Inhibition of adenine nucleotide synthesis: effect on tight junction structure and function of clone 4 MDCK cells.

The formation and maintenance of tight junctions as a barrier to the diffusion of ions and other water-soluble across epithelia is an energy-dependent process. The administration of N-formyl-hydroxyaminoacetic acid (Hadacidin), an analog of aspartate and a competitive inhibitor of adenylosuccinate synthetase, has been shown to inhibit the multiplication of clone 4 MDCK cells and concomitantly reduce the levels of ATP and cAMP (J. Cell. Physiol. 140, 186-194 (1989)). When added to mitotically quiescent confluent cultures of clone 4 MDCK cells, millimolar concentrations of Hadacidin inhibited the generation of transepithelial electrical resistance (TER). In such cultures passive Na+ permeability was similar to controls indicating that the effect of Hadacidin was not on the transcellular pathway. That these cells were viable was demonstrated by their ability to exclude Trypan Blue, and the fact that they remained competent to develop steady state TER upon removal of the inhibitor. Suppression of TER was completely reversed within 48 h of replacing the Hadacidin-supplemented medium with one containing aspartate. Adenosine, but not aspartate, when added simultaneously with the drug, obviated the latter's effect on TER. A mixture of dibutyryl cAMP (db-cAMP) and theophylline was only partially effective in overcoming the effects of Hadacidin on the development of TER and, in fact, markedly delayed its development in control cultures not treated with the drug. When monolayers with established steady state TER were exposed to Hadacidin, no change was noted during the first 24 h. By 48 h, however, TER had decreased to very low values.(ABSTRACT TRUNCATED AT 250 WORDS)

Genomic organization and tissue expression of the murine gene encoding the protein beta-aspartate methyltransferase.

Two overlapping clones containing the entire 684-nucleotide (nt) sequence encoding murine protein beta-aspartate methyltransferase (EC 2.1.1.77) were isolated from a genomic library. Partial nt sequence analysis of the two clones revealed that the protein carboxyl methyltransferase (PCMT)-encoding sequence is distributed among seven exons, ranging from 32 to 339 bp in length, within 25 kb of genomic DNA. Three exons correspond to regions of primary structure which are strongly conserved among a number of eukaryotic and prokaryotic enzymes which utilize S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy). The 5'-flanking region of the PCMT-encoding gene (PCMT) contains an 800-bp G+C-rich region with potential binding sites for transcription factor ETF, but lacks a TATA box and binding sites for other known transcription factors. Multiple PCMT mRNAs were detected on Northern blots of RNA extracted from murine brain, testis, liver and kidney. The overall abundance of PCMT mRNAs in each tissue paralleled the measured specific activity of the PCMT. Comparison of the genomic sequence information with the 3'-untranslated regions (UTRs) of two cDNA clones from a murine testis library indicated that PCMT mRNA precursors undergo alternative splicing. The structure and widespread expression of PCMT are characteristics of vertebrate housekeeping genes.

Protein carboxyl methylation and methyl ester turnover in density-fractionated human erythrocytes.

A widely distributed methyltransferase modifies protein D-aspartyl and L-isoaspartyl residues which arise spontaneously as proteins age. Protein carboxyl methylation reactions were analyzed in human erythrocytes which had been separated on density gradients, a procedure which provides fractions enriched in older cells in the denser areas of the gradient. The total flux of methyl groups through the carboxyl methylation pathway was monitored by incubating cells from each fraction with L-[methyl-3H]methionine and measuring the formation of both protein [3H]methyl esters and [3H]methanol, derived from the hydrolysis of protein [3H]methyl esters in vivo. Cells isolated from denser areas of the gradient showed progressively higher rates of both protein carboxyl methylation and methanol production. In all cases, only 10-20% of the total methyl groups transferred were still present as intact protein [3H]methyl esters, consistent with the rapid hydrolysis of protein methyl esters in erythrocytes of all ages. The total flux of methyl groups through the carboxyl methylation pathway was approximately 3-fold higher in cells isolated from densest areas of the gradient compared to cells isolated from least dense areas of the gradient. Increases of a similar magnitude were observed in the numbers of both membrane protein carboxyl methyl esters and cytosolic protein carboxyl methyl esters. The only protein whose methylation was unchanged in denser cells was a 35,000 Da cytosolic protein. It has been proposed that protein carboxyl methyl esters are intermediates in either the repair or metabolism of structurally damaged proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Dating, mating, and motherhood: identity construction among Mexican maquila workers.

PIP: The authors explore the gender identities among women factory workers in Ciudad Juarez, Mexico. Using data from 3 generations of women, they show that women's participation in the maquila work force is exposing them to new ideologies which challenge traditional images embodied in the marianismo ideal of Mexican womanhood. By focusing upon women's changing experiences of courtship and motherhood, the authors suggest that conventional discourses stressing parentally supervised mate selection and full-time motherhood are being challenged by alternative ones which allow young women to socialize freely with prospective mates in unsupervised contexts, and expand the meaning of responsible motherhood to encompass full-time employment. Women workers' identities are fluid processes in permanent negotiation. ¿^ieng

Identification of a site for carboxyl methylation in human alpha-globin.

