Showing papers by "Kettering University published in 1991"

Peptide-binding specificity of the molecular chaperone BiP.

Abstract: Members of the heat-shock protein family (hsp70s) can distinguish folded from unfolded proteins. This property is crucial to the role of hsp70s as molecular chaperones and is attributable to the amino-acid specificity of the peptide-binding site. The specificity for peptide ligands is investigated using a set of peptides of random sequence but defined chain length. The peptide-binding site selects for aliphatic residues and accommodates them in an environment energetically equivalent to the interior of a folded protein.

A Hypothesis: Nonsteroidal Anti-Inflammatory Drugs Reduce the Incidence of Large-Bowel Cancer

Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin synthesis and tumor growth in the rodent colon. We assessed NSAID use in relation to risk of human large-bowel cancer in a hospital-based, case-control study of 1326 patients with colorectal cancer and 4891 control patients. For regular NSAID use that continued into the year before interview, the multivariate relative risk estimate was 0.5 (95% confidence interval, 0.4 to 0.8); the estimate decreased as the duration of use increased, but the trend was not statistically significant. Similar results were obtained whether cancer or non-cancer controls were used, and the inverse association was apparent for both colon cancer and rectal cancer in men and women and in subjects younger and older than 60 years. Regular NSAID use that had been discontinued at least 1 year previously and non-regular use were not associated with risk. Almost all regular NSAID use was of aspirin-containing drugs. The present data suggest that the sustained use of NSAIDs reduces the incidence of human large-bowel cancer.

A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein beta-adaptin.

Abstract: Four high-molecular-weight proteins form the main subunits of the coat of Golgi-derived (non-clathrin) coated vesicles. One of these coat proteins, beta-COP, is identical to a Golgi-associated protein of relative mass 110,000 (110K) that shares homology with the adaptin proteins of clathrin-coated vesicles. This connection, and the comparable molecular weights of the coat proteins of Golgi-derived and clathrin-coated vesicles, indicates that they may be structurally related. The identification of beta-COP as the 110K protein explains the blocking of secretion by the drug brefeldin A.

The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4+ T cells.

Abstract: Activation via the T lymphocyte cell surface molecule CD28 provides a potent amplification signal for interleukin 2 (IL-2) production in several in vitro systems. The B lymphocyte activation antigen, B7/BB1, is a natural ligand for CD28. Here we investigate the role of CD28 and B7/BB1 in primary activation of CD4+ T lymphocytes stimulated with allogeneic B lymphoblastoid cell lines. A subset of peripheral CD4+ T cells that is unresponsive to crosslinking of CD3/T cell receptor (TCR) with CD3 monoclonal antibody (mAb) does proliferate in response to allogeneic B lymphoblasts. TCR binding to allogeneic major histocompatibility complex antigens was an absolute requirement for activation of these cells because mAbs to either CD3 or human histocompatibility leukocyte antigen (HLA) class II completely inhibited activation. CD28 and B7/BB1 antibodies inhibited T cell proliferation 90% and 84%, respectively. Similar results were obtained with the total CD4+ T lymphocyte population. Crosslinking of HLA-DR antigens on small, resting B cells induced rapid expression of B7/BB1, which peaked at 6 h and returned to baseline levels within 18 h. These data demonstrate that CD28-B7/BB1 binding provides an important early second signal for alloactivation of CD4+ T lymphocyte by B lymphoblasts. The results also suggest that T cells interacting with allogeneic resting B cells may induce B7/BB1 expression in the alloantigen-presenting cell as a consequence of interaction between the TCR and class II molecules.

