Correlation between thyroid hormone status and hepatic hyperplasia and hypertrophy caused by the peroxisome proliferator-activated receptor alpha agonist Wy-14,643

BACKGROUND: The metabolic inhibitor rotenone inhibits hepatocellular proliferation and the incidence of liver cancer resulting from exposure to the PPARalpha agonist Wy-14,643, via unknown mechanisms. Since the absence of thyroid hormones diminishes hepatomegaly, an early biomarker for the hepatocarcinogenicity induced by PPARalpha agonists, this study was undertaken to investigate whether rotenone might interference with the ability of Wy-14,643 to alter the animal thyroid status. METHODS: Male B6C3F1 mice were given Wy-14,643 (100 ppm), rotenone (600 ppm) or a mixture of both, in the feed for 7 days. Bromodeoxyuridine (BrDU), marker of cell replication, was delivered through subcutaneously implanted osmotic mini-pumps. At the end of the experiment, sera were collected and corticosterone and thyroid hormone levels were measured by solid-phase radioimmunoassay kits. In addition, liver tissue samples were stained immunohistochemically for BrDU to determine percentages of labeled cells. Further, cell surface area was determined from images generated by a Zeiss Axioplan microscope equipped with a plan Neofluar x40 0.75 na objective. Tracings of individual hepatocyte perimeters were then analyzed and cell-surface areas were calculated using MicroMeasure FL-4000. RESULTS: Wy-14,643 caused a significant increase in liver weights, hepatocyte BrDU labeling index (LI), and hepatocyte surface area. In animals which received both Wy-14,643 and rotenone simultaneously, all of these effects were significantly less pronounced compared with mice that received Wy-14,643 alone. Rotenone alone decreased liver weights, LI and surface area. The Free Thyroid Index (FTI), which provides an accurate reflection of the animal's thyroid status, was 5.0 +/- 0.3 in control mice. In animals exposed to rotenone, these values decreased to 2.0 +/- 0.9, but in animals which received Wy-14,643, levels increased significantly to 7.7 +/- 0.9. FTI values decreased to 3.4 +/- 0.8 in mice receiving both rotenone and Wy-14,643. CONCLUSION: A strong correlation was observed between the animal thyroid status and both, hepatocyte proliferation (r2 = 0.62), and hepatocyte surface area (r2 = 0.83). These results support the hypothesis that the thyroid status of the animal plays a role in PPARalpha-induced hepatocellular proliferation and liver cell enlargement. Both these events are known to contribute to the expression of liver cancer in response to the activation of PPARalpha.     

Gamma-Irradiated Venezuelan Equine Encephalitis Vaccines

The efficacy of Formalin-inactivated Venezuelan equine encephalitis (VEE) vaccine has been reported to be low for man. Although a live VEE vaccine has been shown to be highly effective for the protection of laboratory workers, local and systemic reactions have occurred in approximately 20% of inoculated individuals. Therefore, studies were initiated in an attempt to produce an inactivated vaccine of high potency with low toxicity. Inactivated VEE vaccines were prepared by exposing virus suspensions to 8 x 10(6) or 10 x 10(6) r of gamma radiation. Irradiated VEE vaccines prepared from virus suspensions produced in Maitland-type chick embryo (MTCE) cell cultures and in monolayer cultures of human diploid strain WI-38 cells were highly immunogenic for mice and guinea pigs. Guinea pigs vaccinated with a series of three inoculations of vaccine (MTCE) survived challenge with at least 10(8.4) mouse intracerebral 50% lethal doses of VEE virus. Irradiated vaccines induced high levels of serum-neutralizing and hemagglutinin-inhibiting antibodies in guinea pigs and rabbits. These findings suggest that ionizing radiation may be effective in the preparation of an inactivated VEE vaccine.     

Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.     

BioGraph: unsupervised biomedical knowledge discovery via automated hypothesis generation

We present BioGraph, a data integration and data mining platform for the exploration and discovery of biomedical information. The platform offers prioritizations of putative disease genes, supported by functional hypotheses. We show that BioGraph can retrospectively confirm recently discovered disease genes and identify potential susceptibility genes, outperforming existing technologies, without requiring prior domain knowledge. Additionally, BioGraph allows for generic biomedical applications beyond gene discovery. BioGraph is accessible at .     

