Placental and fetal cysteine dioxygenase gene expression in mouse gestation.

Nutrient sulfate is important for fetal development. The fetus has a limited capacity to generate sulfate and relies on maternal sulfate supplied via the placenta. The gestational age when fetal sulfate generation begins is unknown but would require cysteine dioxygenase (CDO1) which mediates a major step of sulfate production from cysteine. We investigated the ontogeny of Cdo1 mRNA expression in mouse fetal and placental tissues, which showed increasing levels from embryonic day 10.5 and was localised to the decidua and several fetal tissues including nasal cavities and brain. These findings suggest a role for Cdo1 in sulfate generation from mid-gestation.

Pathogenetics of the human SLC26 transporters.

Over the past decade, 11 human genes belonging to the solute linked carrier (SLC) 26 family of transporters, have been identified. The SLC26 proteins, which include SAT-1, DTDST, DRA/CLD, pendrin, prestin, PAT-1/CFEX and Tat-1, are structurally related and have been shown to transport one or more of the following substrates: sulfate, chloride, bicarbonate, iodide, oxalate, formate, hydroxyl or fructose. Special interest has focused on four members of the SLC26 family that are associated with distinct recessive diseases: (i) Mutations in SLC26A2 lead to four different chondrodysplasias (diastrophic dysplasia, atelosteogenesis type II, achondrogenesis type IB and multiple epiphyseal dysplasia); (ii) SLC26A3 is associated with congenital chloride diarrhea; (iii) SLC26A4 is associated with Pendred syndrome and non-syndromic deafness, DFNB4; and (iv) SLC26A5 is defective in non-syndromic hearing impairment. This review article summarizes current information on the pathophysiological consequences of mutations in the human SLC26A2 to A5 genes.

Expression, transport properties, and chromosomal location of organic anion transporter subtype 3.

The rat and mouse organic anion-transporting polypeptides (oatp) subtype 3 (oatp3) were cloned to further define components of the intestinal bile acid transport system. In transfected COS cells, oatp3 mediated Na(+)-independent, DIDS-inhibited taurocholate uptake (Michaelis-Menten constant approximately 30 microM). The oatp3-mediated uptake rates and affinities were highest for glycine-conjugated dihydroxy bile acids. In stably transfected, polarized Madin-Darby canine kidney (MDCK) cells, oatp3 mediated only apical uptake of taurocholate. RT-PCR analysis revealed that rat oatp3, but not oatp1 or oatp2, was expressed in small intestine. By RNase protection assay, oatp3 mRNA was readily detected down the length of the small intestine as well as in brain, lung, and retina. An antibody directed to the carboxy terminus localized oatp3 to the apical brush-border membrane of rat jejunal enterocytes. The mouse oatp3 gene was localized to a region of mouse chromosome 6. This region is syntenic with human chromosome 12p12, where the human OATP-A gene was mapped, suggesting that rodent oatp3 is orthologous to the human OATP-A. These transport and expression properties suggest that rat oatp3 mediates the anion exchange-driven absorption of bile acids previously described for the proximal small intestine.

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties.

Bile secretion involves the structural and functional interplay of hepatocytes and cholangiocytes, the cells lining the intrahepatic bile ducts. Hepatocytes actively secrete bile acids into the canalicular space and cholangiocytes then transport bile acids in a vectorial manner across their apical and basolateral plasma membranes. The initial step in the transepithelial transport of bile acids across rat cholangiocytes is apical uptake by a Na(+)-dependent bile acid transporter (ASBT). To date, the molecular basis of the obligate efflux mechanism for extrusion of bile acids across the cholangiocyte basolateral membrane remains unknown. We have identified an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT, expressed in rat cholangiocytes, ileum, and kidney. Alternative splicing causes a frameshift that produces a 154-aa protein. Antipeptide antibodies detected the approximately 19 kDa t-ASBT polypeptide in rat cholangiocytes, ileum, and kidney. The t-ASBT was specifically localized to the basolateral domain of cholangiocytes. Transport studies in Xenopus oocytes revealed that t-ASBT can function as a bile acid efflux protein. Thus, alternative splicing changes the cellular targeting of ASBT, alters its functional properties, and provides a mechanism for rat cholangiocytes and other bile acid-transporting epithelia to extrude bile acids. Our work represents an example in which a single gene appears to encode via alternative splicing both uptake and obligate efflux carriers in a bile acid-transporting epithelial cell.

Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells.

This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPARgamma investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPARgamma is expressed in several transformed cells. However, the function of PPARgamma activation in neoplastic cells is unclear. Activation of PPARgamma with the known prostanoid agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) or the thiazolidinedione (TZD) agonist troglitazone (TGZ) attenuated cellular proliferation of the estrogen receptor-negative breast cancer cell line MDA-MB-231, as well as the estrogen receptor-positive breast cancer cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell cycle progression. Addition of 15dPGJ(2) was not associated with an increase in cellular differentiation, as has been seen in other neoplastic cells, but rather induction of cellular events associated with programmed cell death, apoptosis. Video time-lapse microscopy revealed that 15dPGJ(2) induced morphological changes associated with apoptosis, including cellular rounding, blebbing, the production of echinoid spikes, blistering and cell lysis. In contrast, TGZ caused only a modest induction of apoptosis. These results were verified by histochemistry using the specific DNA stain DAPI to observe nuclear condensation, a marker of apoptosis. Finally, a brief exposure of MDA-MB-231 cells to 15dPGJ(2) initiated an irreversible apoptotic pathway that inhibited the growth of tumors in a nude mouse model. These findings illustrate that induction of apoptosis may be the primary biological response resulting from PPARgamma activation in some breast cancer cells and further suggests a potential role for PPARgamma ligands for the treatment of breast cancer.

Mutational analysis of the cystathionine beta-synthase gene: a splicing mutation, two missense mutations and an insertion in patients with homocystinuria. Mutations in brief no. 120. Online.

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA.

Reconstitution of bile acid transport in a heterologous cell by cotransfection of transporters for bile acid uptake and efflux.

The rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP) is a 110-kDa transmembrane phosphoglycoprotein that is thought to have bile acid efflux, ecto-ATPase, and cell adhesion properties. Its extracellular amino-terminal domain is highly homologous to carcinoembryonic antigen (CEA), a glycophosphatidyl inositol-anchored membrane protein with cell adhesion properties and a marker for adenocarcinoma. In the current study, we examined the possibility of more clearly defining the role of CBATP in bile acid efflux by cotransfecting a heterologous cell, the COS cell, with cDNAs for a bile acid importer, the ileal bile acid transporter (IBAT), as well as for CBATP. The results show that when IBAT mediates uptake of [3H]taurocholate to a level 20-fold higher than that achieved previously by nonspecific pinocytosis, CBATP mediates time-, temperature- and concentration-dependent efflux. Efflux of [3H]taurocholate mediated by CBATP in the cotransfected COS cells is saturable and has curvilinear kinetic characteristics (Vmax = 400 pmol/mg protein/min, Km = 70 microM). It is inhibited by 4,4'-diisothiocyanostilbene-2,2-disulfonic acid and dependent on ATP but not dependent on membrane potential. Although CEA could not mediate bile acid efflux in COS cells cotransfected with IBAT and CEA, efflux of [3H]taurocholate was detected in COS cells cotransfected with IBAT and a chimeric molecule having the carboxyl-terminal tail and membrane spanning domain of CBATP and the amino-terminal extracellular tail of CEA. Taken together, these data provide further evidence that CBATP confers bile acid efflux properties on heterologous cells and that its cytoplasmic tail and membrane spanning segment are integral to this property. The data also establish a model system for more clearly defining the molecular determinants of bile acid transport mediated by this molecule.

Characterisation of five missense mutations in the cystathionine beta-synthase gene from three patients with B6-nonresponsive homocystinuria.

Homocystinuria, due to a deficiency of the enzyme cystathionine beta-synthase (CBS), is an inborn error of sulphur-amino acid metabolism. This is an autosomal recessive disease which results in hyperhomocysteinaemia and a wide range of clinical features, including optic lens dislocation, mental retardation, skeletal abnormalities and premature thrombotic events. We report the identification of 5 missense mutations in the protein-coding region of the CBS gene from 3 patients with pyridoxine-nonresponsive homocystinuria. Reverse-transcription PCR was used to amplify CBS cDNA from each patient and the coding region was analysed by direct sequencing. The mutations detected included 3 novel (1058C-->T, 992C-->A and 1316G-->A) and 2 previously identified (430G-->A and 833C-->T) base alterations in the CBS cDNA. Each of these mutations predicts a single amino acid substitution in the CBS polypeptide. Appropriate cassettes of patient CBS cDNA, containing each of the above defined mutations, were used to replace the corresponding cassettes of normal CBS cDNA sequence within the bacterial expression vector pT7-7. These recombinant mutant and normal CBS constructs were expressed in Escherichia coli cells and the catalytic activities of the mutant proteins were compared with normal. All of the mutant proteins exhibited decreased catalytic activity in vitro, which confirmed the association between the individual mutation and CBS dysfunction in each patient.

