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L Graf

Publications and source records attributed to L Graf.

At least 37 records · Page 2Linked to original sources
PubMed1988

Mitochondrial import of rat pre-ornithine transcarbamylase: accurate processing of the precursor form is not required for uptake into mitochondria, nor assembly into catalytically active enzyme.

Mitochondrial uptake of the cytoplasmically synthesized precursor of the mammalian enzyme ornithine transcarbamylase is mediated by an N-terminal leader sequence of 32 amino acids. In the mitochondrial matrix, the precursor form is processed to the mature subunit by proteolytic removal of this pre-sequence and in the enzyme from rat liver it has been suggested that this occurs in a two-step process which involves an intermediate cleavage at residue 24. We show that deletion of residues 20-26 spanning this intermediate cleavage site prevents correct processing to the mature subunit but it does not prevent mitochondrial targeting and internalization or assembly of the incorrectly processed product into a catalytically active enzyme. The incorrectly processed enzyme, which is larger than the normal mature enzyme, is nevertheless more susceptible to proteolytic degradation in permanently transfected human cells than the correctly processed enzyme.

Absorption↗
PubMed1987

Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin.

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.

Amino Acid Sequence↗
PubMed1987

Facilitation of engraftment of DLA-nonidentical marrow by treatment of recipients with monoclonal antibody directed against marrow cells surviving radiation.

Past studies in dogs have suggested that marrow graft rejection was mediated by major histocompatibility complex (MHC) class II antigen-positive non-T cells that survived standard doses of total-body irradiation (TBI). We have now raised 4 monoclonal antibodies (mAbs) against marrow cells harvested 6 days after TBI. The mAbs are highly reactive (greater than 70%) with marrow cells surviving radiation and also bind strongly (greater than 50%) to normal marrow cells, lymphocytes, monocytes, and granulocytes. One of the mAbs (34-S3) reacted strongly with NK-like cells. In vitro treatment of marrow with mAb and rabbit complement (C') did not affect erythroid colony-forming unit (CFU-E) growth, whereas 2 of the 4 mAbs inhibited granulocyte-macrophage colony-forming unit (CFU-GM) growth, and all 3 mAbs tested suppressed autologous marrow engraftment. One of the mAbs, 69-S5 (IgG1), bound to a 95,000 dalton antigen. It crossreacted with human cells, but not with cells from Rhesus monkeys, baboons, and cats. We administered this mAb intravenously at 0.2 mg/kg/day on days -5 to 0 to dogs given 9.2 Gy TBI on day 0 followed by marrow grafts (less than or equal to 4 x 10(8) cells/kg) from DLA-nonidentical unrelated donors. Three of five dogs had sustained grafts. Increasing the dose of mAb ten-fold (2 mg/kg/day) resulted in graft failure (2 of 2 dogs). Treatment with a dose of 0.2 mg/kg/day from day -7 to -2 showed sustained engraftment in 7 of 10 dogs. This result is in contrast to sustained grafts in 3 of 36 dogs not given mAb, and in 1 of 7 dogs treated with an irrelevant mAb (P = 0.0002 and 0.04, respectively). We conclude that treatment of recipients with a mAb raised against marrow cells surviving radiation and not directed at major histocompatibility complex (MHC) class II antigens and NK-like cells can also facilitate engraftment of DLA-nonidentical canine marrow. These results may be relevant for the transplantation of HLA-incompatible marrow in man, particularly after in vivo T cell depletion, where graft failure is frequent.

Animals↗
PubMed1987

Cellular interactions and genetic restriction.

These studies emphasize the limitations of in vitro colony assays utilizing heterogeneous cell populations for the identification of cell-cell interactions that may involve genetic restriction. Obviously, different strategies are required to determine if Class II molecules have any role, alone or in combination with other determinants, in mediating hematopoietic cell-cell interactions. In this report, we present preliminary data derived from two approaches designed to address this issue. First, clonal cell lines developed to mimic Class II expression of normal precursors have been used to study structure-function relationships of HLA-D region gene products. Second, the lymphocyte adhesion assay has been adopted to study binding of precursors to marrow stroma. We hypothesize that the strategies should make it possible to identify hematopoietic cell-cell interactions and determine to what extent Class II molecules participate in these interactions.

