Biosynthesis, intracellular processing and secretion of haptoglobin in cultured rat hepatocytes

Biosynthesis, intracellular processing and secretion of the hetero-tetrameric (alpha 2 beta 2) glycoprotein, haptoglobin, were studied in primary cultured rat hepatocytes. The results obtained from pulse-chase experiments demonstrated that haptoglobin was initially synthesized as a larger precursor (pro-form), a single polypeptide chain comprising both the alpha- and beta-subunits, and immediately cleaved into subunits during intracellular transport, although about 8% of the newly synthesized haptoglobin was secreted as a pro-form. Monensin which impedes the secretory process at the Golgi complex blocked the complete glycosylation of beta-subunit but rather accelerated the conversion of the pro-form to subunits. These results indicate that the proteolytic processing of the haptoglobin precursor takes place at an early stage before the Golgi complex of the intracellular transport.     

Autocatalytic processing of recombinant human procathepsin L. Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro

The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.     

Altered regulation of a proteinase mediated primary secretion processing system as the basic defect in cystic fibrosis

Depsite the high incidence of cystic fibrosis there is no clear cut direction to research into the basic defect responsible for this disorder. This is, at least in part, due to the exceptional amount of irreproducibility surrounding the disease. The defective ion reabsorption in CF sweat glands may be a prototype of a generalized failure to correctly process exocrine gland primary secretions. Such a physiological defect could account for many of the clinical symptoms of CF. It is hypothesised that one or more secreted proteinase(s) may mediate the events of secretion processing. A mutation in the structural gene for such a proteinase or a regulating anti-proteinase or some enzyme carrying out a functionally important post translational modification of the proteinase would then account for the disease.     

Self-activation of recombinant human lysosomal procathepsin D at a newly engineered cleavage junction, "short" pseudocathepsin D

To obtain a recombinant model of human cathepsin D with kinetic properties that are identical with native human liver enzyme, we have addressed the significant differences in structure and catalytic function between naturally occurring enzyme and bacterially derived pseudocathepsin D. Human procathepsin D was expressed in a baculovirus system to obtain correctly folded, glycosylated enzyme that upon acidification completely converts to the active intermediate, pseudocathepsin D. The oligosaccharide moieties of this recombinant enzyme contributed to about 5% of the apparent molecular mass of the enzyme, and the carbohydrate composition was quite similar to the native material. However, specificity constants (kcat/Km) of this glycosylated pseudoform for several synthetic chromogenic substrates were considerably less (33%-50%) than those for the native enzyme and were virtually identical with those observed with nonglycosylated pseudocathepsin D. A cleavable junction suitable for self-processing at the normal maturation point of human cathepsin D was engineered into procathepsin D according to known specificity requirements of this enzyme, and the construct was expressed using baculovirus. Following experiments that demonstrated that the new proenzyme failed to process to the expected point, the new cleavage junction was moved 6 residues toward the amino terminus of procathepsin D and expressed in Escherichia coli. After refolding, the protein containing the newly engineered junction self-processed, generating a shortened mutant form of pseudocathepsin D that is 6 residues longer at the amino terminus than the native material. The kinetic properties of this newly engineered pseudoform proved to be identical with those of the native enzyme, thus establishing an improved recombinant model for this important aspartic proteinase.     

Apolipoprotein(a) synthesis and secretion from hepatoma cells is coupled to triglyceride synthesis and secretion

Apolipoprotein(a) (apo(a)) is synthesized and secreted from liver cells and represents one of the two major protein components of the atherogenic lipoprotein, Lp(a). Little is known, however, of the factors that regulate the secretion of this protein. We have undertaken an analysis of the response to oleate supplementation in stable clones of HepG2 and McA-RH7777 cells expressing either a 6 K-IV or 17 K-IV isoform of apo(a). These cell lines were examined by pulse-chase analysis and each demonstrated an increase (range 2-6-fold) in apo(a) secretion following supplementation with 0.8 mM oleate. Microsomal membranes, prepared from HepG2 cells expressing a 6 K-IV apo(a) isoform, demonstrated that oleate supplementation increased the apparent protection of apo(a) from protease digestion, suggesting that alterations in the translocation efficiency of apo(a) may accompany the addition of oleate. Cells incubated with brefeldin A demonstrated increased recovery of the precursor form of apo(a) with oleate supplementation, suggesting that alterations in post-translational degradation may also contribute to the observed increase in apo(a) secretion following oleate addition. To further characterize the oleate-dependent increase in apo(a) secretion, cells were incubated with an inhibitor of the microsomal triglyceride transfer protein. These experiments demonstrated a dose-dependent decrease in apo(a) secretion from both cell lines. Furthermore, addition of either the microsomal triglyceride transfer protein inhibitor or triacsin C, an inhibitor of acyl-CoA synthase, completely abrogated the oleate-dependent increase in apo(a) secretion. Taken together, these data provide evidence that apo(a) secretion from hepatoma cells may be linked to elements of cellular triglyceride assembly and secretion.     

