Morphological investigation on phenylhydrazine-induced erythropoiesis in the adult mouse liver

In adult mice suffering from a phenylhydrazine (PHZ)-induced hemolytic anemia, erythropoietic islands were observed in the liver. These islands were studied with the light and electron microscope. Within two days after the beginning of four daily injections of PHZ, erythoid elements appeared in the sinusoids and central veins. A maximum number of erythroblasts was found on day 7. Light and electron microscopic observations revealed that the erythropoietic islands consisted of centrally located macrophages(CM) with a Kupffer cell-like morphology, surrounded by erythroblasts, which were often of the same maturation stage. CM in central veins (CM-V) and in sinusoids (CM-S) were found to have a different morphology. The CM-V phagocytized less circulating red blood cells and were in contact with a smaller number of erythroblasts. Furthermore, the contact areas between erythroblasts and CM-S extended for a much longer distance than those between erythroblasts and CM-V. The progenitor cell for the CM-V is most likely a monocyte, since cells which were morphologically determined as monocytes were found to appear on the first day of the PHZ treatment and differentiated into macrophages within about 2 days. The origin of the CM-S population was less clear, but could be monocytic as well. These data are tentatively explained as a migration of a progenitor of a cellular component of the erythroid micro-environment into the liver after appropriate stimuli. In contrast to fetal liver erythropoiesis, erythroblasts in the adult liver occurred only incidentally extrasinusoidally. Furthermore, specialized membrane contacts between erythroblasts and CM or hepatocytes could not be observed in adult liver. Ferritin could not be detected on the erythroid cell membrane or located in coated vesicles. Also, no ferritin could be observed within or attached to the finger-like processes of CM. The observations suggest that the coated vesicles in erythoid elements are partly exocytotic vesicles and are not specific for ferritin transport. The morphological aspects of PHZ-induced extramedullary erythropoiesis is discussed in relation to the hemopoietic microenvironment.     

Characterization of the mouse Myoc/Tigr gene

Mutations in the myocilin (MYOC), also known as Trabecular meshwork-Inducible Glucocorticoid Response (TIGR) gene can lead to juvenile open-angle glaucoma in human and may be responsible for at least 3% of primary open-angle glaucoma. To develop a mouse model of primary open angle glaucoma, and to get deeper insight into the mechanisms of the MYOC/TIGR gene regulation and function, we have isolated and characterized full size mouse Myoc/Tigr cDNA and genomic clones. The mouse and human MYOC/TIGR genes have the same exon-intron structure and contain 3 exons, although the mouse gene is 6 kb shorter than the human gene (10 kb versus 16 kb) due to differences in the length of introns. The MYOC/TIGR gene encodes a moderately conserved protein, which is 82% identical between human and mouse. The encoded protein is 14 amino acids shorter at the N-terminus in the mouse than in the human (490 versus 504 amino acids). Mouse and human MYOC/TIGR genes show a similar pattern of expression in adult ocular and nonocular tissues. The mouse Myoc/Tigr gene was mapped to Chromosome 1 at position 82.8 cM from the centromere. All residues, which were identified in the human MYOC/TIGR protein as critical for glaucoma development, are conserved in the mouse Myoc/Tigr.     

Disruption of redox homeostasis in the transforming growth factor-alpha/c-myc transgenic mouse model of accelerated hepatocarcinogenesis

In previous studies we have demonstrated that transforming growth factor (TGF)-alpha/c-myc double transgenic mice exhibit an enhanced rate of cell proliferation, accumulate extensive DNA damage, and develop multiple liver tumors between 4 and 8 months of age. To clarify the biochemical events that may be responsible for the genotoxic and carcinogenic effects observed in this transgenic model, several parameters of redox homeostasis in the liver were examined prior to development of hepatic tumors. By 2 months of age, production of reactive oxygen species, determined by the peroxidation-sensitive fluorescent dye, 2',7'-dichlorofluorescin diacetate, was significantly elevated in TGF-alpha/c-myc transgenic hepatocytes versus either wild type or c-myc single transgenic cells, and occurred in parallel with an increase in lipid peroxidation. Concomitantly with a rise in oxidant levels, antioxidant defenses were decreased, including total glutathione content and the activity of glutathione peroxidase, whereas thioredoxin reductase activity was not changed. However, hepatic tumors which developed in TGF-alpha/c-myc mice exhibited an increase in thioredoxin reductase activity and a very low activity of glutathione peroxidase. Furthermore, specific deletions were detected in mtDNA as early as 5 weeks of age in the transgenic mice. These data provide experimental evidence that co-expression of TGF-alpha and c-myc transgenes in mouse liver promotes overproduction of reactive oxygen species and thus creates an oxidative stress environment. This phenomenon may account for the massive DNA damage and acceleration of hepatocarcinogenesis observed in the TGF-alpha/c-myc mouse model.     

