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Blepharospasm is traditionally thought to be a movement disorder that results from basal ganglia dysfunction. Recently, accumulating morphometric studies have revealed structural alterations outside the basal ganglia, such as in the brainstem, cerebellum and sensorimotor cortex, suggesting that blepharospasm may result from network disorders. However, the temporal and causal relationships between structural alterations and whether there are disease duration-related hierarchical structural changes in these patients remain largely unknown. Structural MRI was performed in 62 patients with blepharospasm, 62 patients with hemifacial spasm and 62 healthy controls to assess the structural alterations using voxel-based morphology and structural covariance networks. The use of the causal structural covariance network, modularity analysis and functional decoding were subsequently performed to map the causal effect of grey matter change pattern, hierarchical topography and functional characterizations of the structural network throughout the disease duration of blepharospasm. Greater grey matter volume in the left and right supplementary motor areas was identified in patients with blepharospasm compared to that in patients with hemifacial spasm and healthy controls, whereas no significant difference was identified between patients with hemifacial spasm and healthy controls. In addition, increased grey matter volume covariance between the right supplementary motor area and right brainstem, left superior frontal gyrus, left supplementary motor area and left paracentral gyrus was found in patients with blepharospasm compared to healthy controls. Further causal structural covariance network, modularity analysis and functional decoding showed that the right supplementary motor area served as a driving core in patients with blepharospasm, extending greater grey matter volume to areas in the cortico-basal ganglia–brainstem motor pathway and cortical regions in the vision–motor integration pathway. Taken together, our results suggest that the right supplementary motor area is an early and important pathologically impaired region in patients with blepharospasm. With a longer duration of blepharospasm, increased grey matter volume extends from the right supplementary motor area to the cortico-basal ganglia motor and visual–motor integration pathways, showing a hierarchy of structural abnormalities in the disease progression of blepharospasm, which provides novel evidence to support the notion that blepharospasm may arise from network disorders and is associated with a wide range of grey matter abnormalities.

Summary 1. The aim of the present study was to investigate the effect of prostaglandin (PG) E1 on hypoxia/re-oxygenation (H/R) apoptosis and the expression of bcl-2 and bax in cultured neonatal rat cardiomyocytes. 2. The H/R model was made using the first generation of cultured neonatal rat cardiomyocytes. Hypoxia/re-oxygenation apoptosis was studied by electron microscopy and agarose gel electrophoresis. The percentage of apoptotic cells was measured by terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labeling (TUNEL). The expression of bcl-2 and bax was detected by in situ hybridization and immunohistochemical staining. 3. Most cells of the H/R group tested by electron microscopy showed cytoplasmic concentration, nuclear chromatin condensation and margination. Prostaglandin E1 (5, 15 and 45 μg/L) relieved the injury. The results of DNA electrophoresis in the H/R group showed a typical DNA ladder and the DNA ladder decreased gradually corresponding with increasing doses of PGE1. 4. The TUNEL staining showed that the total number of apoptotic cells in the H/R group was much more than that in the PGE1 (45 μg/L) group. 5. The results of in situ hybridization and immunohistochemical staining showed that the bcl-2 content in the H/R group was lower than that in the control group; bax content showed the reverse. Compared with the H/R group, bcl-2 content was significantly higher in the PGE1 (5, 15 and 45 μg/L) groups. However, bax content in the PGE1 (5, 15 and 45 μg/L) groups was significantly lower than that in the H/R group. 6. In conclusion, H/R injury can induce cardiomyocyte apoptosis. Prostaglandin E1 obviously has anti-apoptotic effects on cardiomyocytes and the mechanisms probably involve the inhibition of bax expression and increased expression of bcl-2.

The galactosyltransferase, GalT-4, which catalyses the biosynthesis in vitro of neolactotetraosylceramide, nLcOse4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (Glc NAc beta 1-3Gal beta 1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc beta 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn2+). The K(m) values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 microM, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-alpha 1-acid glycoprotein or N-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc beta 1-1-3Gal beta 1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for an N-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH2 beta 1-3Gal beta 1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while the K(m) and Vmax of the reacetylated substrate (GlcNAc beta 1-3Gal beta 1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.

Twenty-three of the 25 cysteine residues in apolipoprotein B-100 have been isolated directly from tryptic or peptic peptide mixtures. Sixteen cysteine residues exist in disulfide forms: Cys-1-Cys-3, Cys-2-Cys-4, Cys-5-Cys-6, Cys-7-Cys-8, Cys-9-Cys-10, Cys-11-Cys-12, Cys-13-Cys-14, and Cys-20-Cys-21. All of these except Cys-20-Cys-21 are recently discovered disulfide linkages. In addition to Cys-22 and Cys-24, which have been described as sulfhydryls on low density lipoprotein, Cys-15 to Cys-18 and Cys-23 are in the reduced form. Cys-19 and Cys-25 are not yet confirmed. Our results revealed that all identified disulfide linkages are located in the trypsin-releasable regions and that all except Cys-1-Cys-3 and Cys-2-Cys-4 are linked to the neighboring cysteine. We propose a linear model of apolipoprotein B-100 in low density lipoprotein that wraps around the low density lipoprotein molecule.

