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At least 325 records · Page 18Linked to original sources
PubMed1997

S100A1 and S100B expression and target proteins in type I diabetes.

Calcium receptor proteins are an essential link between hormones that alter intracellular calcium levels and the generation of cellular responses. However, there is no information available regarding the role of calcium receptor proteins, in particular the S100 family, in insulin action and/or diabetes. This study examines the effects of streptozotocin-induced type I diabetes on the expression of the individual S100A1 and S100B isoforms as well as their binding proteins. Diabetes did not increase (or initiate) S100B expression in any non-S100B-expressing tissue (skeletal muscle, heart, kidney, liver, spleen, and pancreas). In all S100B-expressing tissues examined (brain, white fat, and testes), S100B protein levels increased approximately 2-fold while steady state S100B messenger RNA (mRNA) levels decreased. S100A1-expressing tissues exhibited increased (kidney and lung), decreased (skeletal muscle), and unchanged (brain and heart) S100A1 protein levels. While noncoordinate changes in S100A1 protein and steady state mRNA levels were observed in heart, other S100A1-expressing tissues (brain, slow twitch skeletal muscle, and kidney) exhibited coordinate changes in S100A1 protein and steady state mRNA levels. Altogether, these results suggest that the effects of diabetes on S100 expression are isoform as well as tissue-specific. Gel overlay analysis of the S100-binding protein profile revealed both increases and decreases in binding proteins in all tissues examined. In summary, changes in the expression of S100A1, S100B, and S100-binding proteins occur in type I diabetes and represent important molecular events in the effects of insulin/insulin insufficiency on cell function.

Animals↗
PubMed1997

Bcl-2 expression in target cells leads to functional inhibition of caspase-3 protease family in human NK and lymphokine-activated killer cell granule-mediated apoptosis.

In the granule exocytosis pathway of cell-mediated cytotoxicity, rapid apoptotic nuclear damage in target cells has been unequivocally linked to granzyme B activity. Direct cleavage and activation of caspase-3 and related proteases by granzyme B have been identified as a central event in apoptosis induction by cytotoxic granules. The Bcl-2 oncoprotein has been recently shown to act at the level or upstream of caspase-3 family activation to inhibit apoptosis induced by various stimuli including Fas ligation, an alternative cell-mediated lytic pathway. In this study, we have investigated whether activation of this caspase family by granzyme B, during human NK and lymphokine-activated killer cell granule-mediated apoptosis, could be influenced by Bcl-2 expression. Bcl-2-overexpressing clones were generated from parental K562 and U937 cell lines (K6 and U4 clones, respectively). Bcl-2 expression abrogated early 125I-DNA release and DNA fragmentation, these defects being compensated for by extended incubation times. Cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 family substrate, was detected in parental K562 cells exposed to lymphokine-activated killer effectors but not in K6 targets, indicating that caspase-3 and related proteases function was inhibited by Bcl-2. Functional inhibition of caspase-3 family with benzyloxycarbonyl-Asp-Glu-Val-Asp(OMe) fluoromethylketone led to similar consequences on apoptotic nuclear events as for Bcl-2 expression. Thus, Bcl-2 antagonizes granzyme B-mediated apoptosis by a mechanism that interferes with caspase-3 activity. Finally, Bcl-2 expression or the Asp-Glu-Val-Asp peptide was much less efficient in preventing phosphatidylserine externalization, suggesting that despite impaired nuclear apoptosis, immediate recognition and elimination of Bcl-2-expressing cells by tissue phagocytes should remain partly unaffected.

Apoptosis↗
PubMed2026

Expression patterns of potential targets for antibody-directed therapy in metastatic castration-resistant prostate cancer patients.

