Differential utilization of calcitonin gene regulatory DNA sequences in cultured lines of medullary thyroid carcinoma and small-cell lung carcinoma.

Regulation of expression of the human calcitonin gene was found to differ between two tumor lines of different tissue origin, medullary thyroid carcinoma (TT line) and small-cell lung carcinoma (DMS53 line). Distal 5' DNA elements between -750 and -2000 exhibited a stronger basal activity in DMS53 than in TT cells, whereas proximal DNA sequences between -132 and -252 mediated a dramatic cyclic AMP response in TT but not DMS53 cells.     

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.     

Decarboxylation of uroporphyrinogen III by erythrocyte uroporphyrinogen decarboxylase. Evidence for a random decarboxylation mechanism.

The isomeric composition of type-III heptacarboxylic porphyrinogens derived from decarbosylation of uroporphyrinogen III by erythrocyte uroporphyringogen decarboxylase was analysed by h.p.l.c. with electrochemical detection. All four possible isomers were identified, and there were little differences in the proportion of isomers formed by erythrocytes from normal subjects and from patients with sporadic porphyria cutanea tarda. The results provide conclusive evidence that the normal decarboxylation pathway is random in nature, and the fourth isomer only increases when enzyme abnormality is found.     

A phase II trial of murine monoclonal antibody 17-1A and interferon-γ: Clinical and immunological data

Summary A group of 15 patients with metastatic colorectal adenocarcinoma received a combination of interferon (0.1 mg/m², days 1–15) and the murine monoclonal antibody 17-1A (400 mg, days 5, 7, 9 and 12). The treatment was tolerated with minimal toxicity. Of the 14 evaluable patients, 13 developed human antibody to murine 17-1A, with 11 patients demonstrating antibody to the variable region of 17-1A (anti-idiotype). Antibody to the variable region was inhibited by 17-1A but not by mouse immunoglobulin. Sera from patients with substantial anti-idiotype reactivity were capable of inhibiting the binding of murine 17-1A to antigen expressing LS174-T cells thus indicating the presence of antibody directed against the 17-1A combining site (mirror-image anti-idiotype). The median survival of the whole group was 56 weeks and there was no correlation between clinical response/survival and the development of anti-idiotype antibody.Supported by the Veterans Administration Medical Center and by Public Health Services grant CA 45 232 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services     

Conformations of the central transforming region (Ile 55-Met 67) of the p21 protein and their relationship to activation of the protein

The GTP-binding p21 protein, encoded by the ras-oncogene, becomes transforming if amino acid substitutions are made at critical positions in the polypeptide chain, e.g., at Gly 12, Gly 13, Ala 59, Gln 61 and Glu 63. Most of these substitutions occur in two phosphate-binding loop regions, Tyr 4-Thr 20, herein designated as segment 1, and Ile 55-Met 67, herein designated, as segment 2. These two segments are homologous to two corresponding regions in the two purine nucleotide binding proteins, bacterial elongation factor (EF-tu) (Val 12-Thr 28 corresponds to segment 1; His 78-Ile 92 corresponds to segment 2) and adenylate kinase (ADK) (Lys 9-Cys 25 corresponds to segment 1 and Tyr 95-Arg 107 corresponds to segment 2). We find that the conformations of the segment 1 region in the p21 protein, EF-tu and ADK are similar to one another and that the conformation of the segment 2 region of EF-tu is superimposable on that of segment 2 of ADK. Furthermore, the relative position of the two segments in EF-tu is strikingly similar to that of the two segments in ADK. In the originally proposed X-ray structure for the p21 protein, the conformation of segment 2 in the p21 protein is not similar to that found for the other two proteins, and its disposition relative to segment 1 and the remainder of the protein is also different from that observed for the other two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products

The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.     

Non-everted oxygenated rat intestinal segments as a measure of neutral detergent fiber effects on iron absorption

Iron absorption in the presence of varying amounts and sizes of dietary fiber was measured. A method using non-everted rat intestinal segments perfused in oxygen was refined. Neutral detergent fiber (NDF), a component of dietary fiber, was extracted from cooked pinto bean (Phaseolus vulgaris). The NDF did not affect iron absorption in intestinal segments from iron replete rats. However, 4 and 6 mg of NDF/ml significantly decreased iron absorption in the intestinal segments from anemic rats. NDF with a smaller particle size of 0.125 mm increased iron absorption relative to that absorbed with 0.180 mm particles. Histological examination validated using non-everted intestinal segments perfused with oxygen as a method for studying dietary effects on iron absorption. Segments which are not everted are less prone to damage. Perfusion with oxygen maintained metabolic activity in the tissue during the experiment.