Characterization of lamin proteins in BHK cells

Lamins are structural proteins found in rat liver nuclear envelope and are major constituents of the nuclear matrix. 2-D gel electrophoresis indicates that BHK cell nuclear matrix is composed of four major proteins (62 kD, 68 kD, 70 kD and 72 kD). Three of these proteins are very similar to lamins A, B and C of rat liver nuclear envelope according to their molecular mass and isoelectric points. An anti-serum specific to BHK matrix proteins has been raised. On 2-D immunoblot, this serum detects all the 62, 68 and 72 kD polypeptide isovariants but only one of the two isovariants of the 70 kD polypeptide. Rat lamins A, B and C react with the anti-BHK matrix serum. However, when a monoclonal antibody to rat liver lamins A, B and C is used (Burke, B, Tooze, J & Warren, G, EMBO j 2 (1983) 361 [23]), only the 72 kD (lamin A-like) and the 62 kD (lamin C-like) BHK polypeptides are detected. Our results suggest that although a strong similarity exists between BHK and rat lamins, there is no identical cross-reactivity between the two species.     

Pancreatic B-cells secrete a range of novel peptides besides insulin

Insulin secretion by a transplantable rat islet B-cell tumour is accompanied by the release of two putative proinsulin cleavage intermediates, four peptides of M_r 9000–12 000 (excluding proinsulin) and peptides of M_r 21 000, 34 000 and 60 000. Granule-enriched subcellular preparations contain major peptides of identical M_r values. Of these peptides seven at least coincide in molecular weight with peptides secreted by isolated rat islets and thus may be constituents of the normal insulin secretory granule.     

Biliary protein output by isolated perfused rat livers. Effects of bile salts.

The output of proteins into bile was studied by using isolated perfused rat livers. Replacement of rat blood with defined perfusion media deprived the liver of rat serum proteins (albumin, immunoglobulin A) and resulted in a rapid decline in the amounts of these proteins in bile. When bovine serum albumin was incorporated into the perfusion medium it appeared in bile within 20 min and the amount in the bile was determined by the concentration of the protein in the perfusion medium. The use of a defined perfusion medium also deprived the livers of bile salts and the amounts of these, and of plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I], in bile declined rapidly. Introduction of micelle-forming bile salts (taurocholate or glycodeoxycholate) to the perfusion medium 80 min after liver isolation markedly increased the output of plasma-membrane enzymes but had no effect on the other proteins. The magnitude of this response was dependent on the bile salt used and its concentration in bile; there was little effect on plasma-membrane enzyme output until the critical micellar concentration of the bile salt had been exceeded in the bile. A bile salt analogue, taurodehydrocholate, which does not form micelles, did not produce the enhanced output of plasma-membrane enzymes. This work supports the view that the output of plasma-membrane enzymes in bile is a consequence of bile salt output and also provides evidence for mechanisms by which serum proteins enter the bile.     

Long term organ culture of rat liver rudiment in a synthetic medium: morphological and biochemical development.

Embryonic rat liver rudiments were grown and maintained in a chemically defined medium for up to 16 weeks. Liver explants grew and developed liver plates, bile canaliculi and ductules. Liver cells differentiated and, by the end of the second culture week, revealed cytological and ultrastructural features of adult rat liver cells. Glycogen synthetase activity appeared and reached a plateau within 2 weeks.Plasma proteins produced by the explants appeared sequentially in the culture medium. Their relative concentrations in the medium increased progressively to a plateau and they persisted throughout the culture period. Albumin was present in trace amounts during early days of culture. Its relative concentration increased and it became the major protein constituent of the medium by the second week.Transferrin, α₁M-globulin, haptoglobin, β₁c- and α₁B-globulin appeared in that order. The three fibrinogen subunits were identified during the first week of culture.Fetal proteins were identified during the early culture period, the most prominent being α-fetoprotein. The relative concentration of fetal proteins declined steadily as the culture progressed.     

