition pore.
This chapter reviews recent advances in the identification of the structural elements of the permeability transition pore. The discovery that cyclosporin A (CsA) inhibits the pore proved instrumental. Various approaches indicate that CsA blocks the pore by binding to cyclophilin (CyP)-D. In particular, covalent labelling of CyP-D in situ by a photoactive CsA derivative has shown that pore ligands have the same effects on the degree to which CsA both blocks the pore and binds to CyP-D. The recognition that CyP-D is a key component has enabled the other constituents to be resolved. Use of a CyP-D fusion protein as affinity matrix has revealed that CyP-D binds very strongly to 1:1 complexes of the voltage-dependent anion channel (from the outer membrane) and adenine nucleotide translocase (inner membrane). Our current model envisages that the pore arises as a complex between these three components at contact sites between the mitochondrial inner and outer membranes. This is in line with recent reconstitutions of pore activity from protein fractions containing these proteins. The strength of interaction between these proteins suggests that it may be a permanent feature rather than assembled only under pathological conditions. Calcium, the key activator of the pore, does not appear to affect pore assembly; rather, an allosteric action allowing pore flicker into an open state is indicated. CsA inhibits pore flicker and lowers the binding affinity for calcium. Whether adenine nucleotide translocase or the voltage-dependent anion channel (via inner membrane insertion) provides the inner membrane pore has not been settled, and data relevant to this issue are also documented.
Animals↗
Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes.
Insect capture and digestion contribute substantially to the nitrogen budget of carnivorous plants. In Nepenthes, insect-derived nitrogenous compounds are imported from the pitcher fluid and transported throughout the plant via the vascular tissue to support growth. Import and distribution of nutrients may require transmembrane nitrogen transporters. Representatives of three classes of genes encoding transporters for the nitrogenous compounds ammonium, amino acids and peptides were identified in Nepenthes pitchers. The expression at the cellular level of an ammonium transporter gene, three amino acid transporter genes, and one peptide transporter gene were investigated in the insect trapping organs of Nepenthes. Expression of the ammonium transporter gene NaAMT1 was detected in the head cells of digestive glands in the lower part of the pitcher where NaAMT1 may function in ammonium uptake from the pitcher fluid. One amino acid transporter gene, NaAAP1, was expressed in bundle sheath cells surrounding the vascular tissue. To understand the locations where transmembrane transport could be required within the pitcher, symplasmic and apoplasmic continuity was probed using fluorescent dyes. Symplasmic connections were not found between cortical cells and vascular bundles. Therefore, the amino acid transporter encoded by NaAAP1 may be involved in transport of amino acids into the vascular tissue. In contrast, expression of the peptide transporter gene NaNTR1 was detected in phloem cells of the vascular tissue within pitchers. NaNTR1 may function in the export of nitrogen from the pitcher by loading peptides into the phloem.
Amino Acid Sequence↗
Import and processing of heart mitochondrial cyclophilin D.
Cyclophilins are a family of cyclosporin-A-binding proteins which catalyse rotation about prolyl peptide bonds. A mitochondrial isoform in mammalian cells, cyclophilin D, is a component of the permeability transition pore that is formed by the adenine nucleotide translocase and the voltage-dependent anion channel at contact sites between the inner and outer membrane. This study investigated the submitochondrial location of cyclophilin D by following the fate of radiolabelled protein following import. Precursor [(35)S]cyclophilin D was expressed in vitro from a PCR-generated cDNA. The precursor was imported by rat heart mitochondria and processed in a single step to a 21-kDa protein that was identical (SDS/PAGE) to an in vitro expressed mature protein and a cyclophilin D purified from rat heart mitochondria. No further modification of the mature protein could be demonstrated. Fractionation of mitochondria following import established that cyclophilin D locates only to the matrix. It is concluded that cyclophilin D binding to the permeability transition pore must occur at the inner face of the mitochondrial inner membrane.
Animals↗
CYP1A2 is not the primary enzyme responsible for 4-aminobiphenyl-induced hepatocarcinogenesis in mice.
4-Aminobiphenyl (4-ABP), a potent carcinogen in rodents (liver cancer) and human (bladder cancer), is found as an environmental contaminant and in tobacco smoke. Hemoglobin adducts and lung DNA adducts of 4-ABP are found in tobacco smokers. In vitro metabolism studies with human and rat liver microsomes have shown that CYP1A2 is primarily responsible for catalyzing N-hydroxylation, the initial step in the metabolic activation of 4-ABP. To determine whether this P450 is a rate limiting pathway for hepatocarcinogenesis, CYP1A2-null mice were analyzed at 16 months of age and were compared with wild-type mice in their response to 4-ABP using the neonatal mouse bioassay and two different doses of the carcinogen. Overall differences in incidences of hepatocellular adenoma, carcinoma and preneoplastic foci were not significant between either genotypes or 4-ABP doses used, whereas small, but significant, differences were found for specific types of foci. These results suggest that while CYP1A2 levels may not be rate limiting for 4-ABP metabolism to produce tumors and foci, it may modulate the induction process of some types of liver foci in either a positive or negative manner. In vitro studies using CYP1A2-null and wild-type mouse liver microsomes revealed that CYP1A2 is not the sole P450 required for 4-ABP N-hydroxylation and that another, yet to be identified, P450 is likely to be involved.