A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1

Abstract: There is evidence to suggest that components of archaebacteria are evolutionarily related to cognates in the eukaryotic cytosol. We postulated that the major heat-shock protein of the thermophilic archaebacterium, Sulfolobus shibatae, is a molecular chaperone and that it is related to an as-yet unidentified chaperone component in the eukaryotic cytosol. Acquired thermotolerance in S. shibatae correlates with the predominant synthesis of this already abundant protein, referred to as thermophilic factor 55 (TF55). TF55 is a homo-oligomeric complex of two stacked 9-membered rings, closely resembling the 7-membered-ring complexes of the chaperonins, groEL, hsp60 and Rubisco-binding protein. The TF55 complex binds unfolded polypeptides in vitro and has ATPase activity-features consistent with its being a molecular chaperone. The primary structure of TF55, however, is not significantly related to the chaperonins. On the other hand, it is highly homologous (36-40% identity) to a ubiquitous eukaryotic protein, t-complex polypeptide-1 (TCP1). In Saccharomyces cerevisiae, TCP1 is an essential protein that may play a part in mitotic spindle formation. We suggest that TF55 in archaebacteria and TCP1 in the eukaryotic cytosol are members of a new class of molecular chaperones.

Cell cycle-specific effects of lovastatin.

Abstract: Lovastatin (LOV), the drug recently introduced to treat hypercholesteremia, inhibits the synthesis of mevalonic acid. The effects of LOV on the cell cycle progression of the human bladder carcinoma T24 cell line expressing activated p21ras were investigated. At a concentration of 2-10 microM, LOV arrested cells in G1 and also prolonged--or arrested a minor fraction of cells in--the G2 phase of the cell cycle; at a concentration of 50 microM, LOV was cytotoxic. The cytostatic effects were reversed by addition of exogenous mevalonate. Cells arrested in the cycle by LOV were viable for up to 72 hr and did not show any changes in RNA or protein content or chromatin condensation, which would be typical of either unbalanced growth or deep quiescence. The expression of the proliferation-associated nuclear proteins Ki-67 and p105 in these cells was reduced by up to 72% and 74%, respectively, compared with exponentially growing control cells. After removal of LOV, the cells resumed progression through the cycle; they entered S phase asynchronously after a lag of approximately 6 hr. Because mevalonate is essential for the posttranslational modification (isoprenylation) of p21ras, which in turn allows this protein to become attached to the cell membrane, the data suggest that the LOV-induced G1 arrest may be a consequence of the loss of the signal transduction capacity of p21ras. Indeed, while exposure of cells to LOV had no effect on the cellular content of p21ras (detected immunocytochemically), it altered the intracellular location of this protein, causing its dissociation from the cell membrane and translocation toward the cytoplasm and nucleus. However, it is also possible that inhibition of isoprenylation of proteins other than p21ras (e.g., nuclear lamins) by LOV may be responsible for the observed suppression of growth of T24 cells.

Cytogenetic analysis of 434 consecutively ascertained specimens of non-Hodgkin's lymphoma: correlations between recurrent aberrations, histology, and exposure to cytotoxic treatment.

Abstract: Cytogenetic abnormalities in non-Hodgkin's Lymphoma (NHL) provide a model system for the analysis of the role of multiple genomic aberrations in human malignancy. In order to define correlations with histology, tumor evolution, and the effects of genotoxic exposure, cytogenetic analysis was performed on 434 specimens of NHL derived from 423 patients consecutively ascertained over a 5-year period (1984-1989). Six recurring translocations (RT) were observed: t(14;18)(q32;q21), t(8;14)(q24;q32), t(11;14)(q13;q32), t(3;22)(q27;11), t(2;5)(p23;q35), and t(1;6)(q21;q25). No translocation was specific to a single histologic subtype. Other structural chromosome abnormalities were analyzed according to break site; groups of related breaks were considered together for statistical analysis. Recurring other structural and numerical aberrations (ROA) encountered in greater than 10% of specimens included rearrangements with breaks at bands 1p32-36, 1q21-23, 6q21-25, and trisomies of chromosomes 7 and 12. ROA with one of these breaks or numerical abnormalities were the sole abnormalities in at least two cases. Correlations were observed among ROA and between ROA and histologic subtypes. Trisomy 7, breaks at 1q21-23, 1p32-36, 6q21-25, and 7q32 were associated with t(14;18); trisomy 18 was associated with trisomy 3; and structural abnormalities of chromosome 17 were associated with breaks at 1p32-36 and 6q21-25. Trisomy 7 and trisomy 12 were more frequent in t(14;18)-bearing intermediate to high grade tumors compared to low grade tumors. Trisomy 12 and breaks at band 1p22 were associated with large cell diffuse lymphomas. Incidence rates of reciprocal translocations, ROA, and measures of karyotypic complexity, including number of breakpoints and marker chromosomes were compared in pretreatment and posttreatment samples. Karyotypic complexity was greater in the posttreatment samples, reflecting an increased frequency of nonrecurring and low incidence aberrations. These results better define the association of genomic aberrations and tumorigenesis, histologic transformation, and tumor progression.

