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Biomedical subjects

L Zhu

Publications and source records attributed to L Zhu.

At least 307 records · Page 17Linked to original sources
PubMed1997

Multicomponent gene therapy vaccines for lung cancer: effective eradication of established murine tumors in vivo with interleukin-7/herpes simplex thymidine kinase-transduced autologous tumor and ex vivo activated dendritic cells.

Multiple antitumor modalities may be necessary to overcome lung tumor-mediated immunosuppression and effectively treat non-small cell lung cancer (NSCLC). To evaluate a multimodality gene therapy approach for control of local tumor growth, a weakly immunogenic murine alveolar cell carcinoma, L1C2, was transduced with either the interleukin-7/hygromycin-herpes simplex thymidine kinase (IL-7/HyHSVtk) internal ribosome entry site (IRES) retroviral vector or a vector containing the HyHSVtk, but not the IL-7 gene. Of the many cytokines available for gene transfer, IL-7 was chosen for these studies because it both stimulates CTL responses and down-regulates tumor production of the immunosuppressive peptide TGF-beta. Following selection in hygromycin, IL-7 transduction was confirmed by ELISA. Clones produced 1.25 to 10 ng of IL-7/ml/10(6) cells per 24 h. In vitro, genetically modified tumor cells were significantly more sensitive to ganciclovir (GCV) than unmodified parental tumor cells. The in vivo growth of ex vivo modified L1C2 cells was evaluated. There was a dose-response relationship between the amount of IL-7 secreted in vitro and the growth of genetically modified murine tumor in vivo. Transduced tumor cells regressed in mice following GCV therapy. Although ex vivo gene modification of tumor cells led to complete resolution of the tumor following implantation in vivo, IL-7 and HSVtk gene modified tumor cells were not effective in treating established parental tumors. However when 5 x 10(5) bone marrow-derived, in vitro activated dendritic cells (DC) were administered in combination with transduced tumor and GCV, 5 day old established tumors were eradicated in 80% of mice. These studies suggest that multicomponent vaccines may facilitate improved host responses by replacing host immune deficits and thus could have a role in adjuvant therapy and local control of NSCLC.

Animals↗
PubMed1997

Sensitive non-radioactive PCR detection of clonal T-cell receptor-gamma gene rearrangements in childhood acute lymphoblastic leukaemia.

Rearrangement of the T-cell receptor gamma (TCRgamma) gene potentially provides a valuable target for monitoring minimal residual leukaemia by the Polymerase Chain Reaction (PCR). However, existing strategies directed at this locus frequently lack sensitivity or specificity. We describe a novel PCR strategy for improved detection of clonal TCRgamma gene rearrangements based on the design of two overlapping clone-specific reverse PCR primers spanning the TCRgamma junctional region, which are used sequentially in conjunction with forward V-region specific primers. Unlike other strategies, specificity is generated from the initial and subsequent round of PCR amplification. This non-radioactive technique is highly specific and sensitive, and should prove effective for monitoring minimal residual leukaemia.

Base Sequence↗
PubMed1997

ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury.

The production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non-covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non-cytotoxic derivative PA103 exsA::omega and several cytotoxic and non-cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70-kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70-kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non-cytotoxic strains or PA103 exsA::omega. To clone the gene encoding this potential cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expressed the 70-kDa extracellular protein but maintained expression of ExoT. PA103 exoU::Tn5Tc was non-cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU::Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co-ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.

ADP Ribose Transferases↗
PubMed1997

Cloning and characterization of a new silver-stainable protein SSP29, a member of the LRR family.

Silver-stainable proteins (SSPs) are aspartic acid-rich nuclear proteins which are silver stained under very specific conditions. Using a degenerate oligodeoxyncleotide probe which codes for acidic amino acid residues, a cDNA for a new SSP, referred to as SSP29, has been isolated. The cDNA-derived amino acid sequence shows SSP29 has a molecular mass of 29 kDa, leucine-rich repeats (LRR) near the NH2-terminal region and acidic clusters at the COOH-terminal portion, indicating that SSP29 is also a member of the LRR subfamily of acidic proteins which have been shown to be involved in antigen-mediated cellular responses, leukemogenesis and differentiation. SSP29 can be stained by Ag-NOR staining. SSP29 is expressed in all human tissues and cell lines tested, localized to nucleoplasm and translocated partially to the nucleoli after heat shock. Its interaction with RNA polymerase I suggests that SSP29 may participate in signal transduction that directs nucleolar activities by regulating ribosomal RNA biosynthesis.