Adenoma↗
Role of CYP1A2 in the toxicity of long-term phenacetin feeding in mice.
The mechanisms underlying phenacetin-induced toxicity and carcinogenicity are not clear. In particular, it is not known whether these effects are mediated by metabolic activation of the drug. CYP1A2 is known to metabolize phenacetin in vitro. To determine the role of this enzyme in vivo, the toxicity and carcinogenicity of phenacetin was examined in Cyp1a2-null mice (that lack CYP1A2). Six- to 8-week-old wild type (+/+) or null (-/-) mice were fed either a control diet, or one containing 1.25% phenacetin, ad libitum for up to 67 weeks. Representative groups of mice were examined for phenacetin-induced toxicity and carcinogenicity after 36, 48, 58, or 67 weeks of feeding. Consistent with the known role of CYP1A2 in phenacetin metabolism, plasma levels of phenacetin were higher and acetaminophen levels lower in the (-/-) mice fed phenacetin compared to phenacetin-fed (+/+) controls. Weight gain was significantly depressed in both groups of phenacetin-fed mice after 4 weeks of feeding, and continued to be lower for the remainder of the experiment, compared to controls. Hepatomegaly and splenomegaly were more severe in (-/-) mice but present in both genotypes fed phenacetin at all time points assessed. Histological analysis of liver, kidney, spleen, and urogenital tract also revealed a differential response in the (-/-) mice fed phenacetin compared to (+/+) mice fed the same diet. Further, mortality was the most severe in the (-/-) mice fed phenacetin than in all other groups. Despite significant toxicity in (-/-) mice fed phenacetin, only one renal carcinoma was found among them. Results from this work demonstrate that, in the absence of CYP1A2, phenacetin is more toxic than in controls. This provides evidence that metabolism of phenacetin by CYP1A2 alters toxicity in vivo, and suggests that alternate CYP1A2-independent metabolic pathways contribute to its toxicity.
Acetaminophen↗
A novel urease-negative Helicobacter species associated with colitis and typhlitis in IL-10-deficient mice.
A spiral-shaped bacterium with bipolar, single-sheathed flagella was isolated from the intestines of IL-10 (interleukin-10)-deficient (IL-10(-/-)) mice with inflammatory bowel disease. The organism was microaerobic, grew at 37 and 42 degrees C, and was oxidase and catalase positive but urease negative. On the basis of 16S rRNA gene sequence analysis and biochemical and phenotypic criteria, the organism is classified as a novel helicobacter. Cesarean section-rederived IL-10(-/-) mice without helicobacter infection did not have histological evidence of intestinal inflammation. However, helicobacter-free IL-10(-/-), SCID/NCr, and A/JNCr mice experimentally inoculated with the novel urease-negative Helicobacter sp. developed variable degrees of inflammation in the lower intestine, and in immunocompetent mice, the experimental infection was accompanied by a corresponding elevated immunoglobulin G antibody response to the novel Helicobacter sp. antigen. These data support other recent studies which demonstrate that multiple Helicobacter spp. in both naturally and experimentally infected mice can induce inflammatory bowel disease. The mouse model of helicobacter-associated intestinal inflammation should prove valuable in understanding how specific microbial antigens influence a complex disease process.
Animals↗
Splenic marginal zone B-cell and thymic T-cell lymphomas in p53-deficient mice.
The molecular pathology and histogenesis of lymphomas in 56 retired breeder male and 14 12-week-old male homozygous p53-deficient (p53-/-) mice (C57BL/6TacfBR-[KO]p53 N4) were evaluated. Lymphomas were assessed by serial morphologic techniques, immunohistochemistry, flow cytometry, and analysis of T cell receptor (TCR) or immunoglobulin heavy chain (IgH) gene rearrangements. We found two common types of lymphomas. T-cell lymphomas arose in the thymus through a sequence of lymphocyte depletion, medullary hyperplasia, and unilateral lymphoma. Tumor cells were CD3+, CD90+, and usually TCRalpha/beta+ and possessed clonal TCRbeta gene rearrangements. Thymic lymphoblastic lymphomas (LL) were highly malignant and quickly metastasized to the splenic white pulp and liver, even when the thymus was only slightly increased in weight. In the spleen, a novel lymphoma was found. Marginal zone hyperplasia led to marginal zone lymphoma (MZL), a well-differentiated lymphoma that usually expressed CD45R (B220) and CD5 at low levels and that had clonal IgH gene rearrangements. IgH gene rearrangements were also seen in spleens with marginal zone hyperplasias only. Hyperplastic and neoplastic marginal zone B cells expressed IgM at low to normal levels, as seen by FACS analysis and immunohistochemistry. These tumors only metastasized to the liver at a later stage, as they became less differentiated. Several mice had both types of tumors present in the spleen. Two B-cell lymphoblastic lymphomas of uncertain origin were also found. In this article, we discuss the possible mechanisms responsible for development of the lymphomas seen in these p53-deficient mice.