Two-dimensional electrophoresis of membrane proteins factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI > 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112–120) gives good resolution of proetins of pI 5–8, including cytochromes P-450, but fails to resolve a major acidic protein of pI < 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R·N⁺(CH₃)₂·CH₂CH₂CH₂SO₃^? are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C₁₄ or C₁₆) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Kupffer cell-derived cyclooxygenase-2 regulates hepatocyte Bcl-2 expression in choledocho-venous fistula rats

We have previously demonstrated that after bile duct ligation hepatocytes express Bcl-2, although the mechanisms regulating Bcl-2 expression were not identified. Our aim was to determine if biliary constituents induce hepatocellular expression of Bcl-2 by a cyclooxygenase-2 (COX-2)-dependent mechanism. We used the choledocho-venous fistula (CVF) rat model for these studies and inhibited COX-2 by feeding the animals nimesulide, a selective inhibitor of COX-2 activity. Serum bile acids were 70-fold greater in CVF animals compared with controls, although liver histology and serum alanine aminotransferase values remained normal for the duration of the study. Neither Bcl-2 nor COX-2 was detected in sham-operated animals. However, Bcl-2 was expressed in hepatocytes but not in other liver cells in the CVF animals. In contrast, COX-2 protein was identified in Kupffer cells but not in hepatocytes of CVF animals. Hepatic Bcl-2 protein expression was fourfold lower in the livers from nimesulide-treated CVF rats. In conclusion, high circulating concentrations of biliary constituents are associated with stimulation of de novo hepatocyte expression of Bcl-2 and Kupffer cell expression of COX-2. These data suggest Kupffer cell-derived prostanoids may regulate Bcl-2 expression in the hepatocyte.     

Heterogeneity of insulin-like growth factor binding proteins between structure and affinity. 2. Forms released by human and rat liver in culture

Since the liver is considered to be the major source both of circulating insulin-like growth factors (IGFs) and of their specific binding proteins (BPs), human and rat liver explants were cultured in serum-free medium with a view to characterizing the binding proteins released into the medium and to comparing them with serum binding proteins. In the culture media, as in the serum, IGFs are associated with their binding proteins in the form of complexes. In gel filtration experiments the liver IGF-BP complexes eluted as a single, homogeneous peak with a relative molecular mass of about 40,000, which is similar to that of the 'small' complex of serum. Their sedimentation coefficient, estimated from sucrose gradient centrifugation, was 2.9 S. Polyacrylamide gel electrophoresis (SDS-PAGE) of human liver culture media, in which the binding proteins were cross-linked to 125I-labelled IGF I revealed molecular heterogeneity. Three specific bands corresponding to Mr 46,000, 40,000 and 37,000 were observed, which resemble those of the serum small complex, but none of the higher-Mr bands seen in serum. SDS-PAGE followed by transfer onto nitrocellulose and incubation with 125I-labelled IGF I (western blot) led to the identification in human liver culture media of five molecular forms of binding protein with Mr of 41,500, 38,500, 34,000, 30,000 and 24,000, identical to those seen in serum. The relative concentrations of the 41,500 and 38,500-Mr forms varied from one medium to another, but the 34,000 and 30,000-Mr forms were regularly more abundant in the liver culture media than in normal serum. The binding proteins produced by the liver therefore represent the native forms in the circulation (although this does not exclude other sources). The absence of high-Mr IGF-BP complexes in the liver culture media, and yet the presence of the 41,500 and 38,500-Mr forms, which are the only binding units of the serum 'large' complex (150,000 Mr), indicates that these two binding proteins are capable of binding IGFs to form 'monomeric' IGF-BP complexes. Western-blot analysis of rat binding proteins revealed a certain analogy with the human proteins, three forms having their Mr between 43,000 and 39,000 and three between 32,000 and 24,000. Liver binding proteins in human adults and foetuses were found to be identical, whereas in the case of serum the 41,500 and 38,500-Mr forms were more abundant in the adult and the 34,000 and 30,000-Mr forms more abundant in the foetus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrophoretic analysis of Campostoma anomalum,Rhinichthys cataractae and their F1 offspring

Campostoma anomalum, Rhinichthys cataractae and their F₁ hybrids were examined electrophoretically for 44 enzymatic loci, general muscle, and serum proteins. Of the 44 loci scored, acid phosphatase (ACP-B), alkaline phosphatase (AKP-A), esterase (EST-B), α-glycerophosphate dehydrogenase (GPD-A), malate dehydrogenase (MDH-A) and phosphoglucomutase (PGM-A) showed hybrid inheritance patterns. Serum proteins also demonstrated additive inheritance patterns, comprising 15 serum proteins in the parents and 18 in the F₁ hybrid. Banding patterns for all mixtures of parental species were identical to those observed in the hybrid.     