Genomic organization and regulation by dietary fat of the uncoupling protein 3 and 2 genes

Uncoupling protein-1 (UCP1) dissipates the transmitochondrial proton gradient as heat. UCP2 and UCP3 are two recently discovered homologues that also have uncoupling activity and thus presumably have a role in energy homeostasis. We now report the genomic structure of murine UCP3 (7 exons) and UCP2 (8 exons). UCP3 is approximately 8 kilobases upstream of UCP2. An UCP3 variant mRNA, UCP3S, was also found and characterized. The effect of a high fat diet (45% versus 10%) on UCP3 and UCP2 mRNA levels was measured. Eating the 45% fat diet for eight weeks caused greater weight gain in AKR and C57BL/6J mice than in the obesity-resistant A/J mice. The high fat diet increased muscle UCP3 expression twofold in C57BL/6J animals. UCP2 expression increased slightly on the 45% fat diet in white adipose of AKR mice, but not in A/J or C57BL/6J mice. In skeletal muscle, UCP2 expression showed little variation with diet. Thus, UCP2 and UCP3 expression levels change in response to diet-induced obesity, but the changes are modest and depend on the tissue and genotype. The data suggest that it is not a reduction in UCP2 or UCP3 expression that causes obesity in the susceptible mice.     

Patch-clamp recording of charge movement, Ca(2+) current, and Ca(2+) transients in adult skeletal muscle fibers

Intramembrane charge movement (Q), Ca(2+) conductance (G(m)) through the dihydropyridine-sensitive L-type Ca(2+) channel (DHPR) and intracellular Ca(2+) fluorescence (F) have been recorded simultaneously in flexor digitorum brevis muscle fibers of adult mice, using the whole-cell configuration of the patch-clamp technique. The voltage distribution of Q was fitted to a Boltzmann equation; the Q(max), V(1/2Q), and effective valence (z(Q)) values were 41 +/- 3.1 nC/&mgr;F, -17.6 +/- 0.7 mV, and 2.0 +/- 0.12, respectively. V(1/2G) and z(G) values were -0.3 +/- 0.06 mV and 5.6 +/- 0.34, respectively. Peak Ca(2+) transients did not change significantly after 30 min of recording. F was fit to a Boltzmann equation, and the values for V(F1/2) and z(F) were 6.2 +/- 0.04 mV and 2.4, respectively. F was adequately fit to the fourth power of Q. These results demonstrate that the patch-clamp technique is appropriate for recording Q, G(m), and intracellular [Ca(2+)] simultaneously in mature skeletal muscle fibers and that the voltage distribution of the changes in intracellular Ca(2+) can be predicted by a Hodgkin-Huxley model.     

Multiple actins in drosophila melanogaster

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.     

Studies of the synthesis,structure and function of the phosphorylated oligosaccharides of lysosomal enzymes

Newly synthesized lysosomal enzymes were found to contain N-acetylglucosamine residues in phosphodiester linkage to the 6 position of the mannose residues on high-mannose type oligosaccharides. The formation of these structures was shown to be catalyzed by a specific N-acetylglucosaminylphosphotransferase enzyme, that utilises UDP-N-acetylglucosamine as a donor. The phosphorylation reaction can take place on any of four or five positions on the high-mannose oligosaccharide. Subsequently an α-N-acetylglucosaminylphosphodiesterase removes the outer blocking N-acetylglucosamine residues to generate the mature phosphomannsoyl recognition signal. This signal is responsible for the targetting of newly synthesized lysosomal enzymes to lysosomes. The human syndromes of I-cell disease (Mucolipidosis II) and pseudo-Hurler polydystrophy (Mucolipidosis III) were shown to be caused by deficiency of the first enzyme in the pathway, the UDP-N-acetylglucosamine: Glycoprotein N-acetylglucosaminylphosphotransferase.