Animals↗
PubMed1987

Evidence for chemical differences in HLA-DR molecules on autologous acute lymphoblastic leukemia and B-lymphoblastoid cell lines.

HLA-DR molecules on autologous acute lymphoblastic leukemia (ALL) and B-lymphoblastoid cell lines from two individuals were compared by immune precipitation and gel electrophoresis. Cells were surface labeled with 125I and proteins immunoprecipitated with specific monoclonal antibodies (MoAb). Two electrophoretically distinct bands were found in the HLA-DR beta chain region on both ALL cell lines in contrast to only one on each of the autologous B-lymphoblastoid cell lines. Differences in the electrophoretic mobility of the alpha chains were also observed with ALL and B-lymphoblastoid cell lines from one individual. Preclearing of radiolabeled cell lysates with MoAb specific for HLA-DQ and -DP molecules demonstrated that the complexity of the HLA-DR pattern is not the result of antibody cross-reactivity with alpha and beta chains from other class II products. Immunoprecipitation experiments indicated that two beta chain bands are observed with each of the parental HLA-DR molecules on the ALL but not the B-lymphoblastoid cell line from an HLA-DR3,7-positive individual. We conclude that the HLA-DR molecules expressed on ALL and B-lymphoblastoid cell lines from the same individual can differ chemically. Neuraminidase treatment reduced these electrophoretic differences, indicating that these molecules differ in their sialic acid content. Since small changes in class II molecules can profoundly alter cellular interactions, the functional significance of these differences requires further investigation.

B-Lymphocytes↗
PubMed1986

HLA-DP can be expressed with or without -DR molecules on a malignant B cell line.

HLA class II molecules (HLA-DR, -DQ, -DP) appear to differ in their ability to serve as corecognition elements in antigen presentation to T lymphocytes. Expression of these molecules on the autologous malignant B cell lines BALM-3, -4, and -5 was studied by binding of alloantisera and by indirect immunofluorescence and immune precipitation performed with well-characterized monoclonal antibodies. The following phenotypes were identified: BALM-5, HLA-DR+, -DP+; BALM-4, HLA-DR-, -DP+; BALM-3, HLA-DR-, -DP-. When treated with phorbol acetate (TPA), all three cell lines synthesize and express both HLA-DR and -DP molecules, indicating that the structural genes for these molecules remain intact. Thus HLA-DP can be expressed with or without HLA-DR molecules. Immune precipitation studies of metabolically labeled BALM-4 cells detect HLA-DR molecules in cells from TPA-treated but not control cultures, suggesting that TPA acts by inducing the transcription and/or translation of the genes for these class II molecules. HLA-DP molecules are detected in cells from both BALM-4 cultures. Recent studies suggest that BALM-5 but not BALM-3 or BALM-4 cells are HLA-DQ positive. TPA also appears to induce expression of HLA-DQ molecules on the latter two cell lines. The unique class II phenotypes of these autologous B cell lines in the resting state therefore appear to reflect differences in their ability to execute discrete steps leading to the surface expression of individual class II molecules. Variable expression of individual class II molecules by different cell populations may affect their ability to present cell-associated antigens to T lymphocytes.

B-Lymphocytes↗
PubMed1986

Differential expression of HLA-DR, -DQ, and -DP antigens on malignant B cells.