INTRODUCTION: Survival in metastatic castration-resistant prostate cancer (mCRPC) patients remains limited and treatment is complicated by tumor heterogeneity. As antibody-based therapeutics emerge, identifying actionable antigen targets and patient subgroups most likely to benefit is essential. MATERIALS & METHODS: Gene expression of 62 antibody-targetable proteins was analyzed in 296 mCRPC biopsies. These genes encode proteins targeted by approved or investigational antibody-based cancer therapeutics. Associations between target expression with genomic classifications and transcriptomic subtypes were evaluated. Target expression was also assessed in tumors with low expression of established mCRPC targets. Subgroup-specific targets were validated in an independent cohort and single-cell transcriptomics. RESULTS: Established targets KLK2, FOLH1 (PSMA) and STEAP1 showed the highest median expression across the cohort. Target expression did not correlate with genomic classifications, including homologous recombination deficiency, microsatellite instability, CDK12, TP53, PTEN or AR alterations Target expression did associate with transcriptomic subtypes: CRPC-AR (driven by androgen receptor-signaling) and CRPC-SCL (stem cell-like features, AP-1/YAP/TAZ-driven), displayed the highest expression of multiple targets, including KLK2, FOLH1, and SLC44A4. CRPC-NE (neuroendocrine phenotype) showed heterogeneous expression, with high CD46 expression, whereas CRPC-WNT (Wnt-signaling driven) generally showed low target expression. Notably, CD46 was highly expressed in tumors with low KLK2, FOLH1, and STEAP1 expression, a subgroup associated with poor prognosis. CONCLUSIONS: Although several antibody targets showed broad expression in mCRPC-tumors, expression varied by transcriptomic subtype. Subgroups such as CRPC-WNT expressed fewer targets, suggesting the need for alternative therapeutic strategies. CD46 emerged as a promising target, with wide expression across multiple subtypes, including clinically challenging CRPC-NE and mCRPC tumors lacking expression of established targets.

Humans↗
PubMed1996

Furin regulates both the activation of Pseudomonas exotoxin A and the Quantity of the toxin receptor expressed on target cells.

Pseudomonas exotoxin A (PE) binds and enters mammalian cells via the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). The toxin then requires proteolytic cleavage to generate an enzymatically active fragment with translocates to the cell cytosol and inhibits protein synthesis. To assess the role of furin in determining toxin susceptibility, CHO cells were transfected with a mouse furin gene (CHO+fur cells) and maintained under neomycin selection. Cells expressing the transfected gene were about two- to threefold more sensitive to PE than were cells expressing only a neomycin resistance gene (CHO+neo cells). Possible reasons for the increased toxin sensitivity include the cleavage of a greater number of PE molecules and/or the conversion of more single-chain LRP to the processed, two-chain form. Processing of LRP appears to be necessary to allow the surface display of this receptor. Results of ligand binding studies indicated that the CHO+fur cells displayed about twofold more surface-expressed LRP than did CHO+neo cells. In addition, the in vitro cleavage of PE by recombinant furin enhanced toxin potency about threefold for CHO+neo cells but enhanced it very little for CHO+fur cells. This suggested that CHO+fur cells were processing PE at close to the maximum usable rate. Together these findings suggest that furin is involved in at least two separate protein processing pathways that each contribute to the sensitivity of cells to PE.

ADP Ribose Transferases↗
PubMed1999

Genetic analysis of the role of protein kinase C signaling pathways in behaviors by direct gene transfer with HSV-1 vectors.

A genetic intervention strategy is described to elucidate the specific biochemical pathways in identified types of neurons that underlie behavioral adaptations. This strategy contains three parts: A Herpes simplex virus (HSV-1) vector is used to obtain localized gene transfer, a cell type-specific promoter is used to target expression to a particular type of neuron, and a constitutively active signal transduction enzyme is expressed to alter neuronal physiology. To enable this approach, a constitutively active protein kinase C (PKC) was developed which causes a long-lasting, activation-dependent increase in neurotransmitter release from cultured sympathetic neurons. This genetic intervention strategy was tested using the nigrostriatal system: Microinjection of HSV-1 vectors that contain the tyrosine hydroxylase promoter targeted expression to dopaminergic nigrostriatal neurons. Expression of the constitutively active PKC in a small percentage of nigrostriatal neurons (approximately 0.1-2%) produced a long-term (> or = 1 month) change in apomorphine-induced rotational behavior, the amount of rotational behavior correlated with the number of affected nigrostriatal neurons, and D2-like dopamine receptor levels were elevated in the striatal regions innervated by the affected nigrostriatal neurons. The strengths and limitations of this genetic intervention strategy are discussed.

