Formation of germ layers and roles of the dorsal lip of the blastopore in normally developing embryos of the newt Cynops pyrrhogaster

Three-dimensional relationships between tissues during the formation of germ layers were studied in sections of normally developing embryos of the newt, Cynops pyrrhogaster. In gastrulae, the inner postinvolution layer was not in direct contact with the outer preinvolution layer as a result of the presence of an intervening layer of cells. Only after the formation of the yolk plug, a narrow strip of primitive notochord, which consisted of columnar cells, established a close contact with the central part of the overlaying presumptive neural plate. The primitive notochord was also linked to endoderm at its right and left margins, facing the archenteron. Mesodermal cells other than notochord cells were mesenchymal until the neurula stage, when primitive somites appeared on both sides of the notochord. From a comparison of the relative locations of tissues in embryos at different stages of development, it was shown that the notochord elongates by a remodeling of the mass of the primitive notochord, and that, as the anteriorly directed translocation of the neural area and the invagination of endoderm occur, these processes keep pace with the elongation of the notochord. These observations suggest organizing or guiding roles for the notochord in the formation of germ layers. A role for the dorsal lip of the blastopore as the organizer is discussed in relation to the origin of the notochord.     

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.     

Phloem Loading and Transport of Endogenously or Exogenously Labelled Photo-Assimilate in Bean, Beet, Maize and Cucurbit

Transport of carbon-11 labelled photo-assimilate was monitoredin Phaseolus vulgaris. Beta vulgaris, Zea mays, and Cucwbitopepo. The region of leaf to be labelled was first abraded anda solution passed over it to gain access to its apoplast andmonitor changes in label therein. With PCMBS in the bathingsolution the rate of washout of label into the bathing solutionincreased, but the effect of phloem loading was very variablefor each species: on some occasions transport was hardly affected,on others it was halted. This was true even for Cucurbito pepo,where a symplastic pathway of loading has been widely acceptedand suggests that PCMBS affects symplastic transport or thatthere is an apoplastic step in Cucurbito pepo. Apoplastic pHhad little effect on transport or label washout unless a veryacid (pH 40) buffer was introduced, contrary to notions of hydrogenion co-transport for sugar uptake. Anoxia caused phloem loadingto decrease immediately and label washout to increase in allspecies. It is suggested that both symplastic and apoplasticpathways can operate in all species but that their proportionvaries according to species and ambient and/or growth conditions. Key words: Phloem loading, photo-assimilate transport     

Localization of immunoreactive epidermal growth factor receptors in human nervous system

Epidermal growth factor is a well-defined peptide which stimulates cell growth and elicits cell responses in a variety of tissues by binding to specific receptors, EGF-R. A specific antiserum against the EGF receptor, which has previously been used to characterize EGF-R in human skin, fibroblasts, and smooth muscle, was used to survey the distribution of EGF-R in human nervous system. Portions of formalin-fixed, paraffin-embedded autopsy specimens were examined by use of immunohistochemical staining (PAP technique) with EGF-R antiserum. Many types of nerve cells, e.g., cerebral cortical pyramidal cells, hippocampal pyramidal cells, Purkinje cells, anterior horn cells, and dorsal root ganglion neurons, contained immunoreactive EGF-R. However, immunoreactive EGF-R were not detected in astrocytes, oligodendrogliocytes, and other small neurons such as granule cells. Intense immunostaining for EGF-R was also detected in ependymal cells from choroidal and extrachoroidal locations. Although immunoreactive EGF-R is widely distributed in human nervous system, the functional role of EGF and its receptor in the nervous system remains unknown.     

Localization of the receptor binding site of IFN-alpha 2b

The receptor binding site of IFN-alpha is not precisely known. To further characterize this site, mAb against IFN-alpha 2b were selected that block the binding of radiolabeled IFN-alpha 2b to its cell surface receptor. These antibodies also neutralized the anti-viral and anti-proliferative properties of IFN-alpha 2b. A subset of these antibodies (group 1) do not recognize IFN-alpha 2a, either in solid-phase immunoassays or functional assays, whereas a second subset (group 2), with no cross-reactivity with group 1, recognizes both IFN-alpha subtypes. Because IFN-alpha 2b and IFN-alpha 2a differ by only alpha Arg23-Lys23 substitution, group 1 antibodies must recognize an epitope within the receptor binding region of IFN-alpha 2b that includes Arg23. Group 2 antibodies recognize a separate and distinct epitope within the binding site that does not include Arg23.     

