Liver-like alkaline phosphatase in the tissue-unspecific type enzyme found in rabbit organs

Rabbit liver and kidney tissues are known to produce an intestinal-like alkaline phosphatase (IAP-like enzyme) as a dominant isozyme, with a minor isozyme of tissue-unspecific type (UAP), unlike humans and other mammalians. We investigated immunohistochemically and biochemically these unique isozymes in the rabbit liver and bone, and compared them with the human isozyme. In rabbit liver, UAP was found to be localized only in the apical part of the membrane of cells lining the bile duct, whereas IAP-like enzyme was found in the sinusoidal membrane of hepatocytes. Rabbit liver UAP was separated from IAP-like enzyme by DEAE-cellulose column chromatography. Rabbit bone tissue contained only one UAP isozyme. The two UAPs were biochemically and physicochemically compared with human liver AP. Both UAPs reacted with an anti-human liver AP monoclonal antibody, not with an anti-human bone AP monoclonal antibody, indicating that both enzymes have the same antigenicity as human liver AP. Rabbit liver and bone UAPs had similar N-linked sugar-chain heterogeneities to the respective human enzymes. In addition, rabbit bone AP also had an O-linked sugar chain, as did human bone AP, unlike rabbit and human liver APs.     

Alkaline phosphatase isoenzymes in human kidney and urine,immunohistochemical, immunological and biochemical characterization

Summary Human kidney contains two antigenetically distinct isoenzymes of alkaline phosphatase (AP): a liver type and an intestinal type. The intestinal type AP is a minor component (1%–4%) of the total AP activity: it is found only in the cytoplasm. Both isoenzymes are located, found by an immunohistochemical technique, in the proximal convoluted tubules. This histochemical result eliminates the possibility that the low intestinal AP content in the kidney might only originate from blood vessels, where the intestinal isoenzyme was also found. The renal isoenzymes contribute to urinary AP. Intestinal type AP in urine of healthy persons, 10%–40% of the total AP activity, was found after high speed centrifugation predominantly in the supernatant (100,000 g), the liver type mainly in the sediment. Biochemical characterization revealed that intestinal type AP in kidney and urine are identical and differ from the isoenzyme of intestinal mucosa only slightly in their electrophoretic mobility.     

Expression of intestinal alkaline phosphatase in human organs

Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.     

Adenylate kinase isoenzyme patterns in normal and neoplastic human lung, and in various adult as compared to fetal rat tissues

The isozyme pattern and total activity of adenylate kinase were studied in normal adult and fetal human and rat tissues using starch gel electrophoresis. Three adenylate kinase isoenzymes were identified in human tissues. Although normal adult lung exhibited higher adenylate kinase activity than did its fetal or neoplastic variant, isozyme patterns in the three types of tissues were indistinguishable from each other and from that in fetal human liver. The pattern of these three isozymes in rat lung (as in spleen) also did not change between fetal and adult life. However, adult kidney and heart of this species did appear to contain isozymes not present in fetal life. Brain (both adult and fetal) was striking different from all the other tissues in that it contained only one adenylate kinase isozyme. The total adenylate kinase activity per gram of adult rat liver, kidney and lung was significantly higher than in the cognate fetal organs, whereas that in brain or spleen did not change with age. The activity in adult heart (similar to the fetal one) was higher than in any other tissue examined.     

Expression of intestinal alkaline phosphatase in human organs.

Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.     

Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relations to a fetal form

The Novikoff hepatoma glycogen phosphorylase b has been purified over 300-fold, free of glycogen synthetase, some of its properties have been studied, and its relationship to fetal forms of rat muscle and liver phosphorylase has been established immunochemically. Its molecular weight is approximately 200,000, and, like the liver but unlike the muscle isozyme, it does not dimerize on conversion to the a form. However, it differs from the liver isozyme in being activated by AMP (K_a = 0.2 mM) and in not being activated by sulfate ion. Antibody to the adult rat muscle phosphorylase did not inhibit the activity of the tumor or liver isozyme. Although antibody to liver or hepatoma phosphorylase had no effect on adult muscle phosphorylase, each of these antibodies partially inhibited the other enzyme. These findings indicate the presence of some liver isozyme in the tumor, and this was confirmed by isoelectric focusing. Rat liver and muscle phosphorylase (and synthetase) were low during embryonal development but rose rapidly at or shortly after birth. Immunochemical studies revealed that both fetal liver and fetal muscle phosphorylases are immunologically identifiable with the tumor enzyme; and the fetal form is also present as a major form in rat kidney and brain.     

Immunohistochemical analysis of rat S-adenosylmethionine synthetase isozymes in developmental liver

Mammalian S-adenosylmethionine (AdoMet) synthetase exists as two isozymes, liver-type and kidney(non-hepatic)-type enzymes. The developmental expression of these two isozyme proteins has been investigated in rat liver using immunohistochemical techniques. The liver-type AdoMet synthetase is expressed only in adult liver, but not in fetal liver. On the other hand, the kidney-type AdoMet synthetase is predominantly expressed in fetal liver and faintly detected in adult liver. It was also found that both isozymes were localized to the hepatocytes of rat liver. These results clearly show that AdoMet synthetase isozymes are developmentally regulated within hepatocytes. In addition, in rat kidney we have shown that the kidney-type AdoMet synthetase is predominantly localized to the distal tubule.     