Amino Acid Sequence↗
PubMed1997

The influence of intersubunit cross-linking on the conformational changes of creatine kinase during denaturation by guanidine hydrochloride.

Intersubunit cross-linked creatine kinase (CK) has been prepared with the cross-linking reagent dithiobis(succinimidyl propionate) (DTSP). Unfolding of cross-linked CK during denaturation by guanidine hydrochloride (GuHCl), as monitored by intrinsic fluorescence, circular dichroism and fluorescence of the hydrophobic probe, 1-aniline-naphthalene-8-sulfonate (ANS), occurs in two stages with increasing GuHCl concentration. The process is similar to that of the unmodified enzyme, but in the second stage, conformational changes of the cross-linked enzyme need higher concentration of GuHCl, suggesting that there is a stable intermediate during its unfolding transition and the intermediate is stabilized by intersubunit cross-linkage.

Anilino Naphthalenesulfonates↗
PubMed1997

Carcinogenicity of benzo[a]pyrene and manufactured gas plant residues in infant mice.

The present study determined tumorigenicity, tumor classification and DNA damage induced in infant mice by benzo[a]pyrene (B[a]P) or Manufactured Gas Plant (MGP) residues after a single exposure. Male and female B6C3F1 mice were exposed to B[a]P or MGP residue from a single environmental site (MGP-4) and males were also exposed to MGP residue composite from seven different sites (MGP-M7). At 26, 39 and 52 weeks after exposure tumorigenesis was assessed in lung, forestomach and liver. Formation and persistence of DNA adducts were quantified by 32P-postlabeling. Exposure of males to B[a]P induced liver tumors in a dose and time dependent manner. MGP induced more advanced tumors than B[a]P. Only a single liver tumor was found in MGP-4 treated females. No forestomach and few pulmonary adenomas were induced in males or females. MGP-4, MGP-M7 or B[a]P induced DNA adducts in males and females. Adducts in liver, lung and forestomach peaked on different days and decreased at different rates. At 24 h post-exposure, no significant differences in initial DNA adduct levels occurred in males and females exposed to MGP-4 or B[a]P. Lack of DNA damage (adducted DNA) did not account for non-responsiveness of lung and forestomach in B6C3F1 genders as well as in liver in females. MGP tumorigenicity could not be accounted for solely by B[a]P content nor did it reflect additivity of B[a]P and other carcinogenic polycyclic aromatic hydrocarbons (PAHs) in MGP. Synergy among MGP-PAHs, presence of unidentified carcinogens and/or promoters in MGP may account for MGP potency. The B6C3F1 infant male model is a convenient and rapid assay for assessing MGP liver tumorigenicity and potency.

Animals↗
PubMed1997

Absence of colony stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice disrupts estrous cycles and ovulation.

Colony stimulating factor-1 (CSF-1) is a hematopoietic growth factor required for the recruitment, proliferation, and differentiation of mononuclear phagocytes. In addition, CSF-1 is expressed in the female reproductive tract coincident with CSF-1 receptor localization on preovulatory oocytes, ovarian and uterine macrophages, decidual cells, and trophoblast. A role for CSF-1 in female reproduction was confirmed by studies on CSF-1-deficient, osteopetrotic (csfmop/csfmop) mice, which suffer from low pregnancy rates and smaller litter sizes compared to wild-type mice. The present study was designed to determine the exact causes of the preimplantation fertility defects in these mutant mice. Female csfmop/csfmop mice have extended estrous cycles compared to wild-type females, and s.c. administration of CSF-1 from birth restores estrous cyclicity. These mice fail to display the characteristic proestrous surge in circulating estradiol-17beta. However, concentrations of this hormone are normal during the remainder of the cycle. Furthermore, csfmop/csfmop females have significantly lower ovulation rates than wild-type mice, but the implantation rates of fertilized oocytes are normal. Serum pregnancy concentrations of progesterone are also normal in csfmop/csfmop females, in line with the relatively normal progression of pregnancy in these mice. Thus, the major effect of CSF-1 on female reproductive function is on the frequency and rate of ovulation, indicating a major role for this growth factor in regulating follicular development and ovulation.