Cloning and characterization of a novel peptidase from rat and human ileum.

A novel 100-kDa ileal brush border membrane protein (I100) has been purified by anionic glycocholate affinity chromatography. Polyclonal antibodies raised against this protein were utilized to clone and characterize I100 in rats. A partial length human I100 cDNA was identified by hybridization screening. In the rat, the I100 protein is a 746-amino acid glycosylated (calculated core molecular mass of 80 kDa) type II integral membrane protein found on the apical surface of ileal villus enterocytes. Its 2.6-kilobase mRNA is expressed in distal small intestine in rats and in humans. The I100 cDNA is homologous to but distinct from human prostate-specific membrane antigen and rat brain N-acetylaspartylglutamate peptidase. It is expressed on both the basolateral and apical surfaces of stably transfected Madin Darby canine kidney cells. Analysis of these stably transfected Madin Darby canine kidney cells and I100 immunoprecipitates of rat ileal brush border membrane vesicles reveals that it has dipeptidyl peptidase IV activity. Future invesitgations will need to determine the exact substrate specificity of this novel peptidase.

Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter.

Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [3H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na+-dependent transcellular transport of [3H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na+-dependent uptake of [3H]taurocholate, with apparent Km and Vmax values of 209+/-45 microM and 1.23+/-0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na+-dependent taurocholate-cotransporting polypeptide and rat ileal apical Na+-dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids.

A pig collagen peptide fraction. A unique material for maintaining biological activity during lyophilization and during storage in the liquid state.

There is frequent use of human and animal proteins as stabilizers during lyophilization of a variety of biological substances with a view to long term stable storage. This report describes the comparative excellent stabilizing effect of a porcine collagen peptide fraction (CPF) during the lyophilization and subsequent storage of three commonly used biological substances, alkaline phosphatase, tissue plasminogen activator and thrombin. The CPF was heated to 150 degrees C for one hour before use. The CPF was shown to have some advantage during lyophilized storage over human serum albumin. Solutions of thrombin stored in CPF at room temperature and at 37 degrees C for one week retained nearly all activity, while storage of thrombin in human serum albumin solution at 37 degrees C lost nearly all biological activity. These preliminary data suggest that porcine CPF is a safe and advantageous stabilizer for addition to biological products with a view to long-term lyophilized storage and short-term liquid storage.

Human homocysteine catabolism: three major pathways and their relevance to development of arterial occlusive disease.

Two separate metabolic pathways that methylate homocysteine to methionine are known in humans, utilizing, respectively, 5-methyltetrahydrofolate and betaine as methyl donors. Deficiency of the folate-dependent methylation system is linked to hyperhomocysteinemia. Our data suggest that this deficiency leads to concurrent metabolic down-regulation of homocysteine transsulfuration that may contribute to hyperhomocysteinemia. By contrast, no instances have been reported of hyperhomocysteinemia resulting from deficiencies of betaine-dependent homocysteine methylation. Long-term betaine supplementation of 10 patients, who had pyridoxine-resistant homocystinuria and gross hyperhomocysteinemia due to deficiency of cystathionine beta-synthase activity, caused a substantial lowering of plasma homocysteine, which has now been maintained for periods of up to 13 years. Betaine had to be taken regularly because the effect soon disappeared when treatment was stopped. In conclusion, depressed activity of the transsulfuration pathway may contribute to hyperhomocysteinemia because of primary deficiencies of enzymes of either the transsulfuration or of the folate-dependent methylation pathways. Stimulation of betaine-dependent homocysteine remethylation causes a commensurate decrease in plasma homocysteine that can be maintained as long as betaine is taken.

Variable hyperhomocysteinaemia phenotype in heterozygotes for the Gly307Ser mutation in cystathionine beta-synthase.