Altered bcl-2 and bax expression and intracellular Ca2+ signaling in apoptosis of pancreatic cells and the impairment of glucose-induced insulin secretion

Apoptosis is the process of cellular self-destruction, and genes such as bcl-2 and bax are known to inhibit and promote apoptosis, respectively. In this study, we show that apoptosis can be induced in pancreatic beta-cell lines, and we investigate the apoptotic pathways through the bcl-2 and bax genes and intracellular Ca2+. Serum deprivation induces apoptosis in the MIN6 and RINm5F pancreatic beta-cell lines, and alters the bcl-2 messenger RNA (mRNA) and protein. KCl, BayK, A23187, and ionomycin elicit an elevation of cytosolic/nuclear Ca2+, which, however, is insufficient to evoke apoptosis or to alter bcl-2 or bax mRNA expression in MIN6 cells. The extracellular Ca2+ chelators, EGTA and 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetrapotassium salt, hydrate, evoke apoptosis and also alter the ratio of bcl-2 to bax mRNA and protein concomitantly with the depletion of cytosolic/nuclear Ca2+. This indicates that there are at least two apoptotic pathways in pancreatic beta-cells: through serum deprivation and through a decrease in cytosolic/nuclear Ca2+. MIN6 cells exhibit reduced insulin secretion induced by glucose regardless of the molecular pathway of apoptosis. Apoptosis in pancreatic beta-cells, therefore, may be closely related to the impairment of insulin secretion in certain pathological conditions such as diabetes mellitus.     

Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae

We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast. Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs. 90%). Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER. These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER. Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins. Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant. Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells. These results contradict the hypothesis advanced by Rudolph et al. [Cell 58 (1989) 133-145] that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.     

Altered glycosylation of the env-sea oncoprotein inhibits intracellular transport and transformation

The transforming gene product of the S13 avian erythroblastosis virus, env-sea, is a member of the growth factor receptor class of tyrosine kinases. The env-sea precursor protein gp155env-sea is proteolytically processed into the mature cleavage products gp85env and gp70env-sea, which are subsequently terminally glycosylated and transported to the cell surface. Previous studies have shown that the abnormal glycosylation of gp155env-sea induced by the carbohydrate processing inhibitor castanospermine blocks the proteolytic cleavage of gp155env-sea and impairs its transforming ability. We have shown recently that an uncleaved but fully glycosylated sea-encoded protein retains the ability to transform chicken embryo fibroblasts, indicating that proteolytic processing is not essential for transformation by the env-sea tyrosine kinase. To address the question of how castanospermine blocks transformation by env-sea, differential sucrose gradient centrifugation was performed on env-sea-transformed cells treated with the inhibitor. This report shows that no surface forms of env-sea could be detected in inhibitor-treated cells, suggesting that castanospermine acts by blocking the transport of sea-encoded proteins to the cell surface.     

Expression of rat cathepsin D cDNA in Saccharomyces cerevisiae: implications for intracellular targeting of cathepsin D to vacuoles

To investigate the intracellular transport mechanisms of lysosomal cathepsin D in yeast cells, we produced cathepsin D in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D. Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuoles with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein. To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells. Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction. Immunofluorescence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles. Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.     

High magnesium concentration inhibits ligand-stimulated calcium influx and hormone secretion in rat pituitary lactotropes with involvement of intracellular free magnesium

The effects of extracellular magnesium concentration ([Mg2+]ex) on thyrotropin-releasing hormone (TRH)-stimulated intracellular free calcium mobilization and prolactin secretion were investigated concomitantly with measurement of the intracellular free magnesium concentration ([Mg2+]i). TRH-stimulated intracellular free calcium mobilization was significantly inhibited when the medium was replaced by high Mg2+ medium ([Mg2+]ex = 10 mM) in normal Ca2+ medium. The inhibitory effects of high Mg2+ became apparent concomitantly with an increase in [Mg2+]i from 0.7 to 1.3 mM. High Mg2+ significantly inhibited TRH-induced PRL secretion in a dose-dependent manner in normal Ca2+ medium. TRH-stimulated inositol triphosphate (IP3) production was rather augmented by the replacement with high Mg2+ medium. In summary, high Mg2+ inhibits Ca2+ influx stimulated by TRH in the rat pituitary lactotropes, possibly with the involvement of [Mg2+]i increase. These results have general importance in relation to high Mg(2+)-induced suppression of the biological functions of cells.     