The complete amino acid sequence of chicken plasma apolipoprotein (apo) A-I was determined by sequencing overlapping peptide fragments produced by trypsin, S. aureus V8 protease, and cyanogen bromide cleavage respectively. All of the peptide fragments were purified on a Waters or on a Beckman HPLC system with a Vydac C18 column using 0.1% TFA in water as buffer A, and 0.08% TFA in 95% acetonitrile and 5% water as buffer B. Most of the peaks separated by these systems were pure. The partially purified fractions were subjected to rechromatography with a Hypersil ODS column using 0.005M sodium phosphate, pH 6.0, as buffer A, and 90% acetonitrile and 10% water as buffer B. The N-terminus of chicken apo A-I was determined to be aspartic acid by directly sequencing the intact protein up to 30 residues, while the C-terminus was identified as alanine by carboxypeptidase Y cleavage. There are 240 amino acid residues in mature chicken apo A-I. By direct analysis of cyanogen bromide peptide, we also determined the sequence of a 6 amino acid prosegment, which is present at approximately 10% of the molar amount of the mature protein in chicken plasma.

The aim of the present study was to investigate the effect of prostaglandin (PG) E1 on hypoxia/re-oxygenation (H/R) apoptosis and the expression of bcl-2 and bax in cultured neonatal rat cardiomyocytes. The H/R model was made using the first generation of cultured neonatal rat cardiomyocytes. Hypoxia/re-oxygenation apoptosis was studied by electron microscopy and agarose gel electrophoresis. The percentage of apoptotic cells was measured by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL). The expression of bcl-2 and bax was detected by in situ hybridization and immunohistochemical staining. Most cells of the H/R group tested by electron microscopy showed cytoplasmic concentration, nuclear chromatin condensation and margination. Prostaglandin E1 (5, 15 and 45 microg/L) relieved the injury. The results of DNA electrophoresis in the H/R group showed a typical DNA ladder and the DNA ladder decreased gradually corresponding with increasing doses of PGE1. The TUNEL staining showed that the total number of apoptotic cells in the H/R group was much more than that in the PGE1 (45 microg/L) group. The results of in situ hybridization and immunohistochemical staining showed that the bcl-2 content in the H/R group was lower than that in the control group; bax content showed the reverse. Compared with the H/R group, bcl-2 content was significantly higher in the PGE1 (5, 15 and 45 microg/L) groups. However, bax content in the PGE1 (5, 15 and 45 microg/L) groups was significantly lower than that in the H/R group. 6. In conclusion, H/R injury can induce cardiomyocyte apoptosis. Prostaglandin E1 obviously has anti-apoptotic effects on cardiomyocytes and the mechanisms probably involve the inhibition of bax expression and increased expression of bcl-2.

To investigate the effect of G(alphaq/11) signaling pathway and ATP-sensitive potassium channels (K(ATP) channels) on prostaglandin E1 (PGE1) induced early and delay-preconditioning protection in rat hearts.Two series of experiments were performed in Wistar rat hearts. In the first series of experiment, all rats were pretreated with PGE1 40 min or 23 h 20 min before the experiment. Ischemia-reperfusion injury was induced by 30 min coronary artery occlusion followed by 90 min reperfusion. Hemodynamics, infarct size, and scores of ventricular arrhythmias were measured. The expression of G(alphaq/11) protein in the heart was measured by Western blot analysis in the second series.Preconditioning with PGE1 (25 microg/kg) markedly reduced infarct size, left ventricular end-diastolic pressure, and scores of ventricular arrhythmia. The effect of PGE1 was significantly attenuated by glibenclamide (1 mg/kg, ip), a nonselective K(ATP) channel inhibitor. PGE1 caused a significant increase in the expression of G(alphaq/11) protein.Activations of G(alphaq/11) signal pathway and K(ATP) channel played significant roles in the cardioprotection of PGE1 preconditioning in rat heart and might be an important mechanism of signal transduction pathway during the PGE1 preconditioning.

The complete amino acid sequence of proapolipoprotein (proapo) A-I of chicken high density lipoproteins was determined by sequencing overlapping peptides produced by trypsin, S. aureus V8 protease, and cyanogen bromide cleavage. There are 240 amino acid residues in mature chicken apoA-I. By direct sequence analysis of a cyanogen bromide peptide, we also determined the sequence of a 6-amino-acid prosegment which is present at approx. 10% the molar amount of the mature peptide in chicken plasma. Sequence comparison among apoA-I from chicken, human, rabbit, dog and rat, and secondary structure analysis indicate that while the degree of sequence homology is only moderate ( < 50% between chicken and man), there is good conservation of apoA-I secondary structure, especially in the N-terminal two-thirds of the protein in these widely separated species.

The primary structure of human apolipoprotein (apo) B-48 has been deduced and shown by a combination of DNA excess hybridization, sequencing of tryptic peptides, cloned complementary DNAs, and intestinal messenger RNAs (mRNAs) to be the product of an intestinal mRNA with an in-frame UAA stop codon resulting from a C to U change in the codon CAA encoding Gln2153 in apoB-100 mRNA. The carboxyl-terminal Ile2152 of apoB-48 purified from chylous ascites fluid has apparently been cleaved from the initial translation product, leaving Met2151 as the new carboxyl-terminus. These data indicate that approximately 85% of the intestinal mRNAs terminate within approximately 0.1 to 1.0 kilobase downstream from the stop codon. The other approximately 15% have lengths similar to hepatic apoB-100 mRNA even though they have the same in-frame stop codon. The organ-specific introduction of a stop codon to a mRNA appears unprecedented and might have implications for cryptic polyadenylation signal recognition and RNA processing.