RESUMO
We have investigated the structure of the photosynthetic membrane in a mutant of barley known to lack a chlorophyll-binding protein. This protein is thought to channel excitation energy to photosystem II, and is known as the "light-harvesting chlorophyll-protein complex." Extensive stacking of thylakoids into grana occurs in both mutant and wild-type chloroplasts. Examination of membrane internal structure by freeze-fracturing indicates that only slight differences exist between the fracture faces of mutant and wild-type membranes. These differences are slight reductions in the size of particles visible on the EFs fracture face, and in the number of particles seen on the PFs fracture face. No differences can be detected between mutant and wild-type on the etched out surface of the membrane. In contrast, tetrameric particles visible on the etched inner surface of wild-type thylakoids are extremely difficult to recognize on similar surfaces of the mutant. These particles can be recognized on inner surfaces of the mutant membranes when they are organized into regular lattices, but these lattices show a much closer particle-to-particle spacing than similar lattices in wild-type membranes. Although several interpretations of these data are possible, these observations are consistent with the proposal that the light-harvesting chlorophyll-protein complex of photosystem II is bound to the tetramer (which is visible on the EFs face as a single particle) near the inner surface of the membrane. The large tetramer, which other studies have shown to span the thylakoid membrane, may represent an assembly of protein, lipid, and pigment comprising all the elements of the photosystem II reaction. A scheme is presented which illustrates one possibility for the light reaction across the photosynthetic membrane.
Assuntos
Proteínas de Transporte/análise , Clorofila , Cloroplastos/ultraestrutura , Grão Comestível/ultraestrutura , Hordeum/ultraestrutura , Fotossíntese , Proteínas de Plantas/análise , Cloroplastos/análise , Técnica de Congelamento e Réplica , Membranas/análise , Membranas/ultraestrutura , MutaçãoRESUMO
The freeze-fracturing technique has been used to investigate membrane architecture in the mesophyll and bundle sheath chloroplasts of Zea mays. The structural organization of mesophyll chloroplasts is virtually identical to that of other species of higher plants which have been investigated with this technique. Characteristic distributions of particles of various sizes are seen on each fracture face after membrane splitting during the fracturing process, and these distributions indicate the differentiation of the membrane system into sacked (grana) and unstacked (stroma) regions, typical of grana-containing chloroplasts. Bundle sheath chloroplasts contain very few grana, and the thylakoids of these plastids are therefore largely unstacked. Analysis of artificially unstacked mesophyll chloroplasts indicates that this difference is not merely related to the presence or absence of adhesion between adjacent thylakoids, but reflects a substantial difference in membrane substructure between mesophyll and bundle sheath photosynthetic membranes. Bundle sheath thylakoids contain virtually the same number of small (P fracture face) particles as mesophyll thylakoids, but contain only 40% as many of the larger (E fracture face) tetrameric particles. These differences, together with biochemical data indicating the comparative deficiency of bundle sheath chloroplasts in Photosystem II activity, suggest that the E face particles are related to the presence or absence of Photosystem II activity.
Assuntos
Cloroplastos/ultraestrutura , Fotossíntese , Membrana Celular/ultraestrutura , Técnica de Fratura por Congelamento , Microscopia Eletrônica , Plantas , Zea maysRESUMO
Brain levels of Ia mRNA, quantified by RNA blot analysis, were found to be 30-50-fold lower than splenic levels in all autoimmune and normal mouse strains examined except MRL/l, whose brain content of Ia mRNA was comparable to normal splenic levels. Prior perfusion to remove blood cells did not alter the amount of Ia mRNA obtainable from MRL/l brain. Elevation of Ia mRNA in MRL/l as compared to control C3H mice was also found in kidney, liver, and spleen, though not in thymus or lung. Results are discussed in relation to an animal model for central nervous system involvement in systemic lupus erythematosus.