The surgical correction of a deviated anterior maxilla in a horse

The surgical correction of facial deformities of the horse have rarely been undertaken. The surgical and medical management of submucous clefting of the anterior maxilla in a young colt is described.     

YM022, a highly potent and selective CCKB antagonist inhibiting gastric acid secretion in the rat, the cat and isolated rabbit glands

OBJECTIVES: To evaluate the implications of the patterns of weaning on the intake of macronutrients, energy intake, food volume and the energy density in healthy infants in the Mediterranean area of Spain. SUBJECTS AND STUDY DESIGN: Cross-sectional study of 120 clinically-healthy, non-breastfed infants at the ages of 4, 6, 9 and 12 months randomly recruited from three pediatric out-patient clinics. Nutrition data were obtained from the infant's food preparer using the 24-hour dietary recall method. RESULTS: Energy intake/kg body weight was within the recommended daily allowance and did not vary significantly with age (423 kJ/kg body weight at 4 months and 443.7 kJ/kg at 12 months). There was a progressive decrease in the intake volume (p < 0.001) in which carbohydrate-rich foodstuffs were the major factors, and an increase in the energy density (p < 0.001) in which the protein-rich items were the principal contributors. Lipid intake diminished progressively (p < 0.01) to a nadir of 26.4% of energy intake at 9 months of age. In each of the meals there was a tendency towards a progressive increase in energy intake with age. This increase was achieved by a significant increase in energy density (p < 0.001 in all meals, except dinner p < 0.05). Conversely, the intake volume of breakfast, lunch and dinner remained essentially unchanged between 6 and 12 months while that of the mid-evening meal decreased markedly. CONCLUSION: Increased energy requirements for growth is achieved, mainly, by an increase in the energy density rather than the intake volume during food-item diversification in the non-breastfed infant. Cereals were the central food item in the weaning diet in our study sample and which adequately compensates, in terms of energy requirement, for the early reliance on the lipids contained in milk.     

Oxidative metabolism in a rat hepatoma (N13) and isolated rat hepatocytes: a flow cytometric comparative study

Recently, we have developed a new and fast kinetic method for assessing mitochondrial membrane potential by flow cytometry, based on the quantitation of the initial rate of rhodamine 123 (Rh123) uptake by living cells. This test has proved suitable to detect metabolic and toxic effects on mitochondria. To characterize energy metabolism in a rat hepatoma cell line (N13), we applied this method to assess several metabolic pathways that eventually generate mitochondrial membrane potential. Using this approach, we found that N13 hepatoma cells retain an oxidative capacity comparable with that observed in isolated hepatocytes under the same conditions. These results show that this cell line may represent an adequate biological model to perform metabolic and toxicological studies in vitro.     

Altered distribution of lysosomal cathepsin D in ischemic myocardium

To determine the influence of cardiac ischemia on the activity and subcellular localization of lysosomal cathepsin D, anesthetized rabbits were subjected to ligation of the circumflex coronary artery. Total enzyme activity remained unchanged throughout the 2-h ischemic period, but the subcellular distribution of cathepsin D, as analyzed by biochemical and immunohistochemical techniques, was altered dramatically. A marked increase in nonsedimentable (i.e., 40,000-g supernate) activity developed by 30-45 min and increased further by 2 h. Simultaneously, the immunofluorescent localization of cathepsin D was also changed significantly. Within 30-60 min after occlusion, the fine, particulate staining observed in control myocytes was replaced by bright fluorescent patches composed of large granules. Many of these structures displayed prominent halos of diffuse fluorescent staining in the neighboring myocytic cytoplasm, apparently outside lysosomes per se. After 2 h, when nonsedimentable activity was maximally elevated, most of the fluorescent particles had disappeared completely. During this same interim there was no detectable change in the distribution of lysosomal cathepsin D within interstitial cells. These results are consistent with the hypothesis that an early feature of cardiac ischemia is the release of cathepsin D from myocytic lysosomes into the cytosol of damaged cells.     