Modulation of in vivo migratory function of alpha 2 beta 1 integrin in mouse liver

We report herein that expression of alpha 2 beta 1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that alpha 2 beta 1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of alpha 2 beta 1 on KX2C2 and RDX2C2 cells using a alpha 2 beta 1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated alpha 2 beta 1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn2+, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that alpha 2 beta 1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that alpha 2 beta 1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.     

A gene apparently determining the extent of sialylation of lysosomal alpha-mannosidase in mouse liver

Organ-specific electrophoretic heterogeneity of lysosomal alpha-mannosidase has been observed within individual strains of inbred mice. Polymorphism between C57BL/6J and CBA/J for liver lysosomal alpha-mannosidase is determined by a single genetic locus on chromosome 5 and appears to be the result of differences in sialylation of the lysosomal enzyme. Two different patterns of expression of development of the liver electrophoretic forms have been observed.     

Cell-density-dependent expression of Cyp1a2 gene in monolayer-cultured adult mouse hepatocytes

The effects of PAF antagonists, of substances which influence the arachidonic acid metabolism, and of dexamethasone and ketotifen were evaluated in an acute PAF-induced mortality model in female NMRI mice. We established a dependence of sensitivity to PAF on strain (AB mice showed no dose dependence) and on sex of the animals as well as on the PAF charges used in our experiments. PAF produced resistance in surviving animals against the PAF-induced death on repeated application. The PAF antagonists, WEB 2170 and WEB 2086, provided the best dose-dependent protection against PAF toxicity, followed by dexamethasone, by the COX/LOX synthetase inhibitor X 86 (a BW 755 C-analogue) and by the PAF receptor antagonist BN 52021. Particularly remarkable was the excellent prevention by aspirin. Aspirin may not only inhibit the cyclooxygenase pathway but also endogenous PAF synthesis. Other drugs, i.e. indomethacin, the thromboxane receptor antagonist, BM 13177, the thromboxane synthetase inhibitor, HOE 944, as well as the lipoxygenase inhibitors (NDGA, esculetin, SHAM and phenidone) exerted a dose-dependent protection only at high doses.     

Lead suppresses chimeric human transferrin gene expression in transgenic mouse liver

The major iron-transport protein in serum is transferrin (TF) which also has the capacity to transport other metals. This report presents evidence that synthesis of human TF can be regulated by the metal lead. Transgenic mice carrying chimeric human TF-chloramphenicol acetyl transferase (CAT) genes received lead or sodium salts by intraperitoneal injections or in drinking water. Transgene expression in liver was suppressed 31 to 50% by the lead treatment. Lead regulates human TF transgenes at the mRNA level since liver CAT enzyme activity, CAT protein, and TF-CAT mRNA levels were all suppressed. The dosages of lead did not alter synthesis of the other liver proteins, mouse TF and albumin, as measured by Northern blot analysis of total liver RNA and rocket immunoelectrophoresis of mouse sera. Moderate levels of lead exposure were sufficient to evoke the human TF transgene response; blood lead levels in mice that received lead acetate in drinking water ranged from 30 micrograms/dl to 56 micrograms/dl. In addition to suppressing expression of TF-CAT genes in transgenic mice, lead also suppressed synthesis of TF protein in cultured human hepatoma HepG2 cells. The regulation of human TF apparently differs from the regulation of mouse TF which is unresponsive to lead exposure.     

