Lamin dimers. Presence in the nuclear lamina of surf clam oocytes and release during nuclear envelope breakdown

The nuclear lamina of surf clam oocytes contains dimers of 67-kDa lamin which are stabilized by both noncovalent interactions and disulfide bonds. The latter can be reduced but re-form when the reducing agent is removed. The cysteine residues involved in these disulfide bonds are inaccessible to alkylating agents unless the protein is unfolded in urea. During nuclear envelope breakdown the lamin is released as a mixture of oligomers in which disulfide-stabilized dimers are associated noncovalently with lamin monomers. Concurrent with solubilization, both dimers and monomers are phosphorylated to a similar extent, indicating that the interactions which maintain these complexes are not destabilized by lamin phosphorylation. Our results suggest the existence of two types of interactions between the lamin molecules in the polymer, which react differently to phosphorylation during nuclear envelope breakdown.     

Evidence for probenecid-sensitive organic anion transporters on polarized thyroid cells in culture

Epithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half-maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen-coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.     

A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.     

Fully differentiated Xenopus eye fragments regenerate to form pattern-duplicated visuo-tectal projections

In order to determine if differentiated Xenopus retina is capable of undergoing regeneration and duplicative pattern formation, we devised a new surgical technique for removal of the temporal two-thirds of the retina. In a series of progressively older larval eyes starting with late tailbud stage embryos (stage 38) and extending to limb-bud stage tadpoles (stage 48), nasal one-third-sized eye fragments successfully regenerated to form nearly normal sized eyes over 75% of the time. Histological preparations showed that early wound healing involved the formation of a neuroepithelium at the ventro-temporal region of the fragment. The pigmented retinal epithelium and associated retinal tissue appeared to be involved in this process. Animals from each stage were reared through metamorphosis and electrophysiologic techniques were employed to determine visuo-tectal projections. Seventy percent of stage 38 animals showed evidence of pattern-duplicated projections. Ninety percent of their responding tectal points showed duplicate innervation from two retinal regions. Older animals (stages 44 to 48) showed less duplication. Only 52% of their responding tectal points duplicated (P less than 0.001). Thus, fully differentiated Xenopus retina can undergo regeneration and duplicative pattern formation similar to that shown by embryonic retinal tissue.     

Do all human urinary infections with Schistosoma mattheei represent hybridization between S. haematobium and S. mattheei?

Enzyme electrophoresis indicated that all Schistosoma mattheei eggs passed in the urine of humans derive from S. mattheei females in copula with S. haematobium males. It appears that S. mattheei males do not reach sexual maturity in man; however, S. haematobiumxS. mattheei males possibly do.     

Elucidation of discontinuous linear determinants in peptides.

Synthetic peptides, made by the method of simultaneous multiple peptide synthesis, were coupled to the protein carrier keyhole limpet hemocyanin and used to raise mAb. Omission and substitution analogs of the original peptides were tested by ELISA to characterize their reactivity with the respective mAb. Linear antigenic determinants were located for 18 different peptides by using omission analogs. The length of the antigenic determinants ranged from 2 to 8 residues, with an average of 6 residues. The three aromatic amino acids, phenylalanine, tryptophan, and tyrosine, the charged hydrophilic amino acids, aspartic acid and lysine, and the neutral amino acid alanine were found to occur most often in the determinant region of the peptides tested, whereas asparagine, cysteine, and histidine occurred the least often. Alanine substitution analogs provided more information than omission analogs by enabling the determination of which side chain groups of the antigenic determinant residues were not critical for binding to the mAb. Detailed, "fingerprint" information about the interaction of the peptide, GASPYPNLSNQQT, and its mAb was obtained by synthesizing a complete series of analogs with individual substitutions for each position of the antigenic determinant, PYPNLS, with the 19 other amino acids. These results suggest that, at the amino acid level, all antigenic determinants of synthetic peptides defined by mAb can be considered discontinuous linear determinants.     

Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation.

Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR. Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb. Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns. Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb. This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line. When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected. The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation. Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation. Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation. The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation. When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate. This occurred in both T cell blasts and in the Jurkat T cell line. Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway.     

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.     

CD18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits

Neutrophil (PMN) migration in the systemic and pulmonary circulation of rabbits was compared by using different inflammatory stimuli to determine the role of the leukocyte adhesion complex, CD11/CD18, in each of these vascular beds. The adhesion complex was blocked by administering the anti-CD18 mAb 60.3. The data show that mAb 60.3 blocks PMN emigration into inflammatory foci in the abdominal wall produced by implanting sponges containing either hydrochloric acid, Streptococcus pneumoniae, Escherichia coli endotoxin, or PMA. mAb 60.3 also inhibited PMN emigration in response to peritoneal instillation of S. pneumoniae. The effect of mAb 60.3 on PMN emigration in the lungs varied depending upon the stimulus. PMN failed to migrate into the PMA-induced pneumonia; however, mAb 60.3 pretreatment only partially inhibited endotoxin-induced pneumonia and did not inhibit S. pneumoniae or hydrochloric acid-induced pneumonias. PMN lavaged from the alveolar spaces in the Streptococcal pneumonia had similar quantities of mAb 60.3 bound to their surfaces as the circulating PMN. We conclude that the CD11/CD18 complex mediates PMN adherence in the systemic circulation. However, PMN adherence in the pulmonary circulation may occur by either CD18-dependent or -independent mechanisms that are specific to the inciting stimulus.     

Metabolism and Translocation of Exogenous Zeatin Riboside in Germinating Seeds and Seedlings of Zea mays

High specific activity [³H]-zeatin riboside (ZR) was suppliedto germinating seed and developing seedlings of Zea mays tostudy its metabolism and translocation The major metabolitesof ZR in endosperm, embryo, and first leaves were adenosine,adenine, and adenine nucleotide When ZR was supplied to theradicle tip a significant proportion of the radioactivity extractedfrom the radicle was identified as zeatin-9-glucoside (Z9G).However, some ZR was also transported to the shoot and vestigialembryo During the initial stage of germination, movement ofzeatin riboside from the embryo to the endosperm was pronouncedbut little movement occurred in the reverse direction Key words: Zea mays cytokinin, zeatin riboside, metabolism, translocation