Ascaris suum: immunoglobulin responses in mice

The immune response in mice to infection with Ascaris suum was characterized by determining (1) changes in serum immunoglobulin levels and (2) changes in the relative proportions of immunoglobulin-containing cells in major lymphoid tissues and sites of possible local immunoglobulin production.     

Distribution of secretory component in hepatocytes and its mode of transfer into bile

Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound ¹²⁵I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [¹⁴C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile.     

Studies on 125I-labeled proteins in rat plasma using an air-driven ultracentrifuge: protein-protein interactions and nonideality

The molecular weights of a number of ¹²⁵I-labeled plasma proteins have been determined from an analysis of their sedimentation equilibrium behavior in an air-driven ultracentrifuge. The values obtained agree well with results obtained by other methods. Molecular weights obtained for ¹²⁵I-labeled bovine serum albumin and the rat serum proteins albumin, α₁-acid glycoprotein, and major acute-phase α₁-protein were unaffected by the addition of 7% rat plasma. Direct evidence for protein-protein interactions was obtained for mixtures of ¹²⁵I-labeled rat α₁-acid glycoprotein and the plant lectin concanavalin A and for mixtures of ¹²⁵I-labeled protein A from Staphylococcus aureus and 7% rat plasma. Interactions of a different type were observed when the sedimentation equilibrium profiles of ¹²⁵I-labeled proteins were determined in concentrated solutions of other proteins. Under these conditions the effects of molecular exclusion or nonideality became significant and low estimates were obtained for the molecular weights of the labeled proteins. Analysis of the data obtained for ¹²⁵I-labeled bovine serum albumin in concentrated solutions of bovine serum albumin (20–80 mg/ ml) yielded nonideality coefficients in good agreement with literature values. Analysis of the behavior of ¹²⁵I-labeled rat serum albumin, transferrin, and α₁-acid glycoprotein yielded nonideality coefficients and hence activities of these proteins in undiluted rat plasma.     

Metabolism of inorganic sulphate in the isolated perfused rat liver. Effect of sulphate concentration on the rate of sulphation by phenol sulphotransferase

1. The metabolism of inorganic [³⁵S]sulphate (Na₂³⁵SO₄) was studied in the isolated perfused rat liver at three initial concentrations of inorganic sulphate in the perfusion medium (0, 0.65 and 1.30mm), in relation to sulphation and glucuronidation of a phenolic drug, harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole). 2. [³⁵S]Sulphate rapidly equilibrated with endogenous sulphate in the liver. It was excreted in bile and reached, at the lowest concentration in the perfusion medium, concentrations in bile that were much higher than those in the perfusion medium; at the higher sulphate concentrations, these concentrations were equal. The physiological concentration of inorganic sulphate in the liver, available for sulphation of drugs, is similar to the plasma concentration. 3. At zero initial inorganic sulphate in the perfusion medium, the rate of sulphation was very low and harmol was mainly glucuronidated. At 0.65mm-sulphate glucuronidation was much decreased and considerable sulphation took place, indicating efficient competition of conjugation by sulphation. At 1.30mm-sulphate the sulphation increased still further. 4. The results suggest that an important factor in sulphation is the relatively high K_m of synthesis of adenosine 3′-phosphate 5′-sulphatophosphate (the co-substrate of sulphation) for inorganic sulphate, which is of the order of the plasma concentration of inorganic sulphate. The steady-state adenosine 3′-phosphate 5′-sulphatophosphate concentration may determine the rate of sulphate conjugation of drugs in the rat in vivo.     