The E beta hot spot of recombination in wild-derived natural recombinant MHC haplotypes. Cross-over site mapping and the identification of a 1.0-kb E beta deletion in the p and w14 haplotypes

Detailed molecular analysis of three wild-derived MHC haplotypes provided evidence for an important role of the E beta recombinational hot spot in the recent evolution of the mouse I region. Examination of RFLP and restriction maps of cloned DNA permitted the mapping of the natural cross-over events in the haplotypes carried by strains B10.GAA37 (w21) and B10.KPB128 (w19) to a fragment of DNA not exceeding 4.1 kb, which lies almost entirely within the intron separating the beta 1 and beta 2 exons of the E beta gene. In the w14 haplotype (strain B10.STC77), which appears to be a natural recombinant between a p-like parental haplotype and another wild-derived haplotype, the site of crossing over can be mapped to a segment between the beta 2 exon of the E beta gene (left border) and the E beta 2 gene (right border). This segment containing the cross-over site in the w14 haplotype includes the E beta hot spot. In addition, the w14 haplotype as well as the standard p haplotype contain a deletion of approximately 1.0 kb in the second intron of the E beta gene, which may represent the product of an unequal cross-over event in a E beta recombinational hot spot.     

Differential effects of glucocorticoids on the proliferation of a murine helper and a cytolytic T cell clone in response to IL-2 and IL-4

The proliferation of murine T cell clones can be supported by IL-2 or by IL-4. We present here evidence that glucocorticosteroids differentially affect these two pathways of proliferation. Dexamethasone (DEX) and other corticosteroids were observed to induce autocrine proliferation of the D10.G4.1 Th cell clone (D10) in the presence of the anti-clonotypic antibody 3D3. This effect was inhibited by the anti-murine IL-4 antibody 11B11, indicating that it is mediated by IL-4. Furthermore, on this cell line, representative of the Th2 group of helper cells, DEX had little effect on the proliferation induced by exogenous IL-4 but completely inhibited the growth-promoting effects of IL-2. In contrast, the effects of DEX on the proliferation of the cytotoxic IL-2-dependent CTLL-2 cell line are completely opposite. DEX blocked the IL-4-driven proliferation of CTLL-2 cells, while leaving unaffected their response to IL-2. It is also shown in this study that the effects of glucocorticoids in this system are totally antagonized by the high affinity anti-glucocorticosteroid RU 38486, indicating that they are mediated through the described intracellular glucocorticoid receptor. These data suggest that the growth effects of IL-2 and IL-4 may be mediated by distinct pathways that are strikingly different in their sensitivity to glucocorticoids. In addition, the regulation of lymphokine-dependent proliferation and the response to glucocorticoids appeared very different in helper and cytotoxic cells.     

Regulation of the CD2 alternate pathway of T cell activation by CD3. Evidence for heterologous desensitization

Normal resting T cells were stimulated through the alternate CD2 pathway. A CD3 mAb VIT3 completely blocked their proliferative response. The time interval for 50% inhibition lasted for 24 h after the onset of CD2 stimulation. Mitogen-activated cloned long term cultured T cells could also be stimulated via CD2. This proliferative response was again inhibitable by VIT3, indicating that CD3 regulates the CD2 pathway not only in resting cells, but also in lymphocytes actively involved in an Ir. T cells were further loaded with Quin2 and their free cytoplasmic Ca2+ levels were monitored in response to CD3 and CD2 stimulation. Antibodies directed against both surface R triggered a rapid elevation of Ca2+ levels. Both responses were abrogated when the cells had been treated overnight with VIT3. The free cytoplasmic Ca2+ levels of VIT3-pretreated cells, however, were not higher than those of control cells. These results point to a functional interaction between CD3 and CD2 possibly at the level of signal transducing proteins. Finally, cholera toxin was found to inhibit the Ca2+ response in Jurkat T cells. Both the CD3 and CD2 stimulation were sensitive to cholera toxin, indicating that a GTP-binding protein may be involved in signal transduction for both surface structures.     

Qa-2 antigen encoded by Q7b is biochemically indistinguishable from Qa-2 expressed on the surface of C57BL/10 mouse spleen cells

Qa-2 was immunoprecipitated from the surface of 125I-labeled C57BL/10 (B10) mouse spleen cells and compared with Qa-2 immunoprecipitated from the surface of R1.1 thymoma cells transfected with Q7b. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that Qa-2 glycoproteins from both of these sources have a relative molecular mass of approximately 37 kDa. After treatment with endoglycosidase F, the Qa-2 polypeptide chains derived from C57BL/10 spleen and Q7b-transfected R1.1 cells displayed identical mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis because of removal of N-linked oligosaccharide residues. Furthermore, treatment of Qa-2 proteins from both sources with cyanogen bromide or alpha-chymotrypsin resulted in identical peptide fragmentation patterns. These results therefore provide a biochemical correlation between a cloned Qa-region gene produce expressed on the surface of transfected cells, and the Qa-2 glycoprotein on spleen cells that was described a decade ago by serologic methods.