HLA class II antigens mediate interactions among cells involved in the immune response and play an important role in the process of self recognition. We made use of conventional alloantisera and six well-characterized monoclonal antibodies (MoAb) to study the HLA class II antigens on CALLA-positive malignant B cell populations and autologous normal B cell lines. Forty additional HLA class II-specific MoAb were also tested for their ability to bind to these cells. By using indirect immunofluorescence and immune precipitation assays, we find that malignant B cells often fail to express one or more of the three known types of HLA class II antigens. Cell lines with the following five phenotypes have been identified: HLA-DR+, -DQ+, -DP+; HLA-DR+, -DQ-, -DP+; HLA-DR-, -DQ+, -DP+; HLA-DR-, -DQ-, -DP+; and HLA-DR-, -DQ-, -DP-. These cell lines have been used to characterize the subregion specificity of MoAb that react with HLA class II antigens. This work confirms the existence of complicated patterns of serologic cross-reactivity between the three different types of HLA class II molecules. It also increases our understanding of the specificity of individual MoAb, thereby facilitating future investigation of the distribution and function of individual antigens. Our studies are consistent with the proposal that altered expression of HLA antigens on tumors might impair recognition of these cells by the immune system of the host, thereby contributing to the proliferation of a malignant clone.

Antibodies, Monoclonal↗
PubMed1985

The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling.

A cloned approximately 5 kb cDNA (human placenta) contains the coding sequences for the insulin receptor. The nucleotide sequence predicts a 1382 amino acid precursor. The alpha subunit comprises the N-terminal portion of the precursor and contains a striking cysteine-rich "cross-linking" domain. The beta-subunit (the C-terminal portion of the precursor) contains a transmembrane domain and, in the intracellular region, the elements of a tyrosine phosphokinase: an ATP-binding site and a possible tyrosine autophosphorylation site or sites. The overall structure is reminiscent of the EGF receptor; the cross-linking domain of the alpha subunit and several regions of the beta subunit exhibit sequence homology with the EGF receptor. The phosphokinase domain also exhibits homology with some oncogenic proteins that have tyrosine phosphokinase activity, in particular, a striking homology with v-ros. Southern blotting experiments suggest that the coding region spans more than 45 kb. The insulin receptor gene is located on chromosome 19.

Amino Acid Sequence↗
PubMed1985

Expression of Ia antigens in a murine T-lymphoma variant.

ESb, a cellular high metastatic variant derived from the murine T-cell lymphoma L5178Y (Eb), was found to synthesize Ia antigens. Ia-specific antibodies reacted with the ESb cells and precipitated Ia-like molecules from them. Two-dimensional gel electrophoretic analysis of immunoprecipitates of metabolically labeled ESb cells indicated that the Ia molecules on ESb were indistinguishable from those on murine B-cells. No Ia antigens were detectable on the parental tumor line Eb. Treatment with recombinant interferon-gamma (IFN-gamma) caused enhancement of class I histocompatibility antigen expression on Eb and ESb tumor lines. In ESb cells the expression of Ia and of Ia-associated invariant chain (Ii) was also increased upon IFN-gamma treatment. No induction of either Ia and Ii antigens was observed upon IFN-gamma treatment of the Eb line. These studies demonstrate a substantial difference between the Eb and ESb tumor lines with respect to: (i) constitutive expression of class II major histocompatibility antigens, and (ii) response to IFN-gamma treatment.

Animals↗
PubMed1985

Studies on interleukin 2 receptor expression and IL-2 production by murine T cell lymphomas.

In order to study the possible role of the T-lymphocyte growth factor, Interleukin 2 (IL-2), and/or of the IL-2 receptor in the autonomous growth of leukaemic cells, 15 mouse leukaemic cell lines of various aetiology were analyzed for (i) IL-2 receptor expression and (ii) for the capacity to secrete IL-2. Several but not all of the cell lines tested were IL-2 receptor positive. The cells constitutively expressing IL-2 receptors at their surface could not be stimulated to secrete IL-2. Cell producing and secreting IL-2 did not express detectable amounts of IL-2 receptors at their surface. It has been demonstrated that proliferation of the leukaemic cells was independent of exogenous IL-2. The monoclonal anti-IL-2 receptor antibody AMT-13 inhibited IL-2 dependent proliferation of activated normal T-lymphocytes but failed to inhibit the growth of IL-2 receptor expressing leukaemic cells. The results argue against the autocrine stimulation hypothesis but do not exclude the possibility of involvement of functionally altered IL-2 receptors on autonomous cell growth.