Selective processing of submandibular rat 1 protein at dibasic cleavage sites. Salivary and bloodstream secretion products

The amino acid sequence of submandibular rat 1 (SMR1) protein, deduced from its cDNA sequence, led to the prediction that the SMR1 gene encodes a hormone-like precursor [Rosinski-Chupin, I., Tronik, D. & Rougeon, F. (1988) Proc. Natl Acad. Sci. USA 85, 8553-8557]. SMR1 contains an N-terminal putative secretory signal sequence and a tetrapeptide (QHNP), located between dibasic amino acids which constitute the most common signal for prohormone processing. We have isolated and characterized from the male rat submandibular gland and its secretions three structurally related peptides, namely an undecapeptide (VRGPRRQHNPR), a hexapeptide (RQHNPR) and a pentapeptide (QHNPR) generated from SMR1 by selective proteolytic cleavages at pairs of arginine residues. The biosynthesis of these peptides is subjected to distinct regulatory pathways depending on the organ, sex and age of the rat. Furthermore, the peptides are differentially distributed in the submandibular gland and in resting or epinephrine-elicited submandibular salivary secretions, suggesting distinct proteolytic pathways for their maturation. The undecapeptide is generated in the gland of both male and female rats, but under basal conditions it is only released into the saliva in male animals. The hexapeptide is produced in large amounts in the gland of adult male rats and released into the saliva in both resting and stimulated conditions. The pentapeptide appears only in the male saliva and is present mostly under stimulated conditions. In addition, administration of epinephrine induces the release of the hexapeptide from the submandibular gland into the bloodstream. The evidence indicates that the rat submandibular gland can function as a dual exocrine and endocrine organ for the SMR1-derived hexapeptide, as has been reported for nerve growth factor, epidermal growth factor, renin and kallikrein. Although the biological activities of the SMR1-derived peptides are not yet known, their high production and adrenergic-induced release only into the saliva and bloodstream of adult male rats, suggest a physiological involvement in some male-specific processes.     

Altered instantaneous and calcium-modulated oscillatory PTH secretion patterns in patients with secondary hyperparathyroidism

The relative contributions of increased parathyroid cell mass and altered control mechanisms of parathyroid hormone (PTH) secretion in secondary hyperparathyroidism are still controversial. In this study, endogenous pulsatile PTH secretion was analyzed by the multiparameter deconvolution technique to differentiate alterations in cell mass-dependent (PTH burst mass) and regulation-dependent (frequency, synchrony, calcium responsiveness) PTH release in uremic patients. PTH concentration versus time profiles were obtained in 13 uremic and 16 healthy adults under baseline conditions and during acute hypo- and hypercalcemia. Plasma PTH half-life was increased in patients compared with control subjects (4.7+/-1.9 versus 2.6+/-0.1 min, P < 0.005). The baseline PTH secretion rate was elevated eightfold in the patients as a result of an increased PTH mass secreted per burst (17.1+/-4.7 versus 2.0+/-0.4 pM, P = 0.0001), higher burst frequency (8.0+/-0.3 versus 6.8+/-0.3 h(-1), P < 0.01), and a higher tonic secretion rate (343+/-99 versus 30+/-4 pM/h, P = 0.0001). Acute hypocalcemia elicited an immediate, frequency- and amplitude-mediated selective increase in the pulsatile secretory component, which was fractionally weaker in patients (+595%) than control subjects (+1755%, P < 0.001). The acceleration and the amplification of PTH bursts were 35 and 60% lower in the patient group. Acute hypercalcemia suppressed total PTH secretion by 79% in control subjects but only by 63% in patients (P < 0.002). PTH burst frequency was reduced during hypercalcemia by 30% in control subjects, but remained unchanged in patients. In conclusion, uremic hyperparathyroidism is mediated by a marked increase in glandular secretion, but also by reduced PTH elimination. The increased spontaneous PTH burst frequency and the blunted responsiveness to changes in Ca2+ indicate partial uncoupling of hyperplastic parathyroid glands from the physiologic regulatory mechanisms that direct pulsatile PTH release.     