Animals↗
Time course of SDS-alkaline lysis of recombinant bacterial cells for plasmid release.
SDS-alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time.
Escherichia coli↗
Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore.
A cyclophilin-D affinity matrix was employed to isolate components of the mitochondrial permeability transition pore. A cDNA encoding cyclophilin-D was cloned from a rat liver library and ligated into pGEX to allow expression of a glutathione S-transferase/cyclophilin-D fusion protein in Escherichia coli XL1 cells. The cyclophilin-D in the fusion was functionally normal as judged by its peptidylprolyl cis-trans-isomerase activity and its inhibition by cyclosporin A. The fusion protein was bound to glutathione-agarose to form the cyclophilin-D affinity matrix. The matrix selectively bound 32-kDa proteins of mitochondrial membrane extracts, but no H2O-soluble proteins were bound. The 32-kDa band on SDS/PAGE resolved into a doublet and reacted with antibodies against the voltage-dependent anion channel (porin) and the adenine nucleotide translocase. These two proteins were also selectively retained by the affinity matrix in the presence of cyclosporin A. The thus-purified voltage-dependent anion channel, adenine nucleotide translocase and the fusion protein were incorporated into phosphatidylcholine liposomes containing fluorescein sulphonate. The proteoliposomes were permeabilized by Ca2+ plus phosphate, and this was blocked completely by cyclosporin A. These properties are identical to those of the permeability transition pore in mitochondria. It is concluded that the basic permeability transition pore structure comprises the voltage-dependent anion channel (outer membrane), adenine nucleotide translocase (inner membrane) and cyclophilin-D, and forms at contact sites between the two membranes.
Animals↗
Ontogeny of the murine glucose-6-phosphatase system.
A deficiency in microsomal glucose-6-phosphatase (G6Pase) activity causes glycogen storage disease type 1 (GSD-1), a clinically and biochemically heterogeneous group of diseases. It has been suggested that catalysis by G6Pase involves multiple components, with defects in the G6Pase catalytic unit causing GSD-1a and defects in the putative substrate and product translocases causing GSD-1b, 1c, and 1d. However, this model is open to debate. To elucidate the G6Pase system, we have examined G6Pase mRNA expression, G6Pase activity, and glucose 6-phosphate (G6P) transport activity in the murine liver and kidney during normal development. In the liver, G6Pase mRNA and enzymatic activity were detected at 18 days gestation and increased markedly at parturition, before leveling off to adult levels. In the kidney, G6Pase mRNA and enzymatic activity appeared at 19 days gestation and peaked at weaning, suggesting that kidney G6Pase may have a different metabolic role. In situ hybridization analysis demonstrated that, in addition to the liver and kidney, the intestine expressed G6Pase. Despite the expression of G6Pase in the embryonic liver, microsomal G6P transport activity was not detectable until birth, peaking at about age 4 weeks. Our study strongly supports the multicomponent model for the G6Pase system.
Age Factors↗
The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast.
Nonessential metal ions such as cadmium are most likely transported across plant membranes via transporters for essential cations. To identify possible pathways for Cd2+ transport we tested putative plant cation transporters for Cd2+ uptake activity by expressing cDNAs in Saccharomyces cerevisiae and found that expression of one clone, LCT1, renders the growth of yeast more sensitive to cadmium. Ion flux assays showed that Cd2+ sensitivity is correlated with an increase in Cd2+ uptake. LCT1-dependent Cd2+ uptake is saturable, lies in the high-affinity range (apparent KM for Cd2+ = 33 microM) and is sensitive to block by La3+ and Ca2+. Growth assays demonstrated a sensitivity of LCT1-expressing yeast cells to extracellular millimolar Ca2+ concentrations. LCT1-dependent increase in Ca2+ uptake correlated with the observed phenotype. Furthermore, LCT1 complements a yeast disruption mutant in the MID1 gene, a non-LCT1-homologous yeast gene encoding a membrane Ca2+ influx system required for recovery from the mating response. We conclude that LCT1 mediates the uptake of Ca2+ and Cd2+ in yeast and may therefore represent a first plant cDNA encoding a plant Ca2+ uptake or an organellar Ca2+ transport pathway in plants and may contribute to transport of the toxic metal Cd2+ across plant membranes.