Animals↗
PubMed1997

Immunohistochemical localization of testin in the female reproductive system of the rat is consistent with its involvement in the turnover of specialized junctional complexes.

The distribution of testin in the female reproductive system of rats throughout the estrous cycle was examined immunohistochemically. In ovarian follicles, immunostainable testin was localized at the junctions between adjacent granulosa cells. During follicular development, immunostainable testin surrounding the granulosa cells increased in every follicle but was reduced drastically when the follicle was undergoing atresia. Testin was also found in the junctions between adjacent germinal epithelial cells that covered the surface of the ovary, at the lower or the lower lateral borders of each cell. In the uterus and oviduct, immunostainable testin was detected only in the luminal and glandular epithelium, where it formed a polygonal network encircling the apical border of the epithelial cells. During the estrous cycle, there was no drastic change in the distribution of testin in the epithelial cells of the ovary. In the vaginal mucosa, testin was found to be localized only at the junction of the epithelial cells on the surface layer of the stratified epithelium; at different stages of the estrous cycle, distinctive staining for testin could be found at proestrus, metestrus, and diestrus, but not at estrus. It is postulated that testin is a cell junction-associated protein in the female reproductive system.

Animals↗
PubMed1997

Statistical analysis of perinatal outcomes in pregnancy complicated with diabetes mellitus.

OBJECTIVE: To evaluate the outcomes of pregnancies complicated with diabetes mellitus (DM) in the maternal and perinatal center of Tokyo Women's Medical College (TWMC), and compare the results with reports of other countries. METHODS: A retrospective study of 482 cases of DM pregnancies were included over a 13-year period in a tertiary care hospital in Tokyo. Information were obtained from medical records of both mothers and babies. RESULTS: The prevalence of pregnancies complicated with DM was 5.1%. Preeclampsia developed in 124 cases at a rate of 25.8%. Incidence of preterm delivery was 16.6%. Caesarean sections were performed in 36.7% patients. There were significant differences of caesarean section rates between diabetic complication group (retinopathy and nephropathy) and non-complication group (69.5% vs 30.5%, p < 0.0001), and between preterm and term deliveries (60.0% vs 32.1%, p < 0.0001). Maternal backgrounds significantly involved with preterm delivery were DM nephropathy and severe preeclampsia. Babies with heavy for gestational age (HGA) were observed at a rate of 21% in all. There were 3 perinatal deaths (intrauterine fetal deaths, IUFD) with the perinatal mortality rate of 6.1/1,000 births. The incidence of caesarean section and perinatal mortality rate of non-DM pregnancies in our center during the same study period were 16.3% and 7.7/1,000 births respectively. CONCLUSIONS: The perinatal outcome in our study was better compared to other worldwide reports. With accurate perinatal management, perinatal mortality rate of DM pregnancies in the current study was almost same as that of non-DM pregnancies in our center. The main factors to promote adverse perinatal outcomes were presence of DM nephropathy and severe preeclampsia.

Adult↗
PubMed1997

A new fundamental bioheat equation for muscle tissue: Part I--Blood perfusion term.

