ABSTRACT
OBJECTIVE@#To study the expressions of the members of HSP110 family in the testis and epididymis of mice at different stages of development and whether they are regulated by hormones.@*METHODS@#The testicular and epididymis tissues of mice at different ages (14, 21, 28, 35, 42, 49, 70, and 90 days after birth, 3 mice at each age) were collected for RT-PCR detection of the expression levels of HSP110 family members. Forty-eight mice were randomized into 3 groups for sham operation, castration, or castration with testosterone injections every other day (starting at 7 days after castration), and at 1, 3, 5, and 7 days after first testosterone injection, the expressions of HSP110 family in the epididymis were detected using RT-PCR.@*RESULTS@#The mRNA expression levels of HSP110 family members underwent obvious variations with the development of the mice: , and expressions in the testicles of the mice first increased and then decreased, and gradually became stable; they also exhibited similar temporal patterns of changes in the epididymis. In the castrated mice, the mRNA expressions of and in the epididymis decreased significantly with the reduction of serum hormone levels ( < 0.05), and became normal after the supplementation of exogenous hormone.@*CONCLUSIONS@#The expression levels of HSP110 family are affected by developmental regulation, and the expressions of and are under the regulation by hormones.
Subject(s)
Animals , Male , Mice , Epididymis , Gene Expression Regulation, Developmental , HSP110 Heat-Shock Proteins , Genetics , Metabolism , Orchiectomy , Testis , Testosterone , PharmacologyABSTRACT
BACKGROUND/AIMS: Most pesticide formulations contain both chief and additive ingredients. But, the additives may not have been tested as thoroughly as the chief ingredients. The surfactant, nonyl phenoxypolyethoxylethanol (NP40), is an additive frequently present in pesticide formulations. We investigated the effects of NP40 and other constituents of a validamycin pesticide formulation on cell viability and on the expression of genes involved in cell damage pathways. METHODS: The effects of validamycin pesticide ingredients on cell viability and of NP40 on the mRNA expression of 80 genes involved in nine key cellular pathways were examined in the human neuroblastoma SK-N-SH cell line. RESULTS: The chemicals present in the validamycin pesticide formulation were cytotoxic to SK-N-SH cells and NP40 showed the greatest cytotoxicity. A range of gene expression changes were identified, with both up- and down-regulation of genes within the same pathway. However, all genes tested in the necrosis signaling pathway were down-regulated and all genes tested in the cell cycle checkpoint/arrest pathway were up-regulated. The median fold-change in gene expression was significantly higher in the cell cycle checkpoint/arrest pathway than in the hypoxia pathway category (p = 0.0064). The 70 kDa heat shock protein 4 gene, within the heat shock protein/unfolded protein response category, showed the highest individual increase in expression (26.1-fold). CONCLUSIONS: NP40 appeared to be particularly harmful, inducing gene expression changes that indicated genotoxicity, activation of the cell death (necrosis signaling) pathway, and induction of the 70 kDa heat shock protein 4 gene.
Subject(s)
Aged , Female , Humans , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Genes, cdc , HSP110 Heat-Shock Proteins/genetics , Inositol/analogs & derivatives , Necrosis , Neurons/drug effects , Nonoxynol/chemistry , Pesticides/chemistry , RNA, Messenger/metabolism , Signal Transduction/drug effects , Surface-Active Agents/chemistryABSTRACT
<p><b>OBJECTIVE</b>To explore the cytotoxic responses of spleen T lymphocytes (CTL) in BALB/c mice induced by recombinant HSP110-HER2/neu ICD complex.</p><p><b>METHODS</b>Tumor-bearing mouse model was immunized by HSP110-HER2/neu ICD complex. The IFN-γ level secreted by activated spleen T lymphocytes was detected by enzyme linked immunospot assay (ELISPOT). The corresponding CTL activity was measured by granzyme release assay.