Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein- serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.      

Bone marrow transplantation for the treatment of leukaemia-results of the Munich Cooperative Group

This report summarizes the results of marrow transplantation from HLA-identical siblings and syngeneic twins for treatment of acute myelogenous leukaemia, chronic myelogenous leukaemia, acute lymphoblastic and undifferentiated leukaemia from 1975 until December 1986. Three conditioning regimens and treatment of the marrow graft in vitro with absorbed antithymocyte globulin or the monoclonal antibody "Campath 1" for prophylaxis of graft-versus-host disease (GVHD) have been studied and analyzed retrospectively. The regimen of total body irradiation in large fractions of 4 Gy and of cyclosphosphamide (200 mg/kg) has achieved the most favorable results. Inactivation of T-cells by treatment of the marrow "in vitro" has decreased the severity of GVHD without improving survival. The antileukaemic effect of the graft may be important for control of the disease and may be improved by better immunosuppression of the recipient.     

Combined prophylactic suppression of graft-versus-host and host-versus-graft reactions following treatment of prospective bone marrow recipients with rat IgG 2b anti-mouse T cell antibodies

A new approach to avoid typical complications from bone marrow transplantation into MHC different mice was studied. Rat monoclonal anti-Thy-1 antibodies of the IgG 2b isotype were identified, which inhibit T lymphocytes in vivo so that transplanted donor T cells as well as residual T cells of the conditioned marrow recipient were suppressed. A single injection of these antibodies after irradiation and before marrow transplantation did not only prevent graft-versus-host mortality but suppressed also host-versus-graft reactivity so that the radiation dose necessary for engraftment of donor cells differing in H-2, IA (both haplotypes) major histocompatibility antigens could be reduced to 6.0 Gy. In addition an anti-T leukemic cell effect from the injected monoclonal T cell antibodies was observed.     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Independent origin of identical beta cardiac myosin heavy-chain mutations in hypertrophic cardiomyopathy.

The origins of the beta cardiac myosin heavy-chain (MHC) gene missense mutations that cause familial hypertrophic cardiomyopathy (FHC) in 14 families have been evaluated. Of eight different mutations, four were present in single families, while four occurred in two or more families. To investigate the origins of the four shared mutations, we defined the beta cardiac MHC haplotypes of each of the mutation-bearing chromosomes by determining the alleles present at three intragenic polymorphic loci. Two of the mutations (Arg453Cys and Val606Met) have arisen independently in each of three families, being found on different chromosomal backgrounds. A third mutation (Gly584Arg) is associated with identical haplotypes in two families with Portuguese ancestors, suggesting a founder effect. Haplotype analysis was uninformative for the fourth mutation (Arg403Gln). Thus, FHC-causing mutations have arisen independently in at least 12 of the 14 families studied, suggesting that the majority have arisen relatively recently as new mutations. This finding predicts the prevalence of disease-causing beta cardiac MHC mutations to be comparable in all population groups.     

Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.     

Surface properties and in vitro functional capacities of human T lymphocyte subsets separated by linear density gradients.

Human lymphocytes derived from various central and peripheral sources were separated on linear density gradients (LDG). Cells from individual density fractions were tested in parallel for: the capacity to form nonimmune rosettes with neuraminidase-treated SRBC, the number of surface-associated HTLA, and in vitro proliferative responses to mitogenic lectins and alloantigens. Heterogeneous density distribution profiles were obtained for all sources of human T cells and revealed an organ specificity. The various T cell density classes obtained from identical organs as well as the identical density classes of different sources revealed to some extent differences in their surface marker patterns and/or their in vitro reactivities. On the basis of the combined techniques at least two major subsets among thymocytes were identified that differed in both surface properties and functional capacities. Density classes of T cells from all peripheral sources were distinguished from thymocytes by a homogeneous lowered HTLA expression. Whereas clear-cut differences in the in vitro functional capacity were observed between the two thymocyte subsets, less striking but still significant differences were found to exist among the various density classes of peripheral T cells.