A new model for muscle tissue heat transfer has been developed using Myrhage and Eriksson's [23] description of a muscle tissue cylinder surrounding secondary (s) vessels as the basic heat transfer unit. This model provides a rational theory for the venous return temperature for the perfusion source term in a modified Pennes bioheat equation, and greatly simplifies the anatomical description of the microvascular architecture required in the Weinbaum-Jiji bioheat equation. An easy-to-use closed-from analytic expression has been derived for the difference between the inlet artery and venous return temperatures using a model for the countercurrent heat exchange in the individual muscle tissue cylinders. The perfusion source term calculated from this model is found to be similar in form to the Pennes's source term except that there is a correction factor or efficiency coefficient multiplying the Pennes term, which rigorously accounts for the thermal equilibration of the returning vein. This coefficient is a function of the vascular cross-sectional geometry of the muscle tissue cylinder, but independent of the Peclet number in contrast to the recent results in Brinck and Werner [8]. The value of this coefficient varies between 0.6 and 0.7 for most muscle tissues. In part II of this study a theory will be presented for determining the local arterial supply temperature at the inlet to the muscle tissue cylinder.

Animals↗
PubMed1997

Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells.

Gene expression from the Moloney murine leukemia retrovirus (Mo-MuLV) is highly restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells. We compared levels of expression in PA317 fibroblasts, F9 (EC) cells, and CCE (ES) cells by Mo-MuLV-based vectors and vectors based on our previously reported MND backbone, which has alterations to address three viral elements implicated as repressors of expression by Mo-MuLV: the enhancer, the primer binding site, and the negative-control region. Expression was evaluated with three reporter genes, the chloramphenicol acetyltransferase (CAT) gene, whose expression was measured by enzymatic assay and by Northern blotting; a truncated nerve growth factor receptor (tNGFR), whose expression was measured by fluorescence-activated cell sorting (FACS) as a cell surface protein; and the enhanced green fluorescent protein (EGFP), whose expression was measured intracellularly by flow cytometry. We found significantly higher levels of CAT activity (5- to 300-fold) and greater quantities of vector-specific transcripts in ES and EC cells transduced with the modified MND-CAT-SN vector than in those transduced with L-CAT-SN. Northern blot analysis indicated that long terminal repeat transcripts from MND-CAT-SN are >80 times more abundant than the L-CAT-SN transcripts. FACS analysis of tNGFR expression from a pair of vectors, L-tNGFR-SN and MND-tNGFR-SN, indicated that only 1.04% of the CCE cells containing the L-tNGFR-SN vector expressed the cell surface reporter, while the MND-tNGFR-SN vector drove expression in 99.54% of the CCE cells. Of the F9 cells containing the L-tNGFR-SN vector, 13.32% expressed tNGFR, while 99.89% of the F9 cells transduced with MND-tNGFR-SN showed expression. Essentially identical results were produced with an analogous pair of vectors encoding EGFP. In unselected pools of F9 cells 48 h posttransduction, the L-EGFP-SN vector drove expression in only 5% of the population while the MND-EGFP-SN vector drove expression in 88% of the cells. After more than 3 weeks in culture without selection, the proportion of cells showing expression from L-EGFP-SN decreased slightly to 3% while expression from the MND-EGFP-SN vector persisted in 80% of the cells. Interestingly, in the few ES and EC cells which did show expression from the L-tNGFR-SN or L-EGFP-SN vectors, the magnitude of reporter expression was similar to that from the MND-tNGFR-SN or MND-EGFP-SN vector in nearly all cells, suggesting that the MND vectors are far less susceptible to position-dependent variegation of expression than are the Mo-MuLV-based vectors. Therefore, the modified retroviral vector, MND, achieves higher net levels of expression due to a greater frequency of expression, which may be useful for the expression of exogenous genes in EC and ES cells.

3T3 Cells↗
PubMed1997

Specific regulation of E2F family members by cyclin-dependent kinases.