Animals↗
PubMed1996

IL-12 p40 messenger RNA expression in target organs during acute graft-versus-host disease. Possible involvement of IFN-gamma.

The onset of acute graft-vs-host disease (aGVHD) is accompanied by macrophage (M phi) priming and the presence of bacteria-derived LPS in the sera of transplanted animals. Priming of M phi occurs during aGVHD despite the suppression of T cell function. We have investigated whether IL-12 mediates the continued production of IFN-gamma during the state of T cell immunosuppression that accompanies aGVHD. Acute GVHD was induced in nonirradiated AxC57BL/6F1 mice by the injection of C57BL/6 lymphoid cells. Despite T cell immunosuppression, M phi became primed, as shown by their expression of inducible nitric oxide synthase mRNA and their production of nitric oxide in response to LPS. Continual exposure to IFN-gamma was required to maintain a primed state in M phi during aGVHD. IL-12 p40 peptide mRNA was increased in M phi purified from animals undergoing aGVHD 14 days after transplantation. Target organs of aGVHD, including thymus, salivary gland, and lung, showed increased IFN-gamma mRNA between days 7 and 14 after transplantation. The increase was accompanied by an induction of mRNA for the p40 peptide of IL-12 and inducible nitric oxide synthase within the target organs. These results provide evidence for localized production of IFN-gamma within aGVHD target organs and suggest that it is mediated by LPS-induced production of IL-12 by M phi. Our data elucidate the mechanism of activation of M phi during aGVHD that results in TNF-alpha and nitric oxide production and delineates the effector role of M phi in the pathology of aGVHD.

Acute Disease↗
PubMed1998

Differential expression and targeting of K+ channel genes in the lobster pyloric central pattern generator.

A molecular analysis of motor pattern generation is an essential complement to electrophysiological and computational investigations. In arthropods, A-channels are posttranslationally modified multimeric proteins containing Shaker family alpha-subunits that may interact with beta-subunits, gamma-subunits, and other auxiliary proteins. One consequence of A-channel structure is that several mechanisms could underlie the cell-specific differences in pyloric IAs including differential gene expression, alternate splicing, and posttranslational modifications. Oocyte expression studies, single-cell RT-PCR, and immunocytochemistry suggest that differential alpha-subunit gene expression is not a mechanism for creating pyloric IA heterogeneity, and that the same gene, shal, encodes the alpha-subunits for the entire family of somatic IAs in the pyloric network. Changes in the level of shal gene expression alter A-channel density between cells, but cannot account for the differences in the biophysical properties of the six pyloric IAs. Preliminary data suggest that the shal gene also encodes the A-channel alpha-subunits for the coarse and fine neuropil but not for most axons. A second gene, shaker, encodes the A-channel alpha-subunits in the majority of axons and at the neuromuscular junction. The distinct properties of the two types of A-channels are consistent with the different roles of IA at the different locations. Both the shaker and shal genes are alternately spliced, and investigations are under way to determine whether alternate splicing is a mechanism for generating pyloric IA heterogeneity.

Animals↗
PubMed2001

Phagocytosis of breast cancer cells mediated by anti-MUC-1 monoclonal antibody, DF3, and its bispecific antibody.