BACKGROUND: A deficiency of cystathionine beta-synthase (CBS) activity is the most frequent cause of homocystinuria, an autosomal recessive disease with multiple clinical manifestations. Mutations in the CBS gene have been reported in several patients with homocystinuria. AIMS: To establish the molecular basis of CBS deficiency in a female patient with pyridoxine non-responsive homocystinuria, and to apply the findings to genetic screening of her family members. METHODS: The entire coding region of the CBS cDNA was amplified by PCR and used for direct sequence analysis. Mutant alleles were confirmed by direct sequence analysis of PCR-amplified genomic DNA, and by a combination of single strand conformation polymorphism and temperature gradient gel electrophoresis analysis. RESULTS: The proband was homozygous for a G919A base transition which predicts the substitution of serine for glycine at codon 307 in the CBS protein (G307S). The parents (both of Irish background) were heterozygotes for the G307S allele, while an asymptomatic sibling had normal CBS sequence, Plasma homocysteine, assessed after an oral methionine load, indicated the mother clearly had moderate hyperhomocysteinaemia, whereas the father had normal concentrations of homocysteine. This is the first report of a normal methionine load test in a proven heterozygote for a CBS mutation which causes severe homocystinuria in the homozygote. Other factor(s) may have contributed to hyperhomocysteinaemia in the mother. The G307S allele has been reported in other patients and appears to be a common allele among families of Celtic origin.

Cloning and expression of the mammalian cytosolic branched chain aminotransferase isoenzyme.

The cDNA for the rat cytosolic branched chain aminotransferase (BCATc) has been cloned. The BCATc cDNA encodes a polypeptide of 410 amino acids with a calculated molecular mass of 46.0 kDa. By Northern blot analysis, BCATc message of approximately 2.7 kilobases was readily detected in rat brain, but was absent from liver, a rat hepatoma cell line, kidney, and skeletal muscle. When expressed in COS-1 cells, the enzyme is immunologically indistinguishable from the native enzyme found in rat brain cytosol. Comparison of the rat BCATc sequence with available data bases identified the Escherichia coli (and Salmonella typhimurium) branched chain aminotransferase (BCAT) and revealed a Haemophilus influenzae BCAT, a yeast BCAT, which is hypothesized to be a mitochondrial form of the enzyme, and the murine BCATc (protein ECA39). Calculated molecular masses for the complete proteins are 33.9 kDa, 37.9 kDa, 42.9 kDa, and 43.6 kDa, respectively. The rat BCATc sequence was 84% identical with murine BCATc, 45% identical with yeast, 33% identical with H. influenzae, 27% identical with the E. coli and S. typhimurium BCAT, and 22% identical with the evolutionary related D-amino acid aminotransferase (D-AAT) (Tanizawa, K., Asano, S., Masu, Y., Kuramitsu, S., Kagamiyama, H., Tanaka, H., and Soda, K. (1989) J. Biol. Chem. 264, 2450-2454). Amino acid sequence alignment of BCATc with D-AAT suggests that the folding pattern of the overlapping mammalian BCATc sequence is similar to that of D-AAT and indicates that orientation of the pyridoxal phosphate cofactor in the active site of the eukaryotic BCAT is the same as in D-AAT. Thus, BCAT are the only eukaryotic aminotransferases to abstract and replace the proton on the re face of the pyridoxal phosphate cofactor. Finally, requirements for recognition of substrate L-amino acid and alpha-carboxylate binding are discussed.

Identification of a mutation in the ileal sodium-dependent bile acid transporter gene that abolishes transport activity.

The ileal Na+/bile acid cotransporter plays a critical role in the reabsorption of bile acids from the small intestine. In the course of cloning and characterizing the human ileal Na+/bile acid cotransporter cDNA, a dysfunctional isoform was identified in a patient diagnosed with Crohn's disease. Expression studies using hamster-human ileal Na+/bile acid cotransporter cDNA chimeras narrowed the location of the defect to the carboxyl-terminal 94 amino acids. Comparison of the sequence of the dysfunctional isoform to that of a wild-type human ileal Na+/bile acid cotransporter genomic clone revealed a single C to T transition resulting in a proline to serine substitution at amino acid position 290. The inheritance of this mutation in the proband's family was confirmed by single-stranded conformation polymorphism analysis and DNA sequencing. In transfected COS-1 cells, the single amino acid change abolished taurocholate transport activity but did not alter the transporter's synthesis or subcellular distribution. This dysfunctional mutation represents the first known molecular defect in a human sodium-dependent bile acid transporter.