Effect of helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin D and on epidermal growth factor degradation

The effect of vacuolating toxin (VacA) from Helicobacter pylori on endosomal and lysosomal functions was studied by following procathepsin D maturation and epidermal growth factor (EGF) degradation in HeLa cells exposed to the toxin. VacA inhibited the conversion of procathepsin D (53 kDa) into both the intermediate (47 kDa) and the mature (31 kDa) form. Nonprocessed cathepsin D was partly retained inside cells and partly secreted in the extracellular medium via the constitutive secretion pathway. Intracellular degradation of EGF was also inhibited by VacA with a similar dose-response curve. VacA did not alter endocytosis, cell surface recycling, and retrograde transport from plasma membrane to trans-Golgi network and endoplasmic reticulum, as estimated by using transferrin, diphtheria toxin, and ricin as tracers. Subcellular fractionation of intoxicated cells showed that procathepsin D and nondegraded EGF accumulate in lysosomes. Measurements of intracellular acidification with fluorescein isothiocyanate-dextran revealed a partial neutralization of the lumen of endosomes and lysosomes, sufficient to account for both mistargeting of procathepsin D outside the cell and the decreased activity of lysosomal proteases.     

Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice

ATP-sensitive K+ (KATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated KATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K+ ion-selective pore of the channel. The homozygous mice (Kir6.2(-/-)) lack KATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca2+]i) of pancreatic beta cells in Kir6.2(-/-) are significantly higher than those in control mice (Kir6.2(+/+)), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca2+]i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2(-/-), as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion, Kir6.2(-/-) show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance test, is increased significantly in Kir6.2(-/-), which could protect Kir6.2(-/-) from developing hyperglycemia. Our data indicate that the KATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest also that the KATP channel in skeletal muscle might be involved in insulin action.     

Effects of nitric oxide on leukotriene D4 decreased bile secretion in the perfused rat liver

Nitric oxide (NO) and leukotrienes are potent vasoactive agents that are involved in the control of portal blood flow. The present study investigated the role of leukotriene D4 and NO in a non-recirculating constant pressure rat liver perfusion model to analyse their interchanges on portal flow and bile secretion. The addition of leukotriene D4 (20 nM) to the perfusate for 5 minutes resulted in a decrease in portal blood flow (-55.3%), in bile flow (-24.4%) as well as bile acid release (-35.2%). In parallel, leukotriene D4 increased glucose output. The administration of a lower dose of leukotriene D4 (5 nM) reduced the respective parameters to a lesser degree, indicating dose-dependence. The addition of NO via the infusion of sodium nitroprusside (0.05 mM, 1 mM) reduced the effect of leukotriene D4 on portal flow, bile flow and bile acid secretion whereas the leukotriene D4 effects on hepatic glucose output remained unaffected. Correlation coefficient between decrease in portal flow and reduction of bile flow by infusing leukotriene D4 was R = 0.91, while in the presence of sodium nitroprusside R = 0.85. These results suggest that the leukotriene D4-induced cholestasis is dependent on portal flow. In contrast, hepatic vasoconstriction does not contribute to glycogenolysis stimulated by leukotriene D4 in the perfused liver.     

Purification and characterization of a DNA binding protein in a nuclear scaffold fraction from rat ascites hepatoma cells

Our most recent work [Hibino et al. (1995) Cancer Lett., 88, 49-55] has shown that the selective binding affinities of highly repetitive DNA components for a nuclear scaffold protein from rat ascites hepatoma cells (P230) depend on the degree of sequence-directed bending of the helix axis. In the present experiment, this protein has been highly purified and isolated by a series of column chromatographic procedures to migrate as a single band to a molecular weight position of 230 kd on a SDS-polyacrylamide gel. A filter binding assay showed that the binding of a repetitive AT-rich component (369 bp XmnI fragment) from the hepatoma nucleus, which has a strongly bent overall structure, to isolated P230 is based on a cooperative mode of interaction. Distamycin A, which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding to this protein. These results suggest that AT-rich regions in highly repetitive DNA cause bending of the helix axis to be recognized by nuclear scaffold protein(s). Moreover, it has been shown that the nuclear scaffold fraction from rat liver or an actively growing hepatocyte cell line (Ac2F cells) does not contain P230, but does have a repetitive bent DNA binding protein (P130), which has an apparent molecular weight of 130 kd. In addition, the immunoblot analysis showed that mouse anti-P130 antiserum reacts with P230. Thus, the results in the present study imply that there is some difference in the higher order structure of the nuclear DNA attachment region between rat liver or actively growing hepatocytes and the hepatoma, although P230 appears to be immunochemically similar to P130.