Antibodies to hepatocyte membrane antigens in chronic liver disease: detection by immunofluorescence after Bouin's fixation

Using a modified indirect immunofluorescent (IF) technique in which cryostat tissue sections were fixed in Bouin's solution for ten minutes prior to reaction with sera under test, we have looked for antibodies to the hepatocyte membrane (HMA) in the sera of patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC). Samples were tested initially in parallel on unfixed and Bouin's-fixed rat composite blocks (kidney, stomach, liver) at a titer of 1:100 and those found to be positive were diluted further and reexamined. Conventional unfixed sections of rat composite block showed no liver membrane immunofluorescence although antinuclear (ANA), mitochondrial (AMA), and smooth muscle antibodies (SMA) were detected as anticipated. By contrast, prior Bouin's fixation abolished most of the fluorescence due to ANA, AMA and SMA but resulted in brilliant fluorescence of rat hepatocyte membranes in eleven of twelve patients with CAH (93%) and all ten patients with PBC. Only one of 22 normals (5%), one of 20 with collagen-vascular diseases (5%), and one of seven with nonimmunologic liver disease (14%) were positive for this hepatocyte membrane antibody. Bouin's fixation prior to IF is a simple technique which appears to alter the hepatocyte membrane so that HMA become detectable. The strong association of HMA with CAH and PBC suggests that this test might be of value and may contribute towards a further understanding of the pathogenesis of these conditions.     

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O₂ levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H₂O₂ reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H₂O₂-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O₂. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.     

Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding

The equilibrium folding of a series of self-complementary RNA duplexes and the unmodified yeast tRNA(Phe) is studied as a function of urea and Mg(2+) concentration with optical spectroscopies and chemical modification under isothermal conditions. Via application of standard methodologies from protein folding, the folding free energy and its dependence on urea concentration, the m value, are determined. The free energies of the RNA duplexes obtained from the urea titrations are in good agreement with those calculated from thermal melting studies [Freier, S. I., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373]. The m value correlates with the length of the RNA duplex and is not sensitive to ionic conditions and temperature. The folding of the unmodified yeast tRNA(Phe) can be described by two Mg(2+)-dependent transitions, the second of which corresponds to the formation of the native tertiary structure as confirmed by hydroxyl radical protection and partial nuclease digestion. Both transitions are sensitive to urea and have m values of 0.94 and 1.70 kcal mol(-)(1) M(-)(1), respectively. Although the precise chemical basis of urea denaturation of RNA is uncertain, the m values for the duplexes and tRNA(Phe) are proportional to the amount of the surface area buried in the folding transition. This proportionality, 0.099 cal mol(-)(1) M(-)(1) A(-)(2), is very similar to that observed for proteins, 0.11 cal mol(-)(1) M(-)(1) A(-)(2) [Myers, J., Pace, N., and Scholtz, M. (1995) Protein Sci. 4, 2138]. These results indicate that urea titration can be used to measure both the free energy and the magnitude of an RNA folding transition.     

Extracellular matrix-mediated tissue remodeling following axial movement of teeth.

Tooth eruption is a multifactorial process involving movement of existing tissues and formation of new tissues coordinated by a complex set of genetic events. We have used the model of the unopposed rodent molar to study morphological and genetic mechanisms involved in axial movement of teeth. Following extraction of opposing upper molars, lower molars supererupted by 0.13 mm. Labeled tissue sections revealed significant amounts of new bone and cementum apposition at the root apex of the unopposed side following supereruption for 12 days. Newly apposited cementum and alveolar bone layers were approximately 3-fold thicker in the experimental vs the control group, whereas periodontal ligament width was maintained. Tartrate-resistant acid phosphatase staining indicated bone resorption at the mesial alveolar walls of unopposed molars and provided in tandem with new bone formation at the distal alveolar walls an explanation for the distal drift of molars in this model. Microarray analysis and semiquantitative RT-PCR demonstrated a significant increase in collagen I, integrin beta5, and SPARC gene expression as revealed by comparison between the unopposed molar group and the control group. Immunohistochemical verification revealed increased levels of integrin beta5 and SPARC labeling in the periodontal ligament of the unopposed molar. Together our findings suggest that posteruptive axial movement of teeth was accomplished by significant formation of new root cementum and alveolar bone at the root apex in tandem with upregulation of collagen I, integrin beta5, and SPARC gene expression.     

Analysis of Drosophila species genome size and satellite DNA content reveals significant differences among strains as well as between species

The size of eukaryotic genomes can vary by several orders of magnitude, yet genome size does not correlate with the number of genes nor with the size or complexity of the organism. Although "whole"-genome sequences, such as those now available for 12 Drosophila species, provide information about euchromatic DNA content, they cannot give an accurate estimate of genome sizes that include heterochromatin or repetitive DNA content. Moreover, genome sequences typically represent only one strain or isolate of a single species that does not reflect intraspecies variation. To more accurately estimate whole-genome DNA content and compare these estimates to newly assembled genomes, we used flow cytometry to measure the 2C genome values, relative to Drosophila melanogaster. We estimated genome sizes for the 12 sequenced Drosophila species as well as 91 different strains of 38 species of Drosophilidae. Significant differences in intra- and interspecific 2C genome values exist within the Drosophilidae. Furthermore, by measuring polyploid 16C ovarian follicle cell underreplication we estimated the amount of satellite DNA in each of these species. We found a strong correlation between genome size and amount of satellite underreplication. Addition and loss of heterochromatin satellite repeat elements appear to have made major contributions to the large differences in genome size observed in the Drosophilidae.     

Homologous desensitization of signalling by the alpha (alpha) isoform of the human thromboxane A2 receptor: a specific role for nitric oxide signalling

Thromboxane (TX) A(2) plays a central role in hemostasis, regulating platelet activation status and vascular tone. We have recently established that the TP beta isoform of the human TXA(2) receptor (TP) undergoes rapid, agonist-induced homologous desensitization of signalling largely through a G protein-coupled receptor kinase (GRK) 2/3-dependent mechanism with a lesser role for protein kinase (PK) C. Herein, we investigated the mechanism of desensitization of signalling by the TP alpha isoform. TP alpha undergoes profound agonist-induced desensitization of signalling (intracellular calcium mobilization and inositol 1,4,5 trisphosphate generation) in response to the TXA(2) mimetic U46619 but, unlike that of TP beta, this is independent of GRKs. Similar to TP beta, TP alpha undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, PKC mechanism where Ser(145) within intracellular domain (IC)(2) represents the key phospho-target. TP alpha also undergoes more profound sustained PKC- and PKG-dependent desensitization where Thr(337) and Ser(331), respectively, within its unique C-tail domain were identified as the phospho-targets. Desensitization was impaired by the nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and PKG inhibitors L-NAME, LY 83583 and KT5823, respectively, indicating that homologous desensitization of TP alpha involves nitric oxide generation and signalling. Consistent with this, U46619 led to rapid phosphorylation/activation of endogenous eNOS. Collectively, data herein suggest a mechanism whereby agonist-induced PKC phosphorylation of Ser(145) partially and transiently impairs TP alpha signalling while PKG- and PKC-phosphorylation at both Ser(331) and Thr(337), respectively, within its C-tail domain profoundly desensitizes TP alpha, effectively terminating its signalling. Hence, in addition to the agonist-mediated PKC feedback mechanism, U46619-activation of the NOS/sGC/PKG pathway plays a significant role in inducing homologous desensitization of TP alpha.