The human erythrocyte protein carboxyl methyltransferase modifies unusual protein D-aspartyl and L-isoaspartyl residues which arise spontaneously from internal rearrangements accompanying asparaginyl deamidation and aspartyl isomerization. A site of methylation associated with alpha-globin in intact cells has been identified by peptide mapping of radiolabeled globin isolated from human erythrocytes previously incubated with L-[methyl-3H]methionine. The site is located in a Staphylococcus V8 peptide containing residues 1-30 of alpha-globin. Two potential sources of methylation sites are present in this sequence at Asp-t and Asn-9.

Methylation of atypical protein aspartyl residues during the stress response of HeLa cells.

A protein carboxyl methyltransferase (PCMT), which specifically modifies atypical protein L-isoaspartyl and D-aspartyl residues, is widely distributed in eucaryotic cells, but the factors that regulate its activity in vivo have not been identified. It has been proposed that the PCMT initiates the repair of structurally damaged proteins. To test the possibility that the concentration of structurally abnormal cellular proteins affects PCMT activity, protein carboxyl methylation reactions were studied in HeLa cells exposed to various stresses that increase the extent of protein unfolding in cells. Protein carboxyl methylation rates increased 70-80% during incubations at 42 degrees C and remained elevated for periods of up to 8 hr. This sustained increase was greater than that predicted from thermal effects on the enzyme alone and may reflect the exposure of atypical aspartyl sites as proteins unfold as well as increased rates of protein deamidation and isomerization at elevated temperatures. Methylation rates showed no increases following 12 hr incubations with the amino acid analogs L-azetidine-2-carboxylic acid or L-canavanine. Northern blot analysis of RNA preparations from control and stressed cells revealed three major transcripts for the PCMT in HeLa cells, which are 1.6, 2.6, and 4.5 kb in length. The concentrations of all three transcripts decreased by approximately 20% from control levels during heat shock. No changes in PCMT transcript concentrations were observed during incubation with the amino acid analogs. By contrast, large increases in the concentrations of hsp70 and ubiquitin transcripts were observed following either heat or chemical stresses. The results demonstrate that the PCMT is a constitutive component of cells whose function is required under normal conditions as well as during stress conditions, which accelerate structural damage to cellular proteins.

Reduction of adenine nucleotide content of clone 4 MDCK cells: effects on multiplication, protein synthesis, and morphology.

The antitumor agent hadacidin (N-formyl-hydroxyamino-acetic acid), at 4 mM, inhibited the multiplication of clone 4 Madin Darby canine kidney (MDCK) cells within 24 hr. Growth resumed rapidly upon replacement of hadacidin with aspartate, an observation consistent with the drug's action as a competitive inhibitor of adenylosuccinate synthetase, an enzyme in adenine nucleotide biosynthesis. Data indicate that the drug-treated cells were arrested in S phase of the cell cycle. Accompanying inhibition of multiplication was a 16-fold increase in the area occupied by the cells and a refractoriness to release by treatment with trypsin. None of these changes occurred when 0.5 mM adenosine was included in the incubation mixture containing the inhibitor. Hadacidin decreased the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) content of the cells as well as the rate at which 3H-leucine was incorporated into protein. In the presence of 1 mM dibutyryl cAMP and theophylline, the drug had no effect on cell division and protein synthesis. The data suggest that, in clone 4 MDCK cells, the effects of hadacidin are mediated by diminishing the level of cAMP.

Expression and secretion of a recombinant ricin immunotoxin from murine myeloma cells.

Expression plasmids carrying a humanized N901 immunoglobulin heavy chain gene (hN901HC) fused to a gene encoding the native B chain of ricin toxin (RTB), hN901HC-RTB, or a sugar binding mutant of RTB, hN901HC-RTB delta gly, were constructed. In each case, the fused gene constructions were co-expressed in murine myeloma cells (Sp2/0) with the gene for humanized N901 immunoglobulin light chain to produce the secreted recombinant products hN901-RTB and hN901-RTB delta gly, respectively. When purified by affinity chromatography, both the hN901-RTB and hN901-RTB delta gly products were found to have an apparent molecular mass of M(r) = 210,000 and to be composed of two hN901 antibody heavy chains each fused to a full-length copy of RTB and two hN901 antibody light chains. In each of the recombinant fusions the hN901 antibody moiety retained the full binding affinity and specificity for its cognate antigen, CD56. Moreover, when mixtures of hN901-RTB and native ricin A chain were incubated in the presence of the antigen-positive target cell line SW-2, antigen-specific potentiation of ricin A chain cytotoxicity was observed. It has been demonstrated previously that lectin activity of the B chain is essential for A chain cytotoxicity, and we conclude that the fused wild-type B chain was properly folded and maintained lectin activity. These data demonstrate that feasibility of using recombinant ricin B chain in an immunotoxin and of using mammalian cell culture for its expression. The use of recombinant hN901-RTB fusion protein to evaluate the contribution of the lectin activity of ricin B chain in the penetration of cell membranes by ricin A chain is proposed.

Folate-maytansinoids: target-selective drugs of low molecular weight.

Folate receptor is over-expressed in a variety of carcinomas. To design a cytotoxic drug that would selectively target these carcinomas, we synthesized folate-maytansinoids. These drugs showed high affinity toward folate receptor, appeared to enter cells exclusively via the folate receptor-mediated caveolar pathway and displayed high cytotoxic potency (in the range of 10[-11] to 10[-10] M) and remarkable selectivity for folate receptor-expressing carcinoma cell lines. Folate-maytansinoids represent a new class of tumor-specific agents in which the targeting and the cytotoxic function can be altered independently.