Base Sequence↗
RET cooperates with RB/p53 inactivation in a somatic multi-step model for murine thyroid cancer.
Mice bred to carry germline Rb and p53 null alleles are associated with a tumor spectrum that overlaps with the inherited multiple endocrine neoplasia-1 (MEN1) and MEN2 syndromes in humans, including medullary thyroid cancer (MTC). To study the genetic basis for these tumors, we microdissected MTC specimens or obtained fresh MTC tissue from nine independent Rb(+/-) p53(+/-) mice, amplified the region of the Ret gene known to be mutated in human MTC, and detected acquired missense Ret mutations in four different mice. These mutations were localized to a group of tandem cysteines which are analogous to activating germline mutations observed in human MEN2A and familial MTC (FMTC). To determine whether the remaining wild type Rb allele was inactivated in these murine MTC samples, we subjected tumor tissue to immunohistochemical staining with an Rb antibody, and demonstrated the absence of RB staining in murine MTC, while normal tissue retained RB nuclear staining. These findings demonstrate the ability of the gene knockout model to recapitulate somatic multi-step tumorigenesis and suggest that the development of a murine neuroendocrine tumor requires mutational dysregulation within both receptor tyrosine kinase and nuclear tumor suppressor gene pathways.
Animals↗
Protection against acetaminophen toxicity in CYP1A2 and CYP2E1 double-null mice.
Acetaminophen (APAP) hepatotoxicity is due to its biotransformation to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), that is capable of binding to cellular macromolecules. At least two forms of cytochrome P450, CYP2E1 and CYP1A2, have been implicated in this reaction in mice. To test the combined roles of CYP1A2 and CYP2E1 in an intact animal model, a double-null mouse line lacking functional expression of CYP1A2 and CYP2E1 was produced by cross-breeding Cyp1a2-/- mice with Cyp2e1-/- mice. Animals deficient in the expression of both P450s developed normally and exhibited no obvious phenotypic abnormalities. Comparison of the dose-response to APAP (200-1200 mg/kg) indicated that double-null animals were highly resistant to APAP-induced toxicity whereas the wild-type animals were sensitive. Administration of 600 to 800 mg/kg of this drug to male wild-type animals resulted in increased plasma concentrations of liver enzymes (alanine aminotransferase, sorbitol dehydrogenase), lipidosis, hepatic necrosis, and renal tubular necrosis. In contrast, when APAP of equivalent or higher dose was administered to the double-null mice, plasma levels of liver enzymes and liver histopathology were normal. However, administration of 1200 mg of APAP/kg to the double-null mice resulted in infrequent liver lipidosis and mild kidney lesions. Consistent with the protection from hepatotoxicity, the expected depletion of hepatic glutathione (GSH) content was significantly retarded and APAP covalent binding to hepatic cytosolic proteins was not detectable in the double-null mice. Likewise, in vitro activation of APAP by liver microsomes from the double-null mice was approximately one tenth of that in microsomes from wild-type mice. Thus, the protection against APAP toxicity afforded by deletion of both CYP2E1 and CYP1A2 likely reflects greatly diminished production of the toxic electrophile, NAPQI.
Acetaminophen↗
Sucrose transport in higher plants.
Presumably due to its physicochemical properties, sucrose represents the major transport form of photosynthetically assimilated carbohydrates in plants. Sucrose synthesized in green leaves is transported via the phloem, the long distance distribution network for assimilates in order to supply nonphotosynthetic organs with energy and carbon skeletons. At least in Solanaceae, sugar export seems to be a tightly regulated process involving a number of specific plasma membrane proteins. Significant progress in this field was made possible by the recent identification of plasma membrane sucrose transporter genes. These carriers represent important parts of the long-distance transport machinery and can serve as a starting point to obtain a complete picture of long-distance sucrose transport in plants. A combination of biochemical studies of transporter properties together with expression and localization studies allow specific functions to be assigned to the individual proteins. Furthermore, the use of transgenic plants specifically impaired in sucrose transporter expression has provided strong evidence that SUT1 transporter function is required for phloem loading. Physiological analyses of these plants demonstrate that sucrose transporters are essential components of the sucrose translocation pathway at least in potato and tobacco.
Amino Acid Sequence↗
Mitochondrial import of cyclophilin-D.
Explore the source record for details and available documents.
Animals↗
Cyclophilin-D binding proteins.
Explore the source record for details and available documents.
Animals↗