The transcription factor E2F-1 interacts stably with cyclin A via a small domain near its amino terminus and is negatively regulated by the cyclin A-dependent kinases. Thus, the activities of E2F, a family of transcription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators: the retinoblastoma protein family and the cyclin-dependent kinase family. To investigate further the regulation of E2F by cyclin-dependent kinases, we have extended our studies to include additional cyclins and E2F family members. Using purified components in an in vitro system, we show that the E2F-1-DP-1 heterodimer, the functionally active form of the E2F activity, is not a substrate for the active cyclin D-dependent kinases but is efficiently phosphorylated by the cyclin B-dependent kinases, which do not form stable complexes with the E2F-1-DP-1 heterodimer. Phosphorylation of the E2F-1-DP-1 heterodimer by cyclin B-dependent kinases, however, did not result in down-regulation of its DNA-binding activity, as is readily seen after phosphorylation by cyclin A-dependent kinases, suggesting that phosphorylation per se is not sufficient to regulate E2F DNA-binding activity. Furthermore, heterodimers containing E2F-4, a family member lacking the cyclin A binding domain found in E2F-1, are not efficiently phosphorylated or functionally down-regulated by cyclin A-dependent kinases. However, addition of the E2F-1 cyclin A binding domain to E2F-4 conferred cyclin A-dependent kinase-mediated down-regulation of the E2F-4-DP-1 heterodimer. Thus, both enzymatic phosphorylation and stable physical interaction are necessary for the specific regulation of E2F family members by cyclin-dependent kinases.

CDC2-CDC28 Kinases↗
PubMed1997

Immunoglobulin light chain alters mesangial cell calcium homeostasis.

This study examined the hypothesis that certain immunoglobulin light chains directly altered mesangial cell calcium homeostasis. Intracellular Ca2+ concentration (intracellular [Ca2+]) signaling was determined in suspensions of rat mesangial cells using the acetoxymethyl ester of fura 2 with a calcium removal/replacement protocol. Pretreatment of cultured rat mesangial cells with a glomerulopathic kappa-light chain (gle) produced reversible dose- and time-dependent attenuation of ATP- and thrombin-evoked [Ca2+] transients (189 +/- 24 vs. 126 +/- 10 nM, P < 0.05 with ATP; 198 +/- 5 vs. 117 +/- 3 nM, P < 0.05 with thrombin) and capacitative calcium influx (199 +/- 14 vs. 142 +/- 17 nM, P < 0.05 for ATP; 252 +/- 19 vs. 198 +/- 18 nM, P < 0.05 for thrombin). Mesangial cells treated with gle and supplemented with myo-inositol (450 microM) did not demonstrate the attenuation of the ATP-evoked [Ca2+] transient and capacitative calcium influx. Gle also decreased mean [Ca2+] transient (80 +/- 7 vs. 56 +/- 1 nM, P < 0.05) and capacitative calcium influx (306 +/- 10 vs. 241 +/- 4 nM, P < 0.05) in response to thapsigargin, a Ca2+-adenosinetriphosphatase inhibitor. This inhibition was not reversed by exogenous myo-inositol. Another kappa-light chain (10 microg/ml) did not affect mesangial cell calcium signaling. Deranged mesangial cell calcium homeostasis by certain light chains may play a central pathogenetic role in glomerulosclerosis associated with deposition of immunoglobulin light chains.

Adenosine Triphosphate↗
PubMed1997

Upregulation of alpha 9 integrin and tenascin during epithelial regeneration after debridement in the cornea.

Stratified epithelia are exposed to abrasive forces and are required to respond rapidly to injury to minimize fluid loss and the risk for microbial infection. Healing involves a cell migratory phase to reestablish barrier function and cell proliferation to restratify the epithelium. Cell migration during re-epithelialization involves cell sliding, termed sheet movement, during which cells retain their cell-cell junctions while dynamically altering their shape and cell-substrate interactions to permit movement across the exposed wound bed. Proteins of the integrin family of receptor molecules modulate cell shape, cell migration, and signal transduction in many cell types. In epithelial cells, integrins of the beta 1 family have been implicated in regulating cell proliferation and differentiation, alpha 9 beta b1 is one of the newer members of the integrin beta family and has been recently shown to function as a tenascin receptor. Although little is known about its function in vivo, studies in developing mouse cornea and eyelid suggest that it may play a role in epithelial differentiation. Using a debridement wound model in the mouse cornea, we show in this study that (a) in response to small debridement wounds that close without cell proliferation, alpha 9 integrin protein and mRNA are not induced during migration but are induced during restratification, (b) larger debridement wounds that require cell proliferation to generate the cells necessary for sheet movement result in a dramatic induction of alpha 9 protein and its mRNA during both migration and restratification, and (c) tenascin, an alpha 9 beta 1 ligand, accumulates beneath epithelial cells during restratification but not during cell migration. Therefore, alpha 9 integrin protein production and tenascin accumulation are dynamically regulated in response to corneal epithelial injury.