Animals↗
PubMed1985

The primary structure of the imported mitochondrial protein, ornithine transcarbamylase from rat liver: mRNA levels during ontogeny.

Ornithine transcarbamylase, one of the enzymes of the urea cycle in ureotelic organisms, is synthesized in the cytoplasm of hepatocytes as a precursor larger than the mature form found in the mitochondrial matrix. We deduced the amino acid sequence of the precursor of ornithine transcarbamylase from rat liver from the nucleotide sequence of overlapping cDNA clones spanning the complete coding region, 3' untranslated region, and most of the 5' untranslated region of the mRNA. The mature enzyme consists of 322 amino acids and is derived from the larger precursor by proteolytic removal of 32 amino acids from the amino-terminus. The amino-terminal extension contains eight basic and no acidic residues. This highly basic character appears to be a feature of presequences on cytoplasmically synthesized mitochondrial proteins. Comparison of the amino acid sequence determined for the enzyme from rat with that from human liver (Horwich et al., 1984) shows that there is a high degree of homology between the sequences of the mature protein (93%) and relatively less homology between the sequences of the amino-terminal extension (72%). The ornithine transcarbamylase from rat liver also shows a considerable degree of amino acid homology (44%) with the enzyme from Escherichia coli (Van Vliet et al., 1984) and leads to suggestions about residues involved in substrate binding and catalysis. An analysis of levels of RNA in fetal and neonatal liver shows that ornithine transcarbamylase mRNA levels increase from about 40% of adult levels at day 14 of gestation to a peak at day 20 of gestation, and, after a drop around the time of birth, rises to adult levels during the second week after birth.

Age Factors↗
PubMed1984

Suggestive evidence that the highly metastatic variant ESb of the T-cell lymphoma Eb is derived from spontaneous fusion with a host macrophage.

Two lines of evidence are reported which suggest that the highly metastatic variant ESb of the T-cell lymphoma Eb is derived from spontaneous fusion with a host macrophage. Firstly, ESb cells are shown to express the macrophage differentiation antigen Mac-1 which was not found on Eb cells or on any other tumor cells tested except the macrophage tumor line Pu5. Secondly, the progression from low to high metastatic capacity could be reproduced in vitro following hybridization of thioguanine-resistant Eb cells (EbTGR) with syngeneic bone-marrow-derived macrophages. Two HAT medium-selected hybrid tumor lines (Eb-F1 and Eb-F2) could be established. They were found to express cell surface markers of both parental lines: T lymphoid differentiation antigens from T-lymphoma and macrophage antigens (Mac-1, class II MHC antigens) from the normal cell fusion partner. The antigens were identified on the hybrids and subclones thereof by means of monoclonal antibodies and 3 different detection assays: cytofluorography, complement-dependent cytotoxicity and immunoprecipitation followed by gel electrophoresis. Animals inoculated s.c. with the parental line EbTGR developed local tumors but not metastases and survived for more than 40 days. In contrast, animals inoculated similarly with Eb-F1 or Eb-F2 cells quickly developed metastases in visceral organs and died as early as 10-14 days following inoculation. In many but not all respects, the in vitro-derived T-lymphoma-macrophage hybrids resembled the spontaneous in vivo-derived variant ESb. These findings, together with the presence of Mac-1 antigen on ESb cells, suggest (1) that ESb variant cells may be derived from spontaneous fusion with a host cell, most likely a macrophage and (2) that somatic cell fusion may be an important mechanism of genetic rearrangements leading to metastatic variants. The new highly metastatic tumor lines which were developed under well-defined in vitro conditions, and their subclones, may become very useful tools for studying the contribution of specific genetic traits and of membrane-related structures to various steps of the metastatic process.

Animals↗
PubMed1984

A highly basic N-terminal extension of the mitochondrial matrix enzyme ornithine transcarbamylase from rat liver.