Human epithelial mucin, MUC-1, is commonly expressed in adenocarcinoma including 80% of breast cancers. erbB-2 is overexpressed in approximately 30% of breast cancers. Expression of MUC-1 and erbB-2 may be partially overlapping but discoordinate. Therefore, combined use of antibodies directed against these two antigens might increase the number of patients who benefit from immunotherapy. Monoclonal antibody (MAb) DF3 recognizes the MUC-1 tandem repeat. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by monocyte-derived macrophages mediated by MAb DF3 and its bispecific antibody (BsAb) DF3xH22 with the second epitope directed against the Fc component of phagocytic cells. Purified monocytes from healthy donors were cultured with granulocyte macrophage colony-stimulating factor with or without IFN-gamma. antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) assays were performed with these macrophages and MUC-1-expressing target cells (ZR75-1) in the presence of MAb DF3 and BsAb DF3xH22. ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and R-phytoerythrin (RPE) (red)-conjugated MAb against human CD14 and CD11b and was confirmed by confocal microscopy. ADCC was measured by (51)Cr release assay. Immunohistochemical staining studies of MUC-1 and erbB-2 were performed on 67 primary breast cancer tissues. Expression of MUC-1 and erbB-2 was partially overlapping but discoordinate in 67 consecutive breast cancers. Both MAb DF3 and BsAb DF3xH22 mediated ADCP. However, ADCP mediated by MAb DF3 was greater than that mediated by BsAb DF3xH22. ADCC as detected by (51)Cr release was not seen with either antibody. The addition of IFN-gamma to monocyte-derived macrophage cultures inhibited ADCP compared to granulocyte macrophage colony-stimulating factor alone. Given the partially overlapping but discoordinate expression of MUC-1 and erbB-2 in breast cancer, therapy directed toward both antigens should be considered. MAb DF3 and the BsAb DF3xH22, can effectively mediate phagocytosis of MUC-1-expressing target cells. Further investigations are needed to determine whether this antibody-induced phagocytosis results in long-term specific T-cell activation against MUC-1.

Adenocarcinoma↗
PubMed1999

Changes in gene expression and targets for therapy.

A better understanding of the molecular changes associated with the onset and progression of prostate cancer may provide us with a rational basis for the development of new diagnostic and therapeutic tools. Likewise, the recent identification of critical biochemical pathways, including angiogenesis, programmed cell death, cell adhesion and signal transduction, provide us with promising targets for therapeutic approaches. Furthermore, the identification and characterization of new tumor-specific antigens or prostate-cancer-specific gene promoters could be instrumental for the development of new treatment modalities. Many research groups are trying to identify genes that are involved in prostate cancer development and which may serve as new tumor markers and potential targets for therapy. In addition to prostate-specific antigen, prostate-specific membrane antigen and human kallikrein-2, the recently identified prostate stem cell antigen may also provide us with a new tool for the diagnosis and treatment of prostate cancer. Our own studies led to the identification of DD3, a gene that is strongly overexpressed in human prostatic cancers and the expression of which appears to be restricted to the prostate. Further studies are necessary to establish the clinical usefulness of these new prostate-cancer-specific genes for the management of prostate cancer patients.

Antineoplastic Agents↗
PubMed1999

Regulators of G protein signaling exhibit distinct patterns of gene expression and target G protein specificity in human lymphocytes.

The newly recognized regulators of G protein signaling (RGS) attenuate heterotrimeric G protein signaling pathways. We have cloned an IL-2-induced gene from human T cells, cytokine-responsive gene 1, which encodes a member of the RGS family, RGS16. The RGS16 protein binds Gialpha and Gqalpha proteins present in T cells, and inhibits Gi- and Gq-mediated signaling pathways. By comparison, the mitogen-induced RGS2 inhibits Gq but not Gi signaling. Moreover, the two RGS genes exhibit marked differences in expression patterns. The IL-2-induced expression of the RGS16 gene in T cells is suppressed by elevated cAMP, whereas the RGS2 gene shows a reciprocal pattern of regulation by these stimuli. Because the mitogen and cytokine receptors that trigger expression of RGS2 and RGS16 in T cells do not activate heterotrimeric G proteins, these RGS proteins and the G proteins that they regulate may play a heretofore unrecognized role in T cell functional responses to Ag and cytokine activation.

Cells, Cultured↗
PubMed1998

CXCR4 and CCR5 expression delineates targets for HIV-1 disruption of T cell differentiation.