Characterization of the promoter of the human gene encoding the high-affinity IgG receptor: transcriptional induction by gamma-interferon is mediated through common DNA response elements

Abstract: Expression of the high-affinity receptor for IgG (Fc gamma RI) is restricted to cells of myeloid lineage and is induced by gamma-interferon (IFN-gamma) but not by IFN-alpha/beta. The organization of the human Fc gamma RI gene has been determined and the DNA elements governing its cell type-restricted transcription and IFN-gamma induction are reported here. A 39-nucleotide sequence (IFN-gamma response region, or GRR) is defined that is both necessary and sufficient for IFN-gamma inducibility. Sequence analysis of the GRR reveals the presence of promoter elements initially defined for the major histocompatibility complex class II genes: i.e., X, H, and gamma-IRE sequences. Comparison of a number of genes whose expression is induced selectively by IFN-gamma indicates that the presence of these elements is a general feature of IFN-gamma-responsive genes. Our studies suggest that the combination of X, H, and gamma-IRE elements is a common motif in the pathway of transcriptional induction by this lymphokine.

A subunit common to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions.

Abstract: The zeta subunit is a component of the Fc gamma receptor of natural killer cells (Fc gamma RIII or CD16), as well as the multimeric T-cell receptor/CD3 complex, and is required for assembly of both native receptors. The role of the zeta subunit in human Fc gamma RIIIA assembly differs from its role in T-cell receptor/CD3 complex assembly. The transmembrane domain of the Fc gamma RIIIA alpha subunit forms noncovalent interactions with the comparable domain of the zeta subunit and is sufficient for surface expression of the Fc gamma RIIIA complex. In the absence of these interactions, sequences in the transmembrane domain of the Fc gamma RIIIA alpha subunit signal its degradation. Leu-46, present in the transmembrane domain of the human zeta subunit, is important for assembly with the Fc gamma RIIIA alpha subunit. Substitution of this leucine with an isoleucine, as found in the mouse zeta subunit, significantly reduces this interaction. In contrast, the mouse and human zeta subunits interact with the pentameric T-cell receptor/CD3 complex, resulting in surface expression of this receptor.

Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific

Abstract: Specialized type I topoisomerases catalyze DNA strand transfer during site-specific recombination in prokaryotes and fungi. As a rule, the site specificity of these systems is determined by the DNA binding and cleavage preference of the topoisomerase per se. The Mr 32,000 topoisomerase I encoded by vaccinia virus (a member of the eukaryotic family of "general" type I enzymes) is also selective in its interaction with DNA; binding and cleavage occur in vitro at a pentameric motif 5'-(C or T)CCTT in duplex DNA. Expression of vaccinia virus DNA topoisomerase I in a lambda lysogen of Escherichia coli promotes int-independent excisive recombination of the prophage. To address whether the topoisomerase directly catalyzes DNA strand transfer in vivo, the recombination junctions of plaque-purified progeny phage were cloned and sequenced. In five of six distinct excision events examined, a topoisomerase cleavage sequence is present in one strand of the DNA duplex of both recombining partners. Recombination entails no duplication, insertion, or deletion of nucleotides at the crossover points, consistent with excision via conservative strand exchange at sites of topoisomerase cleavage. Three of these five recombination events are distinguished by the presence of direct repeats at the parental half-sites that extend beyond the pentameric cleavage motif, suggesting that sequence homology may facilitate excision. The data are consistent with a model in which vaccinia topoisomerase catalyzes reciprocal strand transfer, leading to the formation of a nonmigrating Holliday junction, the resolution of which can lead to excisive recombination.

Emmetropisation, squint, and reduced visual acuity after treatment.