Assuntos
Doenças Autoimunes/imunologia , Encéfalo/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Lúpus Eritematoso Sistêmico/imunologia , Animais , Doenças Autoimunes/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Haplótipos , Lúpus Eritematoso Sistêmico/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Poli A/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Immune response genes of the murine major histocompatibility complex encode cell-surface glycoproteins that are expressed predominantly on B cells and macrophages and regulate immune responsiveness by restricting antigen recognition by T cells. The two classes of immune response molecule, termed I-A and I-E, are each comprised of two polymorphic chains (alpha and beta), and nucleotide sequence analysis of genomic or cDNA clones has revealed that most of the amino acid differences between allelic I-A alpha or beta chains occur in the first extracellular domain. The mutant mouse strain B6.C-H-2bm12 (bm12), which differs from its parental strain C57BL/6 (B6) at the I-A beta locus, exhibits an immune response profile markedly different from that of B6. Here we present the nucleotide sequence of the mutant bm12 I-A beta gene. Sequence comparison within the coding regions reveals three productive nucleotide differences between the I-A beta genes of B6 and bm12 mice, all three differences occurring within a stretch of 14 nucleotides in the exon encoding the first extracellular domain. The clustered nature of the bm12 mutation, as well as the specific amino acid changes it engenders, suggest a possible mechanism for the generation of polymorphism in class II antigens.
Assuntos
Genes MHC da Classe II , Mutação , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Antígenos HLA/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Polimorfismo Genético , Especificidade da EspécieRESUMO
Our results demonstrate that expression of neuropeptide tyrosine, one of the most abundant and widespread peptides in the mammalian nervous system, occurs in non-neuronal cells, in keeping with the emerging view that neuropeptide synthesis is not restricted to cells of the nervous system. RNA blot analyses and radioimmunoassays detected both NPY mRNA and NPY peptide in rat and mouse spleen, bone marrow, and peripheral blood cells. Immunohistochemical staining of sections from rat bone marrow with an NPY-specific antiserum revealed NPY-like immunoreactivity in megakaryocytes. In situ hybridization confirmed that the NPY-like peptide detected in megakaryocytes was synthesized de novo from NPY mRNA present in these cells. Megakaryocytes, the platelet-forming cells, originate from pluripotent hematopoietic stem cells present in the bone marrow as well as in the spleen of rodents. During microvascular damage, platelets aggregate at the damaged site and release bioactive substances. NPY is known to be a potent vasoconstrictor. Therefore, we propose that megakaryocyte-derived NPY is stored in platelets and released during platelet aggregation, resulting in a long-lasting vasoconstriction. Greatly elevated levels of megakaryocyte-derived NPY, as compared to the level found in BALB/C mice, were found in several mouse strains (NZB, NZB x W, and BXSB) which develop an autoimmune disease resembling systemic lupus erythematosus. Whether the elevation of megakaryocyte-derived NPY plays a role in the autoimmune disease progression in these mice or whether it merely reflects a related hematopoietic abnormality remains to be determined. Subtractive hybridization was used to isolate two cDNA clones that are predominantly expressed in the brain and the immune system. These and similarly derived cDNA clones will be used as molecular probes to study the mechanisms governing tissue-specific expression in the nervous and immune systems. Discovering the function of the proteins encoded by such cDNA clones may reveal evolutionary mechanisms shared by the nervous and immune systems, as well as a molecular basis for the interaction between these systems.
Assuntos
Genes , Sistema Imunitário/fisiologia , Neuropeptídeo Y/genética , Sistemas Neurossecretores/fisiologia , Animais , Humanos , Transcrição GênicaRESUMO
Thymus-leukemia (TL) antigens have been sequentially immunoprecipitated from glycoprotein pools prepared from lysates of biosynthetically labeled ASL-1w leukemia cells with a monoclonal antibody and a standard alloantiserum. Results suggest that on leukemia cells from Tlaa mice, as previously reported for thymocytes from these mice, all the alloantiserum-defined TL specificities (TL.1, 2, 3, 5, 6) as well as the specificity defined by the monoclonal antibody (TL.m3) are carried by a single molecular species. The degree of structural homology between the 45,000-m.w. heavy chains of TL and H-2 was investigated by the technique of comparative tryptic peptide mapping. Results indicate that TL is more distantly related at the primary structural level to H-2 than H-2 antigens are to one another.