T cell receptor alpha gene rearrangement and transcription in adult thymic gamma delta cells

T cells belong to two separate lineages based on surface expression of alpha beta or gamma delta T cell receptors (TCR). Since during thymus development TCR beta, gamma, and delta genes rearrange before alpha genes, and gamma delta cells appear earlier than alpha beta cells, it has been assumed that gamma delta cells are devoid of TCR alpha rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic gamma delta cells undergo VJ alpha rearrangements more frequently than immature alpha beta lineage thymic precursors. Sequence analysis shows VJ alpha rearrangements in gamma delta cells to be mostly (70%) nonproductive. Furthermore, VJ alpha rearrangements in gamma delta cells are transcribed normally and, as shown by analysis of TCR beta-/- mice, occur independently of productive VDJ beta rearrangements. These data are interpreted in the context of a model in which precursors of alpha beta and gamma delta cells differ in their ability to express a functional pre-TCR complex.     

Mapping of a liver phosphorylase kinase alpha-subunit gene on the mouse X chromosome

Phosphorylase kinase (PHK) is a regulatory enzyme of the glycogenolytic pathway composed of a complex of four subunits. We recently mapped the muscle alpha-subunit gene (Phka) to the mouse X chromosome in a region syntenic with the proximal long arm of the human X chromosome and containing the human homologue of this gene, PHKA. We now report the mapping of the liver alpha-subunit gene to the telomeric end of the mouse X chromosome. This mapping position would suggest a location for the human liver alpha-subunit gene on the proximal short arm of the X chromosome, a region recently implicated in X-linked liver glycogenosis (XLG).     

Disruption of the gene encoding the mitogen-regulated translational modulator PHAS-I in mice

PHAS-I is the prototype of a group of eIF4E-binding proteins that can regulate mRNA translation in response to hormones and growth factors. To investigate the importance of PHAS-I in the physiology of the intact animal, we disrupted the PHAS-I gene in mice. Tissues and cells derived from the knockout mice contained no detectable PHAS-I protein. A related protein, PHAS-II, and eIF4E were readily detectable in tissues from these animals, but neither appeared to be changed in a compensatory manner. Mice lacking PHAS-I appeared normal at birth. However, male knockout mice weighed approximately 10% less than controls at all ages, whereas female weights were similar to those of controls. Both males and females were fertile. Tissues from adult animals appeared to be normal by routine histological staining techniques, as were routine blood cell counts and chemistries. Fibroblasts derived from PHAS-I-deficient mouse embryos exhibited normal rates of growth and overall protein synthesis, responded normally to serum stimulation of ornithine decarboxylase activity and cell growth, and rapamycin inhibition of cell growth. Under these experimental conditions, PHAS-I is apparently not required for the normal development and reproductive behavior of female mice, but is required for normal body weight in male mice; the mechanisms responsible for this phenotype remain to be determined.     

Differential expression of five laminin alpha (1-5) chains in developing and adult mouse kidney

The nature of the laminin alpha chains in the embryonic and adult kidney is still being debated. The present study attempted to clarify this issue by immunofluorescence study using monoclonal antibodies against mouse alpha1, alpha2, and alpha5 chains and in situ hybridization for the alpha2, alpha3B, alpha4, and alpha5 mRNAs. Novel alpha1 chain-specific monoclonal antibodies against E8 fragment revealed a restricted distribution of alpha1 chain in a subset of epithelial basement membranes in the embryo, in agreement with previous mRNA data. The alpha2 mRNA was produced by mesenchyme, although the protein was deposited in epithelial basement membranes. The alpha3B mRNA was found only in a small subset of endothelial cells. The alpha4 mRNA was found transiently in embryonic mesenchyme, with particularly high levels in condensed mesenchyme, close to the tips of the ureteric tree where tubulogenesis is initiated. The alpha5 mRNA was strongly expressed by ureter epithelium but not expressed at early stages of tubulogenesis. Immunofluorescence verified low levels of the alpha5 chain in the early stages of tubulogenesis. However, during the capillary loop stage, the alpha5 chain became strongly expressed in the developing glomerular basement membrane, which matches the in situ hybridization results. During subsequent maturation of the kidney, the alpha5 chain became ubiquitously expressed in basement membranes. Overall, the alpha5 chain exhibited the broadest pattern of expression, followed by the alpha1 chain, particularly in the adult stage. These chains were the only ones produced by epithelial cells. Although some basement membranes contained several alpha chains, we failed to detect any of the five studied chains in some basement membranes. Thus, the identity of the alpha chains of many embryonic kidney blood vessels and several basement membranes in the inner medulla in the developing and adult kidney remain unclear.