We have deduced the amino acid sequence of the N-terminal leader peptide of the mitochondrial enzyme ornithine transcarbamylase from a cDNA clone obtained from a rat liver cDNA library. The sequence is remarkable in being highly basic, having 4 arginine, 3 lysine and 1 histidine with no acidic residues in a total of 32 residues. The leader sequence has no extensive hydrophobic stretches, has 72% homology with the leader peptide of human ornithine transcarbamylase [1], and in terms of its basic character resembles the N-terminal extensions on a number of fungal mitochondrial [2-5] and pea chloroplast [6] proteins. Thus the basic nature of these leader peptides may constitute the signal for mitochondrial import.

Amino Acid Sequence↗
PubMed1984

A carbamylphosphate synthetase deficiency with no detectable immunoreactive enzyme and no translatable mRNA.

A lethal carbamylphosphate synthetase (CPS: EC 6.3.4.16) deficiency (McKusick 23730) was found in a newborn girl; who presented on the second day of life with acute hyperammonaemia, hypotonia, seizures and who died in a coma 6 days after birth. The activity of the mitochondrial urea cycle enzymes, CPS and ornithine transcarbamylase (OTC: EC 2.1.3.3) were measured on a needle biopsy sample taken from liver and showed that CPS was 1.4% of the normal mean (0.09 nmol/min/mg protein) whereas OTC activity was normal (110 nmol/min/mg protein). Immunological analysis of the liver sample showed no detectable immunoreactive CPS and confirmed the presence of normal levels of OTC. RNA was extracted from postmortem liver and in vitro translation experiments showed that there was no translatable CPS mRNA and confirmed that no CPS protein was synthesized in this child. The absence of translatable mRNA is explicable in terms of a genetic defect which results in a failure to synthesize mRNA for CPS, or synthesis of a defective form of mRNA which is not translated.

Amino Acid Metabolism, Inborn Errors↗
PubMed1983

Nucleotide sequence of a 'truncated rRNA operon' of the Euglena gracilis chloroplast genome.

An extra 16S rRNA gene (s-16S rDNA) from the Euglena gracilis chloroplast genome and several hundred positions of its flanking regions have been sequenced. The structural part has 1486 positions and is to 98% homologous in its sequence with the 16S rRNA gene in functional chloroplast rRNA operons. Sequences of about 200 positions upstream and 15 positions downstream of the structural part of the s-16S rRNA gene region are highly homologous with corresponding parts in the functional operon. Neither tRNA genes (A1a, I1e) nor parts of the 23S and 5S rRNA genes are found within 557 positions after the 3' end of the s-16S rRNA gene, i.e., the 330 bp homology, observed in electron microscopic studies of heteroduplexes (4), between the s-16S rDNA downstream region and the 6.2 kb repeated segment containing the functional rRNA operon, must be due to a DNA stretch in the interoperon spacer. A structural model of the "truncated rRNA operon" is presented. Results from S-1 endonuclease analysis suggest that the s-16S rDNA region is probably not transcribed into stable s-16S rRNA.

Base Sequence↗
PubMed1983

Regulation of UDPG pyrophosphorylase in Acetabularia mediterranea.

The kinetic properties of UDPG pyrophosphorylase (glucosyl-1-phosphate uridyl transferase, EC 2.7.7.9) suggest that it may play a key role in the regulation of metabolism in Acetabularia mediterranea. The enzyme-catalyzed reaction is readily reversible in vitro, and has been assayed in both directions. The enzyme shows substrate inhibition by UDPG and UTP at substrate concentrations in excess of 2 mM. The kinetic behavior of the enzyme is consistent with the hypothesis that it catalyzes an ordered bisubstrate biproduct reaction in which G-1-P is the leading substrate, and UTP is the leading product. A plot of initial velocity vs. PPi concentration is sigmoid, indicating a cooperative homotropic effect. PGAL inhibits the reaction in the direction: UTP + G-1-P leads to UDPG + PPi It has no effect on the reverse reaction. The responses of the enzyme may serve to regulate the allocation of G-1-P between anabolic and catabolic pathways.

Acetabularia↗