Characterization and immunocytochemical demonstration of glucocorticoid receptor using antisera specific to transformed receptor

Cytosolic and nuclear forms of the glucocorticoid receptor were characterized using immunochemical techniques. Antibodies were raised in rabbits to an M_r 58,000 fragment of the transformed (DNA-binding) glucocorticoid receptor purified from rat liver cytosol by DNA-cellulose chromatography and polyacrylamide gel electrophoresis. Antibodies reacted with the transformed receptor form in a radioimmunoassay for glucocorticoid receptor. Western blot analysis of antibody reactivity revealed a single M_r 185,000 receptor form in rat liver cytosol but a smaller M_r 85,000 form in nucleosol, indicating the M_r 85,000 form is the transformed receptor. Furthermore, western blot analysis indicates that the M_r 185,000 receptor undergoes proteolysis during receptor purification and in vitro transformation processes by generating immunochemically similar proteins of smaller molecular weights. An identical M_r 185,000 glucocorticoid receptor was detected in cytosols of four rat tissues; liver, brain, adrenal medulla, and thymus. The glucocorticoid receptor was localized to the cytoplasm and nucleus of rat adrenal medulla cells by immunohistochemistry, demonstrating the existence in vivo of the transformed receptor and translocation of the receptor from cytoplasm to nucleus.     

Chemical synthesis of bile acid acyl-adenylates and formation by a rat liver microsomal fraction

In mammals, unconjugated bile acids formed in the intestine by bacterial deconjugation are reconjugated (N-acylamidated) with taurine or glycine during hepatocyte transport. Activation of the carboxyl group of bile acids to form acyl-adenylates is a likely key intermediate step in bile acid N-acylamidation. To gain more insight into the process of bile acid adenylate formation, we first synthesized the adenylates of five common, natural bile acids (cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and lithocholic acid), and confirmed their structure by proton NMR. We then investigated adenylate formation by subcellular fractions of rat liver (microsomes, mitochondria, cytosol) using a newly developed LC method for quantifying adenylate formation. The highest activity was observed in the microsomal fraction. The reaction required Mg²⁺ and its optimum pH was about pH 7.0. In term of maximum velocity (V_max) and the Michaelis constant (K_m), the catalytic efficiency of the enzyme under the conditions used was highest with cholic acid of the bile acids tested. The formation of cholyl-adenylate was strongly inhibited by lithocholic and deoxycholic acid, as well as by palmitic acid; ibuprofen and valproic acid were weak inhibitors. In cholestatic disease, such adenylate formation might lead to subsequent bile acid conjugation with glutathione or proteins.     

Cellular immunolocalization of alpha-fetoprotein in rat liver

Increased synthesis of alpha-fetoprotein (AFP) was induced in rat liver by the administration of 3'-methyl-4-dimethyl-aminoazobenzene. The indirect immunoperoxidase technique was used to detect AFP. Cellular localization of AFP was studied using a number of different fixation procedures. Serial sections stained with immunoglobulin served to determine the extent of diffusion of serum proteins into liver cells during fixation. Background staining was minimized when Lillie's neutral buffered formalin plus acetic acid was used as the fixative. After 3'-methyl-4-dimethylaminoazobenzene ingestion, bile duct cell proliferation occurred. The serum AFP was positive in all rats after 17 days on the diet. In rats with AFP-positive sera the immunohistochemical reaction in mature hepatocytes was positive while bile duct cells and small hepatocytes were negative for AFP.     

Bile and liver metallothionein behavior in copper-exposed fish

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2 mg L⁻¹) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC–ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.     

Amelioration of hyperglycaemia and hyperlipidaemia by adjusting the interplay between gut microbiota and bile acid metabolism: Radix Scutellariae as a case