</p><p><b>RESULTS</b>The BALB/c mouse model of human mammary tumor highly expressing HER2/neu was established. HSP110-HER2/neu ICD complex immunization led to a significantly higher level of INF-γ than that in HSP110-P(789-797) immunized and HER2/neu ICD immunized mice. HSP110-HER2/neu ICD complex immunized animals also show significant CTL activity. The results of immunohistochemical staining showed that the number of blue spots in the PBS group was 4.57 ± 1.33, HSP110 group 6.83 ± 2.08, HER2/neu ICD group 16.17 ± 2.86, HSP110-P(789-797) group 43.67 ± 4.78, and SP110-HER2/neu ICD group 76.51 ± 8.17. The number of IFN-γ-secreting spleen lymphocytes in the HSP110-HER2/neu ICD group was significantly higher than that in the HSP110-P(789-797) group, and that of HSP110-P(789-797) group was significantly higher than that of HER2/neu ICD group (P < 0.01). The target cell-killing rate of the PBS group was (8.15 ± 1.27)%, HSP110 group (9.51 ± 1.51)%, HER2/neu ICD group (14.03 ± 2.45)%, HSP110-P(789-797) group (25.99 ± 3.04)% and HSP110-HER2/neu ICD group (38.15 ± 3.95)% (all P < 0.01).</p><p><b>CONCLUSIONS</b>HSP110-HER2/neu ICD complex can promote the proliferation and maturation of T lymphocytes into CTLs, and might be used as anti-tumor vaccine to induce potent cytotoxic T lymophocyte immunoresponse against specific tumor cells.</p>
Subject(s)
Animals , Female , Humans , Mice , Breast Neoplasms , Metabolism , Pathology , Cancer Vaccines , Allergy and Immunology , Cell Line, Tumor , Cell Proliferation , HSP110 Heat-Shock Proteins , Allergy and Immunology , Interferon-gamma , Metabolism , Lymphocyte Activation , Mice, Inbred BALB C , Neoplasm Transplantation , Random Allocation , Receptor, ErbB-2 , Allergy and Immunology , Metabolism , Recombinant Proteins , Allergy and Immunology , Spleen , Cell Biology , Allergy and Immunology , T-Lymphocytes , Cell Biology , Allergy and Immunology , Metabolism , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Vaccines, Synthetic , Allergy and ImmunologyABSTRACT
The heat shock protein ClpB is a member of the Clp family and functions as molecular chaperones. ClpB is related to the acquired thermotolerance in organisms. A cDNA of 3144 bp was screened out of a tomato cDNA library. The polypeptide deduced from the longest ORF contains 980 amino acid residues, and was classified into HSP100/ClpB family based on the result of molecular phylogenesis analysis. Thus it was named as LeHSP110/ClpB according to its calculated molecular weight. LeHSP110/ClpB was characteristic of heat-inducibility but no constitutive expression, and was demonstrated to locate in chloroplastic stroma. An antisense cDNA fragment of LeHsp110/ClpB under the control of CaMV 35S promoter was introduced into tomato by Agrobacterium tumefactions-mediated method. At high temperature, the mRNA levels of LeHsp110/ClpB in antisense transgenic plants were lower than those in control plants. The PS II of transgenic plants is more sensitive to high temperature than that of control plants according to data of Fv/Fm. These results clearly showed that HSP110/ClpB plays an important role in thermotolerance of high plants.
Subject(s)
Adaptation, Physiological , Genetics , Chloroplasts , Metabolism , Cloning, Molecular , Genes, Plant , Genetics , HSP110 Heat-Shock Proteins , Genetics , Metabolism , Hot Temperature , Solanum lycopersicum , Genetics , Physiology , Photosystem II Protein Complex , Metabolism , Plant Proteins , Genetics , Metabolism , Plants, Genetically Modified , Genetics , PhysiologyABSTRACT
BACKGROUND: The ischemia responsive protein 94 kDa(irp94) gene belongs to the heat shock protein 110 family and was isolated in 1999 from rat brain by transiently induced forebrain ischemia. The PC12 cell is the pheochromocytoma cell line of rat, which is differentiated to a sympathetic neuron-like cell by the stimulation of a nerve growth factor. This study is to determine whether irp94 is expressed when an ischemia-like condition is induced by ATP depletion in cultured PC12 cells in vitro. METHODS: PC12 cells were maintained as monolayer cultures in RPMI-1640 medium(Sigma) supplemented with 10% horse serum, 5% fetal bovine serum, 5 mg/ml transferrin, and 1 mg/ml insulin in a humidified 5% CO2 incubator at 37degrees C. The ATP depleting agent antimycin A was added at concentrations of 1, 2.5, and 5 microM to simulate ischemia, and 10 microgram/ml of tunicamycin, which is expected to express heat shock protein maximally, was used as a positive control. The cells were harvested after a 60-minute incubation, and the total RNA was extracted. The reverse transcription polymerase chain reaction(RT-PCR) was performed to use 501 bp irp94 cDNA as a molecular probe, and the expression of irp94 mRNA was analyzed by northern blotting. RESULTS: The irp94 mRNA expression was enhanced, compared to the negative control group, as the concentration of antimycin A was increased. CONCLUSION: This study suggests that irp94 mRNA expression is enhanced as the severity of ischemia is increased. Thus, it is possible to investigate the mechanism of ischemic neuronal injury indirectly by using this in-vitro model of neuronal ischemia.