Differentiation of human adult and fetal intestinal alkaline phosphatases with monoclonal antibodies

Two forms of intestinal alkaline phosphatase have been recognized in humans. They are very similar in a number of biochemical and immunologic characteristics, but the exact genetic relationship between them remains unclear. To further study this problem, six monoclonal antibodies and a polyclonal rabbit antiserum to human fetal intestinal alkaline phosphatase have been produced. All of the monoclonal antibodies and the rabbit antiserum crossreact with adult intestinal alkaline phosphatase and with the intestinal-like alkaline phosphatase found in D98/AH-2 human tissue-culture cells. Four of the monoclonal antibodies and the rabbit antiserum crossreact with placental alkaline phosphatase, while none of the antibodies or the antiserum recognize liver or kidney alkaline phosphatase. Four of the monoclonal antibodies can distinguish between adult and fetal intestinal alkaline phosphatase in electrophoretic titration-binding studies, with the relative binding of adult enzyme being significantly greater than that of the fetal enzyme in each case. One of these antibodies, which also reacts with placental alkaline phosphatase, can distinguish the type 3 allelic variant of the placental enzyme from types 1 and 2. This indicates that the antibody detects a structural difference in the protein moiety of one of the allelic forms of the enzyme. These data suggest that adult and fetal intestinal alkaline phosphatases represent structurally distinct proteins, either encoded for by different genes or produced by differential processing of a common precursor molecule determined by a single gene.     

Two types of aspartic proteinases from buckwheat seed--gene structure and expression analysis

Two types of aspartic proteinase (AP) genes have been isolated from the cDNA library of developing buckwheat seeds. Analysis of their sequences showed that one of these, FeAP9, resembled the structure and shared high homology with the so-called typical plant APs characterized by the presence of a plant-specific insert (PSI), an element unique among APs. The other cDNA, FeAPL1, encoded an AP-like protein lacking that domain. Different expression profiles were observed for FeAP9 and FeAPL1. FeAPL1 mRNAs were restricted to the seeds only, whereas FeAP9 mRNAs were also present in the other plant tissues - leaves, roots, and flowers. Higher levels of FeAP9 were observed in senescent leaves compared with green leaves. The differential expression pattern of these two unique APs raises the interesting possibility that these proteinases have unique substrate specificity and may have different roles in plant development and other physiological processes.     

Pyruvate kinase isozymes in adult and fetal tissues of chicken.

Tissues of fetal and adult chickens were examined for pyruvate kinase activity. Two electrophoretically distinguishable and noninterconvertible isozymes were found. One of these, designated as type K (for kidney), is the sole pyruvate kinase in the early fetus and is found in appreciable quantities in all adult tissues except striated muscle. The second isozyme, type M, appears shortly before hatching in striated muscle and brain. These two isozymes correspond in their developmental pattern, tissue distribution, electrophoretic, immunological, and kinetic propertiesto similarly designated mammalian pyruvate kinases. However, no kinetic, immunological, or electrophoretic evidence could be found for a chicken isozyme corresponding to the mammalian type L pyruvate kinase. As the latter isozyme seems to be limited in its distribution mostly to highly differentiated gluconeogenic tissues (notable liver, kidney, and small intestine), our results support the proposition that the mammalian type L pyruvate kinase is a specilized isozyme that is present in mammals but not in birds.     

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

Laminin α1-Chain Shows a Restricted Distribution in Epithelial Basement Membranes of Fetal and Adult Human Tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) α1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln α1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16–19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln α1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln α1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln α1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

Intestinal and renal origin of trehalase activity in rabbit amniotic fluid

To utilize specific fetal markers in amniotic fluid for prenatal detection of fetal anomalies, it is necessary to determine the precise tissue origin of these markers. In rabbit fetuses, we distinguished between intestinal and renal forms of trehalase (alpha,alpha'-trehalose-1-D-glucohydrolase, EC 3.2.1.28) in amniotic fluid on the basis of differences in net electric charges. Trehalase was solubilized from purified brush-border membranes of fetal rabbit kidney and intestine by Triton X-100 treatment, whereas the trehalase activity in amniotic fluid was soluble. The kinetic properties of trehalase from intestine, kidney and amniotic fluid were very similar. The Mr of the soluble amniotic fluid trehalase was between 72,600 and 66,300 from hydrodynamic parameters, depending on the amount of sugar bound to the enzyme, and 48,500 by radiation inactivation, a method which detects only the protein part of the enzyme. For membrane-bound trehalase from kidney and intestine in situ the radiation inactivation method also gave a molecular size of around 49,000. Isoelectric focusing of freshly solubilized membranes allowed us to distinguish between renal and intestinal forms of trehalase in rabbit fetuses on the basis of different isoelectric points. Each trehalase form was also present in the amniotic fluid but in varying proportions depending on the gestational age at which the amniotic fluid was collected. The results suggest that early in gestation amniotic fluid trehalase activity originates exclusively from the fetal kidney but that more and more intestinal enzyme is released into the amniotic cavity as the fetus develops. Similar results were also obtained when ion-exchange chromatography was used to separate the various trehalase forms. The development of trehalase activity in rabbit fetal kidney and intestine correlates well with its occurrence in the amniotic fluid; trehalase activity in the kidney develops early in gestation whereas the intestinal trehalase activity develops just before term.     