Abstract: In a sample of children used to assess the value of optical correction of hypermetropia from the age of 6 months the refraction of the most hypermetropic meridian frequently became less than 3.5 D as the children grew. When this occurred, the incidence of squint was significantly less (p less than 0.001) and the last known acuity after treatment was significantly better (p less than 0.001) than when it did not. This process of emmetropisation appears to have been impeded by the consistent wearing of hypermetropic spectacle correction from the age of 6 months.

Metatarsus primus varus. A statistical study

Abstract: A survey of 6000 schoolchildren discovered 36 cases of unilateral and 60 cases of bilateral hallux valgus, defined as a metatarsophalangeal angle of more than 14.5 degrees, measured on standing radiographs. Metatarsus primus varus was found not only in the early stages of hallux valgus but in the unaffected feet of children with unilateral hallux valgus. Adduction of the first metatarsal is not due to differential growth of the cortices of the first metatarsal nor is it a consequence of malalignment of the metatarsocuneiform joint. The intermetatarsal angle did not correlate with the angle of metatarsus adductus nor with the intercuneiform angle.

Four distinct regulatory regions of the cut locus and their effect on cell type specification in Drosophila.

Abstract: The cut gene in Drosophila is necessary in at least one cell type, the external sensory organs, for proper cell type specification and morphogenesis. It is also expressed in a variety of other tissues, where its function is less well characterized. Previous work has demonstrated that mutations affecting all the tissues map in the transcribed and translated portion of the gene, while mutations that are tissue specific in their effects map in the 140 kb upstream of the most 5' exon known. Within that 140 kb, the mutations fall into four subregions, two of which contain mutations affecting unique sets of tissues and the other two of which contain mutations that affect a third set. Our examination of the defects of mutants, their complementation behavior, and their effect on the distribution of the cut protein in embryos, alters the picture in three important ways. First, some mutations convert the cells of the Malpighian tubules into what appear to be gut cells, suggesting that cut is necessary for cell type specification and morphogenesis in a variety of tissues. Second, mutations in each of the four subregions in the 140 kb of upstream DNA cause a different set of phenotypes, suggesting that the regulatory region contains at least four separate units with different tissue specific functions. And third, mutations have now been identified that map in the transcribed and translated portion of the gene but that have tissue specific effects.

Ectopic expression of a c-kitW42 minigene in transgenic mice: recapitulation of W phenotypes and evidence for c-kit function in melanoblast progenitors.

Abstract: The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor that is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and adult life, and they result from the partial or complete loss of c-kit function. The W42 allele is a W mutation with severe effects in both the homozygous and the heterozygous states. Previous analysis of the W42 allele identified a missense mutation in an essential amino acid of the c-kitW42 kinase domain that abolishes the in vitro kinase activity of the c-kitW42 protein but does not affect its normal expression. These results suggested that the c-kitW42 allele was a dominant negative mutation within the context of c-kit-mediated signal transduction. To further explore the dominant negative characteristics of the W42 mutation, we have generated transgenic mice in which ectopic expression is driven by the human beta-actin promoter (hAP). Two mouse lines carrying the hAP-c-kitW42 transgene show an effect on pigmentation and the number of tissue mast cells. The patchy coat color pattern of the line 695 mice may reflect variable expression of the transgene in melanoblast progenitors and their descendants and, consequently, is indicative of a function for c-kit in early melanoblasts. Germ cell development and erythropoiesis, however, do not appear to be affected by the transgene. Mice expressing the c-kitW42 transgene therefore recapitulate some of the phenotypes of mice with W mutations. These results are therefore in agreement with the molecular basis of the W42 mutation and the dominant-negative characteristics of the c-kitW42 protein product.

Abnormalities of 2q: A common genetic link between rhabdomyosarcoma and hepatoblastoma?