Animals↗
PubMed1997

Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11.

Apoptosis and survival of diverse cell types are under hormonal control, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regions that mediate the formation of homo- or heterodimers important for enhancing or suppressing apoptosis. Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-2 associated death promoter), a death enhancer, has no C-terminal transmembrane domain for targeting to the outer mitochondrial membrane and nuclear envelope. We hypothesized that BAD, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identified multiple cDNA clones encoding different isoforms of 14-3-3, a group of evolutionally conserved proteins essential for signal transduction and cell cycle progression. Point mutation of BAD in one (S137A), but not the other (S113A), putative binding site found in diverse 14-3-3 interacting proteins abolished the interaction between BAD and 14-3-3 without affecting interactions between BAD and Bcl-2. Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD, we used this mutant to screen for additional BAD-interacting proteins in the yeast two-hybrid system. P11, a nerve growth factor-induced neurite extension factor and member of the calcium-binding S-100 protein family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, transient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-derived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14-3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14-3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14-3-3 family of proteins could interact with key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the present findings suggest that BAD plays an important role in mediating communication between different signal transduction pathways regulated by hormonal signals and the apoptotic mechanism controlled by Bcl-2 family members.

14-3-3 Proteins↗
PubMed1997

Neuron degeneration induced by verapamil and attenuated by EGb761.

Calcium channel blockers are used as neuroprotective agents, as glutamate antagonists. However, it has been found that calcium channel blockers may compromise neuronal survival after long-term exposure. To explore the mechanisms of the toxicity of calcium channel blockers on neurons, we studied the morphological characteristics and biochemical changes of cultured cortical neurons treated with verapamil, a calcium channel blocker. We now report that cerebral cortical cultures exposed to verapamil for 48 h undergo neuronal degeneration in both concentration-dependent and time-dependent fashion, possibly partially through the activation of apoptosis. On the other hand, it was found that Ginkgo biloba extract (EGb761) attenuated verapamil-induced neuronal injury, suggesting the possibility of using verapamil combined with EGb761 clinically. Furthermore, both B-50 immunoactivity (BIA) and the concentration of intracellular calcium in single neurons ([Ca2+]i) decreased after a 48-h exposure to verapamil, suggesting that the mechanisms of verapamil-induced degeneration may be associated with the disruption of intracellular calcium homeostasis and the inhibition of normal axonal elongation.

Animals↗
PubMed1997

Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system.

Here we demonstrate the use of a mammalian two-hybrid system to study protein-protein interactions. Like the yeast two-hybrid system, this is a genetic, in vivo assay based on the reconstitution of the function of a transcriptional activator. In this system, one protein of interest is expressed as a fusion to the Gal4 DNA-binding domain and another protein is expressed as a fusion to the activation domain of the VP16 protein of the herpes simplex virus. The vectors that express these fusion proteins are cotransfected with a reporter chloramphenicol acetyltransferase (CAT) vector into a mammalian cell line. The reporter plasmid contains a cat gene under the control of five consensus Gal4 binding sites. If the two fusion proteins interact, there will be a significant increase in expression of the cat reporter gene. Previously, it was reported that mouse p53 antitumor protein and simian virus 40 large T antigen interact in a yeast two-hybrid system. Using a mammalian two-hybrid system, we were able to independently confirm this interaction. The mammalian two-hybrid system can be used as a complementary approach to verify protein-protein interactions detected by a yeast two-hybrid system screening. In addition, the mammalian two-hybrid system has two main advantages: (i) Assay results can be obtained within 48 h of transfection, and (ii) protein interactions in mammalian cells may better mimic actual in vivo interactions.

Antigens, Polyomavirus Transforming↗