Pediatric chest pain: case report of a malignant cause.

Pediatric chest pain is not an uncommon presenting complaint in the emergency department. Although the majority of cases are benign, the authors report on a patient with a fetal case of myocarditis. A discussion of myocarditis and its management as well as differential diagnosis is included.

The primary structures of four subunits of the human, high-molecular-weight proteinase, macropain (proteasome), are distinct but homologous.

Macropain (proteasome) is a high-molecular-weight proteinase complex composed of at least 13 electrophoretically distinct subunits. Previous work, including peptide mapping and limited amino acid sequencing, suggested that most of the subunits belong to an evolutionarily related group of different gene products (Lee et al. (1990) Biochim. Biophys. Acta. 1037, 178-185). In order to define the extent and pattern of subunit relatedness, and to determine the structural basis for possible similarities and differences in subunit functions, we are deducing the primary structures of macropain subunits by cDNA cloning and DNA sequence analysis. We report here the primary structures of four subunits. The data clearly demonstrate that the proteins represent different, but homologous gene products. Surprisingly, no evidence for homology with any other protein, including proteinases, was obtained. These results suggest that macropain is comprised of a previously unidentified family of evolutionarily related polypeptides. Because biochemical data indicate that macropain contains several different proteinase activities, the current results raise the possibility that the macropain complex is composed of a group of novel proteinases, distinct from those of other structurally identifiable proteinase families.

Incomplete sulphomucin-secreting intestinal metaplasia for gastric cancer. Preliminary data from a prospective study from three centres.

A joint prospective long term study of gastric biopsies has been undertaken to survey intestinal metaplasia Types I, II, and III in terms of their incidence, distribution and value in the selection of high risk cancer patients. This study is based on protocols agreed between three centres for endoscopy, histological interpretation, and mucin histochemistry. The results on the first 1350 gastric biopsies examined during 1981-1982 are presented. Intestinal metaplasia was found in 267 biopsies (20%), being common in association with carcinoma (71%), less so in benign conditions such as gastric ulcer (39%), and chronic gastritis (24%), but rare in 'normal' (3%). Type I intestinal metaplasia was predominant (73%) in all the biopsies with intestinal metaplasia and was most common in benign conditions, 70% in gastric ulcer and 76% in chronic gastritis. Type III intestinal metaplasia (incomplete sulphomucin-secreting intestinal metaplasia) was recorded in only 9.8% of all the biopsies with intestinal metaplasia and had a higher incidence in carcinoma (35%), than in benign conditions (7%) (p less than 0.0001). These results suggest that intestinal metaplasia types may have different malignant potential and their identification may be useful in screening patients for early detection of cancer.

Uptake of [35S]sulphate in human colonic mucosa associated with carcinoma: an autoradiographic analysis at the ultrastructural level.

The uptake of [35S]sodium sulphate, as a sulphomucin precursor, was studied at the ultrastructural level as part of our investigation into alterations in glycoprotein synthesis occurring in colonic mucosa adjacent to carcinoma ('transitional' mucosa). The sulphate labelling was analysed statistically after incorporation and pulse labelling studies to show the amount of uptake into cellular organelles and the rate of transport through the cells. Uptake studies showed the following changes in the 'transitional' mucosa as compared with normal mucosa. (1) In the middle crypt, sulphate incorporation from the Golgi apparatus into the mucin droplets of the goblet cells was reduced in contrast with an increased uptake into the vesicles of the absorptive and 'intermediate' cells. (2) In the upper crypt, more sulphate was incorporated in the goblet cells. These results correlate well with previous histochemical findings of a larger proportion of sulphomucins in the vesicles of columnar cells and a predominance of sialomucins in the goblet cells in the colonic mucosa associated with carcinoma. The pulse labelling studies revealed that in the middle crypt region of the 'transitional' mucosa, the rate of movement of label was faster through the Golgi body but less labelling was reaching the mucin droplets. The data suggest that either an inadequate supply of acceptor molecules was available at the Golgi level or a blockage in the attachment mechanism of the free sulphate to the completing glycoproteins was occurring at this level. Alternatively both mechanisms may be operating simultaneously.