Assuntos
Antígenos de Neoplasias , Glicoproteínas , Leucemia Experimental/imunologia , Glicoproteínas de Membrana , Timo/imunologia , Sequência de Aminoácidos , Animais , Precipitação Química , Glicoproteínas/imunologia , Antígenos H-2 , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptídeos/metabolismoRESUMO
Neuropeptide tyrosine (neuropeptide Y, NPY) is a potent vasoconstrictor with a wide distribution in the central and peripheral nervous systems. Here we show that high levels of rat NPY mRNA are also found in peripheral blood cells, bone marrow, lung, and spleen. Furthermore, radioimmunoassay revealed high levels of NPY-like peptide in these tissues. In mice, the levels of splenic NPY mRNA and immunoreactive peptide differed extensively between strains and were greatly elevated in several strains (NZB, NZBxW, and BXSB) that develop a disease resembling human systemic lupus erythematosus. Like the rat, the NZB mouse showed a high content of NPY mRNA in peripheral blood cells and bone marrow. Immunohistochemical staining revealed NPY-like immunoreactivity in large cells morphologically identifiable as megakaryocytes in rat bone marrow and in the spleen of the NZB mouse strain. Expression of NPY mRNA in megakaryocytes in rat bone marrow and NZB mouse spleen was confirmed by in situ hybridization. These results indicate that NPY is synthesized in megakaryocytes, implying that NPY can be released from platelets and function as a vasoconstrictor during blood-vessel damage. In addition, the increase in splenic NPY in certain autoimmune mouse strains adds to the list of abnormalities associated with these strains.
Assuntos
Doenças Autoimunes/genética , Megacariócitos/análise , Neuropeptídeo Y/genética , Animais , Doenças Autoimunes/patologia , Células da Medula Óssea , Histocitoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Neuropeptídeo Y/análise , Ratos , Ratos Endogâmicos , Baço/citologiaRESUMO
Lysates of radioiodinated thymocytes have been sequentially immunoprecipitated with monoclonal antibodies specific for thymus-leukemia (TL) antigens. The TL antigenic specificities defined by the monoclonal antibodies appear not to correspond precisely to specificities previously defined with conventional alloantisera. Nonetheless, each of two monoclonal antibodies specific for a single TL determinant was found to deplete lysates of B6.Tlaa thymocytes of all molecules precipitable by an alloantiserum specific for the conventionally defined determinants TL.1,2,3,5, and 6. These results suggest that on B6.Tlaa thymocytes, all the conventional TL antigenic specificities (TL. 1,2,3,5,6) as well as the specificities defined by the monoclonal anti-TL antibodies (TL.m2,m3) are carried by a single molecular species.
Assuntos
Anticorpos , Isoantígenos , Animais , Especificidade de Anticorpos , Precipitação Química , Células Clonais/imunologia , Leucemia/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Timo/imunologiaRESUMO
A20.2J B lymphoma cells have been co-transfected with the A alpha b, A beta b or with the A alpha b, A beta bm12 and neomycin resistance genes. The transfected cell lines constitutively express the I-Ab or I-Abm12 class II molecules at a level comparable with that of the endogenous I-Ad antigen. The I-Ab antigens expressed on three independently transfected B cell clones (A20.Ab.1, A20.Ab.2, and A20.Ab.3) are serologically and functionally indistinguishable from the I-Ab molecules expressed by control H-2bxd B hybridoma cells (LB cells). These transfected cell lines were potent I region-restricted antigen-presenting cells to a large panel of antigen-specific, autoreactive and alloreactive T cell hybridomas, as well as normal T cell clones. There were not significant differences in the efficiency of antigen presentation by the Ia molecules encoded by the transfected, as compared with the endogenous, I-A genes. The expression of a functional I-Ab antigen on the surface of cells transfected with A beta bm12 and A alpha b genes is consistent with previous work that implicated the A beta-chain alone in the bm 12 mutation. Furthermore, because the transfected A20.Ab and A20.Abm12 cells display the serologic and functional properties of normal spleen cells from the wild-type and mutant mouse strains, respectively, it is clear that class II genes do not undergo unexpected and unpredictable alterations after transfection in this system. This system permits us to investigate the structural requirements for interactions between class II major histocompatibility complex antigens, a foreign antigen, and the T cell receptor by in vitro site-directed mutagenesis coupled with DNA-mediated gene transfer.