Shakuntla Seetharam

Molecular cloning and expression of a cDNA encoding the membrane-associated rat intestinal alkaline phosphatase.

Rat intestinal alkaline phosphatase (IAP) has been purified and proteolytic fragments sequenced. A cDNA library was constructed from duodenal poly(A) + RNA and screened for IAP positive clones by a full-length cDNA clone-encoding human IAP. A full length rat IAP clone (2237 bp) was isolated and sequenced, revealing a predicted primary sequence of 519 amino acids (61.974 kDa) with an additional signal peptide of 20 amino acids. 80% of amino acids from residues 1-474 were identical when compared with the human IAP, but there was only 31% identity in the COOH-terminal 45 amino acids. The homology diverges just before the putative binding site for the phosphatidylinositol-glycan (PI-glycan) anchor. The resulting peptide in rat AP contains five hydrophilic amino acids not present in the primary structure of human IAP. Binding of a synthetic 48-mer encoding a portion of this unique and divergent region (residues 476-491) was compared with that of the full-length clone on Northern blots of rat intestinal RNA. Two mRNAs, 3.0 and 2.7 kb, were detected by both probes, confirming earlier results, but the 48-mer bound preferentially to the 3.0 kb mRNA. The protein product of the full-length cDNA in a cell-free system was 62 kDa, corresponding with the smaller of the two IAP proteins produced by rat duodenal RNA. The cDNA transfected into COS-1 cells produced a membrane-bound IAP that was released by phosphatidylinositol-specific phospholipase (PI-PLC). These data provide definitive evidence that IAP is anchored by PI-glycan and conclusively demonstrate that the unique COOH-terminal structure encoded by this rat mRNA supports the addition of a PI-glycan anchor.

Human transcobalamin II receptor binds to Staphylococcus aureus protein A: Implications as to its structure and function☆

Purified human placental transcobalamin II receptor (TC II-R) dimer of molecular mass 124 kDa bound to Sepharose-linked bacterial immunoglobulin (IgG) binding proteins protein A, protein G, and protein A/G. TC II-R dimer was detected directly, by blotting human placental and rabbit and rat kidney membrane proteins with 125I-protein A, or indirectly, using antiserum to TC II-R or IgG-Fc region and 125I-protein. TC II-R antiserum, but not protein A, protein G, protein A/G, or antiserum to the IgG-Fc region, when added to culture medium of human intestinal epithelial Caco-2 cells or umbilical vein endothelial cells, inhibited ligand binding. However, protein A, protein G, protein A/G, or antiserum to the Fc region inhibited the internalization of the ligand TC II-[57Co]cyanocobalamin. Taken together, these studies strongly suggest TC II-R is an IgG-like molecule that contains an Fc-like region which is important in ligand internalization but not binding.

Fat feeding stimulates only one of the two mRNAs encoding rat intestinal membranous and secreted alkaline phosphatase

We have identified two mRNAs in rat intestinal mucosa by Northern blot analysis, using cloned cDNAs encoding human placental alkaline phosphatase (PLAP). Probes from both the NH2- and COOH-terminal ends of the human PLAP coding region identified, in rat intestine (especially duodenum), an mRNA of nearly identical size (3 kb) to that found in human placenta. A smaller mRNA (2.7 kb), detected only with the COOH-terminal probe, was more prevalent in jejunum. Following feeding of triacylglycerols, the prevalence of the 2.7 kb mRNA increased over 2-fold. The tissue distribution and response of the 2.7 kb mRNA to fat feeding corresponds exactly with the known behavior of the secreted alkaline phosphatase.

In vitro translation of intestinal sucrase-isomaltase and glucoamylase

It is has been proposed that both sucrase-isomaltase and glucoamylase are initially synthesized as large single-chain precursors which are then processed to heterodimers. We have tested this hypothesis by in vitro translation of their mRNAs. The primary translation product of sucrase-isomaltase mRNA was a single polypeptide of Mr = 190,000. Similar experiments using antiserum against glucoamylase revealed a single polypeptide of Mr = 145,000. These results are consistent with the single chain precursor hypothesis for sucrase-isomaltase. However, the glucoamylase product (145 kDa) is too small to be processed to a heterodimer of Mr = 230,000. Moreover, the mature subunits (Mr = 135,000 and 125,000) probably are derived from the 145 kDa precursor by proteolytic trimming and must include and share most of the precursor protein.

Expression of endodermally derived and neural crest-derived differentiation antigens by human lung and colon tumors

Recent studies of the plasma membrane antigens of a human lung tumor (an oat cell carcinoma) indicated that the tumor expressed endodermally derived and neural crest‐derived differentiation antigens undetectable in normal respiratory epithelium, where the cell of origin of this histologic type of tumor is believed to reside. The present studies were designed to assess whether or not such differentiation antigens were regularly expressed by other oat cell carcinomas of the lung, by other lung tumor types, and by a nonlung tumor type. Rabbit antisera were prepared to the plasma membrane fractions of five different oat cell carcinomas, two adenocarcinomas, and two epidermoid carcinomas of the lung, and two adenocarcinomas of the colon. After absorption with normal lung and liver, the antisera were tested against the immunizing tumor and normal lung, colon, and peripheral nerve frozen section substrates by the indirect immunofluorescence method. The results indicated that the neural crest‐derived differentiation antigen(s) was expressed by all five oat cell carcinomas, but none of the other tumors studied except for one adeno‐carcinoma of the lung. The endodermally derived differentiation antigen(s) was expressed by all of the tumors studied except for one epidermoid carcinoma of the lung. It was concluded that expression of at least some differentiation antigens which are undetectable in normal respiratory epithelium occurs not infrequently among lung carcinomas. Furthermore, expression of the neural crest‐derived differentiation antigen(s) is highly associated with, but not absolutely restricted to, oat cell carcinoma of the lung.

Rat transcobalamin: cloning and regulation of mRNA expression.

Transcobalamin (TC) has been cloned and used for studying its gene expression in the rat. TC mRNA is distributed widely in adult rat tissues, but at different levels (kidney > liver > lung > yolk sac > intestine > heart > brain > spleen > muscle). TC mRNA levels were 4‐fold higher in the jejunum and ileum compared to its levels in the duodenum. During postnatal development, TC mRNA levels in the ileum declined 4‐fold from day 4 to day 12, but increased by 5‐fold between days 12 and 24. In contrast, TC mRNA levels increased by 2.5‐fold in the kidney from day 4 to day 12 and then declined by 2‐fold by day 24. Adrenalectomy of adult rats resulted in a 4‐fold decline in ileal levels of TC mRNA and a 50% decline in the ileal mucosal formation of the TC‐[57Co] cobalamin (Cbl) complex following oral administration of [57Co]Cbl complexed to gastric intrinsic factor (IF). Cortisone treatment reversed these changes noted in the ileum. In contrast to ileum, kidney TC mRNA levels were not altered significantly in adrenalectomized rats before and after cortisone treatment. Taken together, this study has provided evidence for the regulation of TC gene expression in the rat kidney and intestine during their postnatal development, and cortisone selectively regulates ileal but not kidney TC mRNA levels.