BackgroundOur previous clinical research showed that the interaction between gut microbiota and bile acids (BAs) in patients with type 2 diabetes mellitus (T2DM) changed significantly. We hypothesized that T2DM could be improved by adjusting this interaction mediated by farnesoid X receptor (FXR). T2DM belongs to the category of “xiaoke” in traditional Chinese medicine. Radix scutellariae has the effects of clearing away heat and eliminating dampness, curing jaundice and quenching thirst and is widely used alone or in combination with other medicines for the treatment of T2DM in China and throughout Asia. Additionally, the interaction between Radix scutellariae and gut microbiota may influence its efficacy in the treatment of T2DM.PurposeThis study chose Radix scutellariae to validate that T2DM could improve by adjusting the interaction between gut microbiota and bile acid metabolism.Study design and methodsRadix scutellariae water extract (WESB) was administered to a T2DM rat model established by a high-fat diet combined with streptozotocin. The body weight and blood glucose and insulin levels were measured. The levels of serum lipids, creatinine, uric acid, albumin and total bile acid were also detected. Changes in the pathology and histology of the pancreas, liver and kidney were observed by haematoxylin-eosin staining. The 16S rRNAs of gut microbiota were sequenced, and the faecal and serum BAs were determined by liquid chromatography tandem mass spectrometry. The expression levels of BA metabolism-associated proteins in the liver and intestine were evaluated by immunoblot analysis.ResultsThe results showed that WESB improved hyperglycaemia, hyperlipaemia, and liver and kidney damage in T2DM rats. In addition, the abundances of key gut microbiota and the concentrations of certain secondary BAs in faeces and serum were restored. Moreover, there was a significant correlation between the restored gut microbiota and BAs, which might be related to the activation of liver cholesterol 7α-hydroxylase (CYP7A1) and the inhibition of FXR expression in the intestine rather than the liver.ConclusionsThis study provided new ideas for the prevention or treatment of clinical diabetes and its complications by adjusting the interaction between gut microbiota and bile acid metabolism.     

Synthesis of alpha-fetoprotein and albumin by fetal mouse liver cultured in chemically defined medium

Fetal mouse liver synthesizes two major secretory proteins: α-fetoprotein and albumin. The relative proportions of these proteins change during development. Fetal mouse liver secretes predominantly α-fetoprotein, and the neonatal mouse liver secretes predominantly albumin. α-Fetoprotein and albumin synthesis were studied in vitro using an organ culture system and a chemically defined medium (BGJ_b). This medium does not support hepatocellular replication but maintains protein synthesis at high levels for prolonged periods of culture. Patterns of protein synthesis were analyzed as functions of gestational age and time in culture. The ratio of α-fetoprotein/albumin decreases from 1.4 on gestational Day 14 to 0.4 on gestational Day 18. However, when liver cultures were begun on any given gestational day, the ratio of α-fetoprotein/albumin remains constant for as long as 8 days in culture. Thus, developmental changes in α-fetoprotein and albumin synthesis are arrested under the conditions of this culture system. Fetal mouse liver secretes two electrophoretically distinguishable forms of albumin. One form is similar in mobility to albumin from adult mouse serum; the other more electropositive species is similar in mobility to proalbumin isolated from adult mouse liver microsomes and can be converted to albumin by mild trypsin treatment.     

Proteins of guinea-pig bile: selective resorption in the gall bladder

We have examined and compared the proteins present in guinea-pig bile as collected either from the common hepatic duct or from the gall bladder. Guinea-pig bile, collected from the common bile duct, has a rather low concentration of protein. Detailed examination shows that the concentrations of actively transported proteins such as immunoglobulin A and haptoglobin.haemoglobin complexes are markedly lower than in rats although the concentrations of proteins which, like albumin, leak non-specifically into bile are similar in the two species. We also find that the protein composition of guinea-pig bile is extensively and selectively modified by resorp-tion of protein in the gall bladder.     

Biosynthesis of transcobalamin II by adult rat liver parenchymal cells in culture.

The biosynthesis of transcobalamin II was investigated in primary cultures of adult rat liver parenchymal cells maintained in serum-free media. The data indicate that these hepatocytes secrete a vitamin B₁₂-binding substance into the culture medium which is identical to rat serum transcobalamin II as judged by the following criteria: (i) gel filtration on columns of Sephadex G-200; (ii) ion-exchange chromatography on columns of diethyl aminoethyl cellulose and carboxymethyl cellulose; (iii) polyacrylamide-gel electrophoresis at pH 9.5; and (iv) the ability to facilitate cellular vitamin B₁₂ uptake by HeLa cells and mouse L-929 fibroblasts in culture. The secretion of transcobalamin II by the liver parenchymal cells was blocked by cycloheximide, puromycin, and p-fluorophenylalanine. The inhibition by cycloheximide, but not that of the other inhibitors, was partially reversed upon removal of the drug. The liver parenchymal cells incorporated radioactive amino acids into transcobalamin II which was absorbed from the growth medium using affinity chromatography on Sepharose containing covalently linked B₁₂. Collectively, these data indicate that rat liver parenchymal cells, in culture, are capable of the biosynthesis de novo of transcobalamin II and the subsequent secretion of this protein into the culture media.