HIV-1 disease is often associated with CD4+ T lymphopenia as well as quantitative reductions in naive CD8+ T cells and cytopenias involving nonlymphoid hemopoietic lineages. Studies in HIV-1-infected humans as well as in animal models of lenti-virus disease indicate that these effects may be secondary to infection and destruction of multilineage and lineage-restricted hemopoietic progenitor cells. To define the stages of T cell differentiation that might be susceptible to HIV-1, we performed flow cytometric analysis of the surface expression of CXCR4 and CCR5 on T cells and their progenitors from fetal tissue, cord blood, SCID-hu Thy/Liv mice, and adult peripheral blood. We found that CXCR4 is expressed at low levels on hemopoietic progenitors in the bone marrow, is highly expressed on immature (CD3-CD4+CD8-) T cell progenitors in the thymus, and then is down-regulated during thymocyte differentiation. As thymocytes leave the thymus and enter the peripheral circulation, the expression of CXCR4 is again up-regulated. In contrast, CCR5 is undetectable on most hemopoietic progenitors in the bone marrow and on intrathymic T progenitor cells. It is up-regulated when thymocytes coexpress CD4 and CD8, then down-regulated either in the thymus (CD4+ cells) or during exit from the thymus (CD8+ cells). These results indicate that discrete, lineage-related populations of T cell progenitors may vary widely in their potential to respond to chemokines and to be infected by HIV-1, and that T lymphoid differentiation is particularly vulnerable to CXCR4-using viruses.

Adult↗
PubMed2000

In vitro generation of regulatory CD8+ T cells similar to those found in mice with anterior chamber-associated immune deviation.

PURPOSE: When injected intravenously into naive mice, peritoneal exudate cells (PECs) incubated with ovalbumin (OVA) in the presence of transforming growth factor (TGF)-beta2 induce immune deviation similar to that evoked by injection of OVA into the anterior chamber of the eye. Intraocular antigen injection elicits two distinct populations of regulatory T cells that impair delayed hypersensitivity (DH) by two different mechanisms: a CD4+ T cell that suppresses the induction of DH (afferent) and a CD8+ T cell that inhibits DH expression. In an effort to understand the origin and mechanism of action of these regulatory cells, CD8+ T cells from OVA-specific T cell receptor (Tcr) transgenic mice (OT-1) were used. METHODS: CD8+ T cells were harvested from Tcr transgenic OT-1 mice whose Tcr recognize an OVA peptide in the context of the class I major histocompatibility complex molecule Kb. These cells were stimulated in vitro with OVA-pulsed PECs exposed (or not) to TGF-beta2, then analyzed for their capacity to proliferate, to secrete various cytokines, to lyse OVA-expressing target cells, and to regulate bystander T cells in vitro and in vivo. RESULTS: When OVA-pulsed PECs were used in vitro as stimulators, responding OT-1 T cells proliferated and preferentially secreted interferon (IFN)-gamma, interleukin (IL)-2, and tumor necrosis factor (TNF)-alpha, rather than IL-4 and IL-10. When the stimulator PECs were pretreated with TGF-beta2 and then pulsed with OVA, responding OT-1 T cells proliferated even more swiftly, but they secreted significantly less IFN-gamma, IL-2, and TNF-alpha, and no IL-4 or IL-10. OT-1 T cells, which constitutively display cytotoxicity toward OVA-expressing target cells, lost this activity when stimulated with OVA-pulsed, TGF-beta2-pretreated PECs. Moreover, OT-1 T cells stimulated in this manner displayed the capacity to inhibit proliferation of OVA-primed T cells exposed to OVA in vitro and to suppress in vivo the expression of OVA-triggered DH. CONCLUSIONS: OVA-pulsed PECs, pretreated with TGF-beta2, coerce naive OVA-specific CD8+ T cells to become efferent regulators of DH similar to the regulatory T cells evoked by intraocular injection of OVA.

Adoptive Transfer↗
PubMed2002

Pag, a putative tumor suppressor, interacts with the Myc Box II domain of c-Myc and selectively alters its biological function and target gene expression.