Expression and Activation of Caspase-6 in Human Fetal and Adult Tissues

Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues.     

Mouse retinal dehydrogenase 4 (RALDH4), molecular cloning,cellular expression,and activity in 9-cis-retinoic acid biosynthesis in intact cells

This study describes cDNA cloning and characterization of mouse RALDH4. The 2.3-kb cDNA encodes an aldehyde dehydrogenase of 487 amino acid residues, about two-orders of magnitude more active in vitro with 9-cis-retinal than with all-trans-retinal. RALDH4 recognizes as substrate 9-cis-retinal generated in transfected cells by the short-chain dehydrogenases CRAD1, CRAD3, or RDH1, to reconstitute a path of 9-cis-retinoic acid biosynthesis in situ. Northern blot analysis showed expression of RALDH4 mRNA in adult mouse liver and kidney. In situ hybridization revealed expression of RALDH4 in liver on embryo day 14.5, in adult hepatocytes, and kidney cortex. Immunohistochemistry confirmed RALDH4 expression in hepatocytes and showed that hepatocytes also express RALDH1, RALDH2, and RALDH3. Kidney expresses the RALDH4 protein primarily in the proximal and distal convoluted tubules of the cortex but not in the glomeruli or the medulla. Kidney expresses RALDH2 in the proximal convoluted tubules of the cortex but not in the distal convoluted tubules or glomeruli. Kidney expresses RALDH1 and RALDH2 in the medulla. The enzymatic characteristics of RALDH4, its expression in fetal liver, and its unique expression pattern in adult kidney compared with RALDH1, -2, and -3 suggest that it could meet specific needs for 9-cis-retinoic acid biosynthesis.     

Immunohistochemical distribution of type VI collagen in developing human kidney

Summary The distribution of type VI collagen was investigated immunohistochemically in the developing human kidney from 15 to 32 weeks gestational age and it was compared with that observed in the normal infantile and adult human kidney. In fetal kidney, type VI collagen was widely distributed as a fibrillar network in the subcapsularly undifferentiated mesenchyme and intertubular interstitium, and as a basement membrane-like structure around the ureteral bud branches, tubules, and collecting ducts. During nephrogenesis, type VI collagen disappeared from the induced mesenchyme close to the tips of ureteral branches, while it formed a distinct basement membrane-like structure around the early stages of nephron differentiation (comma-shaped and S-shaped bodies) and later along Bowman's capsule of capillary loop and maturing glomeruli. A strong immureactivity for type VI collagen was also found in the glomerular basement membrane and mesangial areas of capillary loop and maturing glomeruli. In infantile kidney, type VI collagen showed a distribution pattern similar to that observed during the fetal period. In adult human kidney, glomerular basement membrane showed a weak positivity for type VI collagen and the basement membrane-like staining around Bowman's capsule, tubules, and collecting ducts was less evident than in fetal and infantle kidney. Our immunohistochemical findings suggest that type VI collagen is a normal component of the glomerular and extraglomerular extracellular matrix of developing human kidney and that it undergoes changes in the expression during maturation.     

Isozyme differentiation of aldolase and pyruvate kinase in fetal,regenerating, preneoplastic,and malignant rat hepatocytes during culture

Summary Aldolase and pyruvate kinase isozymes were investigated in cultured hepatocytes from fetal, regenerating, and 2-acetyl-aminofluorene-fed rat liver as well as in some epithelial liver cell lines. Our results show that: (a) cell proliferation and prolonged expression of specific isozymes were found only in cultured hepatocytes from 17-day old fetuses; (b) the fetal type of pyruvate kinase expressed in regenerating and carcinogen-treated liver was temporarily lost only in cultured hepatocytes from regenerating liver; (c) the adult type of aldolase and pyruvate kinase was absent in one epithelial cell line derived from a carcinogen-treated liver and in the hepatoma tissue cell (HTC) line but was found in the Faza clone of the Reuber H₃₅ cell line during the 50 first passages in vitro; and (d) the isozyme pattern of pyruvate kinase was always more strongly shifted than that of aldolase. The observations suggest that: (a) hepatocytes from carcinogen-treated liver exhibit the same lack of ability to proliferate in primary culture as normal adult hepatocytes; (b) adult hepatocytes can produce fetal isozymes without prior cell division; (c) pyruvate kinase is a stronger marker of dedifferentiation (retrodifferentiation) than aldolase; and (d) regulatory processes of isozyme expression are different during ontogenesis, regeneration, and hepatocarcinogenesis. This work was supported by the “Institut National de la Santé et de la Recherche Médicale” and the “Fondation pour la Recherche Medicale Fran?aise”