CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.     

Further studies on aspartate aminotransferase of thermophilic methanogens by analysis of general properties, bound cofactors, and subunit structures.

Aspartate aminotransferase (AspAT) [EC 2.6.1.1] of thermophilic methanogen was further characterized with the enzyme from Methanobacterium thermoautotrophicum strain FTF-INRA as well as M. thermoformicicum strain SF-4. AspAT of strain FTF-INRA was similar in the amino donor specificity to the enzyme of M. thermoformicicum strain SF-4, in that it was active on L-cysteine and L-cysteine sulfinate in addition to L-glutamate and L-aspartate. The enzymes gave similar absorption spectra having maxima at around 326 and 415 nm with no pH-dependent shift but were found to contain 1 mol of tightly bound pyridoxal 5'-phosphate (PLP) per subunit. Reconstitution of each apoenzyme with added PLP resulted in partial recovery of the original enzymatic activity, suggesting a significant conformational change of the active site region upon removal of the cofactor. Polyacrylamide gel electrophoresis (PAGE) and gel filtration analyses revealed a tetrameric structure (180 kDa) of identical subunits with a molecular mass of 43 kDa for each of these enzymes. Electric current was found to affect the interaction or affinity of each subunit, promoting dissociation of the native enzyme into the monomeric form. Alkaline treatment was effective only for dissociation of the enzyme from strain SF-4. They were distinguishable by the more rapid reassociation of the monomer to the native aggregated form in the enzyme of strain FTF-INRA.     

Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts

Rat 3Y1 cells acquire metastatic potential when transformed with v-src, and this potential is enhanced by double transformation with v-src and v-fos (Taniguchi, S., T. Kawano, T. Mitsudomi, G. Kimura, and T. Baba. 1986. Jpn. J. Cancer Res. 77:1193-1197). We compared the activity of cadherin cell adhesion molecules of normal 3Y1 cells with that of v-src transformed (SR3Y1) and v-src and v-fos double transformed (fosSR3Y1) 3Y1 cells. These cells expressed similar amounts of P-cadherin, and showed similar rates of cadherin-mediated aggregation under suspended conditions. However, the aggregates or colonies of these cells were morphologically distinct. Normal 3Y1 cells formed compacted aggregates in which cells are firmly connected with each other, whereas the transformed cells were more loosely associated, and could freely migrate out of the colonies. Overexpression of exogenous E-cadherin in these transformed cells had no significant effect on their adhesive properties. We then found that herbimycin A, a tyrosine kinase inhibitor, induced tighter cell-cell associations in the aggregates of the transformed cells. In contrast, vanadate, a tyrosine phosphatase inhibitor, inhibited the cadherin-mediated aggregation of SR3Y1 and fosSR3Y1 cells but had little effect on that of normal 3Y1 cells. These results suggest that v-src-mediated tyrosine phosphorylation perturbs cadherin function directly or indirectly, and the inhibition of tyrosine phosphorylation restores cadherin action to the normal state. We next studied tyrosine phosphorylation on cadherins and the cadherin-associated proteins, catenins. While similar amounts of catenins were expressed in all of these cells, the 98-kD catenin was strongly tyrosine phosphorylated only in SR3Y1 and fosSR3Y1 cells. Cadherins were also weakly tyrosine phosphorylated only in the transformed cells. The tyrosine phosphorylation of these proteins was enhanced by vanadate, and inhibited by herbimycin A. Thus, the tyrosine phosphorylation of the cadherin-catenin system itself might affect its function, causing instable cell-cell adhesion.     

Cloning and sequencing of the human homeobox gene HOX4A.

The HOX4A gene, one of the homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with a HOX4B cDNA probe. The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. According to conceptual translation, the HOX4A protein is predicted to be composed of 416 amino acid residues. Interestingly, the HOX4A protein has a sequence, Pro-Ala-Ser-Gln-Ser-Pro-Glu-Arg-Ser, eight amino acids downstream from the homeodomain, which is similar to that containing a phosphorylation site in pp60c-src, Pro-Ala-Ser-Gln-Thr-Pro-Asn-Lys-Thr. However, the HOX2G protein, which exhibits a paralogous relationship with the HOX4A protein, does not possess the sequence which is similar to that in pp60c-src. A comparison of the predicted HOX4A protein with the HOX2G protein revealed four regions of amino acid sequence similarities: an N-terminal tetrapeptide, a pentapeptide (pre-box) upstream of the homeodomain, the homeodomain and a C-terminal octapeptide.     

Immunocytochemical studies on the pancreatic endocrine cells in the Japanese newt (Cynopus pyrrhogaster)]

Pancreatic endocrine cells were examined by light and electron microscopic immunocytochemistry to discuss the co-localization of peptides in one cell type. A cells were irregular in shape with an occasional long cytoplasmic process, and contained glucagon-immunoreactive granules with various contours. These granules were 160-300nm in diameter with various density, and also immunoreactive to anti-human pancreatic polypeptide (PP) serum. A part of them were further immunoreactive to anti-somatostatin serum. B cells were round to elliptical in shape, and often aggregated around the capillaries. Granules of B cells were round to irregular in shape, 270-410 nm in diameter, and immunoreactive to anti-insulin serum. D cells were irregular in shape with meager cytoplasm, and contained somatostatin-immunoreactive granules. These granules were ovoid or teardrop in shape, 140-250nm in longitudinal diameter, and immunoreactive to both anti-somatostatin and anti-human PP sera. PP cells were round to spindle-shaped, and contained human PP-immunoreactive round granules 150-35nm in diameter. These findings reveal the existence of at least 4 types of endocrine cells secreting glucagon, insulin, somatostatin, and PP, respectively, in the newt pancreas, and suggest the co-localization of some of these peptides in one cell type.     

Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.     

Expression of retinoic acid receptor genes in neural crest-derived cells during mouse facial development

Retinoic acid (RA) is known as a teratogen that induces abnormalities in facial structures which are made up mainly of neural crest-derived mesenchyme. We investigated expression patterns of RA receptor (RAR) genes (subtypes alpha, beta, gamma) during mouse facial development. The expression of the RAR beta gene is specific for the mesenchyme around developing eyes and nose, whereas the RAR gamma gene is expressed in the mesenchyme differentiating to facial cartilages and bones. In contrast, the RAR alpha gene is expressed weakly and uniformly over the facial region. These results suggest that crucial roles of endogenous RA in facial development depend on differential functions of the RAR subtypes.