The highly conserved Myc Box II (MBII) domain of c-Myc is critically important for transformation and transcriptional regulation. A yeast two-hybrid screen identified Pag as a MBII-interacting protein. Pag, a member of the peroxiredoxin family, has been reported previously to bind to and inhibit the cytostatic properties of the c-Abl oncoprotein. We now show that Pag promotes increased cell size and confers a proapoptotic phenotype, two hallmark features of ectopic c-Myc overexpression. Pag and c-Myc also confer resistance to oxidative stress, a previously unrecognized property of the latter protein. In contrast, Pag inhibits tumorigenesis by c-Myc-overexpressing fibroblasts and causes a broad but selective loss of c-Myc target gene regulation. Pag is therefore an MBII-interacting protein that can either mimic or enhance some of the c-Myc properties while at the same inhibiting others. These features, along with the previously identified interaction with c-Abl, provide support for the idea that Pag functions as a tumor suppressor.

3T3 Cells↗
PubMed1998

Target-specific expression of presynaptic mossy fiber plasticity.

Mossy fiber synaptic transmission at hippocampal CA3 pyramidal cells and interneurons was compared in rat brain slices to determine whether mossy terminals are functionally equivalent. Tetanic stimulation of mossy fibers induced long-term potentiation in pyramidal neurons but was either without effect or it induced depression at synapses onto interneurons. Unlike transmission onto pyramidal neurons, transmission onto interneurons was not potentiated after adenosine 3',5'-monophosphate (cAMP) activation. Furthermore, metabotropic glutamate receptor depression of transmission onto interneurons did not involve cAMP-dependent pathways. Thus, synaptic terminals arising from a common afferent pathway do not function as a single compartment but are specialized, depending on their postsynaptic target.

Afferent Pathways↗
PubMed1995

Thermostability of a nuclear-targeted luciferase expressed in mammalian cells. Destabilizing influence of the intranuclear microenvironment.

Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock. There are some indications that the nucleus is one of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvironment might be detrimental to the heat stability of proteins, we compared the in situ thermal stability of a reporter protein localized in the nucleus or in the cytoplasm. A recombinant firefly (Photynus pyralis) luciferase carrying a point mutation in the C-terminal domain remains in the cytoplasm (cyt-luciferase). A nuclear localization sequence was fused to the N-terminal domain of cyt-luciferase; the resulting nuc-luciferase was efficiently targeted to the cell nucleus. In both cases, decreased luciferase activity and solubility were found in lysates from heat-shocked cells. These characteristics were taken as an indication of thermal denaturation in situ. The heat-inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplasmic and nuclear compartments to reassemble proteins from an aggregated state. Although both the nuc- and the cyt-luciferases were heat inactivated at similar rates in vitro, nuc-luciferase was more susceptible to thermal denaturation in situ compared to cyt-luciferase. This observation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microenvironment affecting the heat-stability of proteins. In cells made thermotolerant by a priming shock, the thermal inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal sensitivity was less pronounced for the nuc-luciferase (threefold) than for the cyt-luciferase (sevenfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chaperones in the nucleus.

Base Sequence↗
PubMed1997

Mutant molecular motors disrupt neural circuits in Drosophila.

A dominant negative mutation, Glued1, that codes for a component of the dynactin complex, disrupted the axonal anatomy of leg sensory neurons in Drosophila. To examine neuron structure in mutant animals, a P[Gal4] enhancer trap targeted expression of lacZ to the sensory neurons and thereby labeled neurons in the femoral chordotonal organ and their axons within the central nervous system. When these sensory axons were examined in the Glued1 mutant specimens, they were observed to arborize abnormally. This anatomical disruption of the sensory axons was associated with a corresponding disruption in a reflex. Normally, the tibial extensor motor neurons were excited when the femoral-tibial joint was flexed, but this resistance reflex was nearly absent in mutant animals. We used the P[Gal4] insertion strains to target expression of tetanus toxin light chain to these sensory neurons in wild-type animals and showed that this blocked the resistance reflex and produced a phenocopy of the Glued result. We conclude that disruption of the dynein-dynactin complex disrupts sensory axon path finding during metamorphosis, and this in turn disrupts synaptic connectivity.

Animals↗