Abstract: Cytogenetic and restriction fragment length polymorphism (RFLP) analyses were performed on a mediastinal germ cell tumor comprising distinct teratoma and embryonal rhabdomyosarcoma components in a 31-year-old male and a hepatoblastoma in a 2 month-old male child. Clonal relationship between the teratoma and rhabdomyosarcoma of the germ cell tumor was established by the presence in both of i(12p), the characteristic marker of germ cell tumors. Both the rhabdomyosarcoma component of the mediastinal germ cell tumor and the hepatoblastoma exhibited rearrangements of 2q. These data suggest that malignant differentiation of a teratoma is accompanied by the development of chromosome abnormalities specific for the transformed histology and further suggest that 2q abnormalities may be the common genetic link in the development of the two histologically unrelated tumor types, embryonal rhabdomyosarcoma and hepatoblastoma.

DNA binding properties of the vitamin D3 receptor zinc finger region.

Abstract: The DNA binding domains of the nuclear receptor superfamily are highly conserved and consist of residues that fold into two zinc finger-like motifs, suggesting that the structures of this region among the members of the superfamily are likely to be very similar. Furthermore, the response elements that these receptors bind to are similar in sequence and organization. Nevertheless, these receptors selectively recognize target response elements and differentially regulate linked genes. In order to study the details of receptor:DNA binding, we have over-expressed and purified the vitamin D3 receptor DNA binding domain (VDRF) and have begun characterizing its DNA binding properties. We find that the VDRF protein binds strongly and specifically to direct repeats constituting a vitamin D response element from the mouse osteopontin (Spp-1) promoter region but weakly to the human osteocalcin vitamin D response element. Unlike receptors that recognize hormone response elements oriented as inverted repeats, such as ...

Detection and analysis of origin of i(12p), a diagnostic marker of human male germ cell tumors, by fluorescence in situ hybridization.

Abstract: The i(12p) chromosome marker has been shown to be a diagnostic and prognostic marker of human male germ cell tumors (GCTs). An analysis of the i(12p) and chromosome 12 aneuploidy was performed in five primary cell cultures and three established cell lines derived from human male GCTs by fluorescence in situ hybridization (FISH) with a chromosome 12 centromere-specific alpha-satellite DNA probe. Distinct differences in the centromeric signals originating from the i(12p) and normal chromosome 12 were detected, which were found to be useful for unambiguous distinction between the i(12p) and normal chromosomes 12 at interphase as well as at metaphase in these cultures. This method can be used for rapid screening of large numbers of interphase cells, eliminating the main limitation of conventional karyotypic analysis, namely, frequent inability to obtain sufficient numbers of dividing cells in direct preparations or in short-term culture of fresh biopsies. Our analysis of chromosome 12 centromeric signal size along with karyotypic data and results of analysis of restriction fragment length polymorphisms (RFLPs) on 12q in four GCTs suggested that the i(12p)s are formed by nonreciprocal centromeric interchanges between nonsister chromatids of homologous chromosomes.

Alternatives in the Management of Gastric Lymphoma.

Abstract: Advances in endoscopic technology now enable non-surgical diagnosis of gastric lymphoma, while combination chemotherapy and modern radiotherapy techniques offer an alternative to gastrectomy as the primary treatment of gastric lymphoma. Cumulative data challenge the traditional concept that non-operative treatment of gastric lymphoma often leads to fatal complications, such as perforation and hemorrhage. The reported risk of major side effects is reviewed and the alternative approaches to the management of gastric lymphoma are analyzed. In addition, pathologic considerations unique to lymphomatous involvement of the stomach are discussed.

FcγRIII (CD16) on human macrophages is a functional product of the FcγRIII-2 gene

Abstract: The low-affinity Fc receptor for immune-complexed IgG (FcγRIII; CD16) present on in vitro cultured human monocytes are encoded by an FcγRIII-2 gene that, by cDNA sequence analysis, is identical to that expressed on tissue macrophages and on natural killer cells. In macrophages, FcγRIII-2 encodes a glycoprotein of 52-62 kDa, with a peptide backbone of 33 kDa identical to that of the homologous receptor on natural killer cells. Like this and unlike in polymorphonuclear neutrophils, FcγRIII (CD16) on cultured monocytes is insensitive to phosphatidylinositol-specific phospholipase C, is not allelic for the neutrophil NA alloantigens NA-1/NA-2, is not recognized by a monoclonal antibody (1D3) detecting an epitope present only on neutrophil FcγRIII (CD16) and functions to trigger cytotoxicity upon ligand binding.