Hoechst Fluorescence Staining Research Articles

Study on the immune-enhancing and inhabiting transmissible gastroenteritis virus effects of polysaccharides from Cimicifuga rhizoma

Cimicifugae rhizoma is a traditional Chinese herbal medicine in China, and modern pharmacological research showed that it has obvious antiviral activity. Many polysaccharides have been proved to have immune enhancement and antiviral activity, but there are few studies on the biological activity of Cimicifuga rhizoma polysaccharide (CRP). The aim was to explore the character of CRP and its effects on improving immune activity and inhibiting transmissible gastroenteritis virus (TGEV). The monosaccharide composition, molecular weight, fourier transform infrared spectra and electron microscopy analysis of CRP was measured. The effect of CRP on immune activity in lymphocytes and RAW264.7 cells were studied by colorimetry, FITC-OVA fluorescent staining and ELISA. The effect of CRP on TGEV-infected PK-15 cells was determined using Real-time PCR, Hoechst fluorescence staining, trypan blue staining, acridine orange staining, Annexin V-FITC/PI fluorescent staining, DCFH-DA loading probe, and JC-1 staining. Network pharmacology was used to predict the targets of CRP in enhancing immunity and anti-TGEV, and molecular docking was used to further analyze the binding mode between CPR and core targets. The results showed that CRP was mainly composed of glucose and galactose, and its molecular weight was 64.28 kDa. The content of iNOS and NO in CRP group were significantly higher than the control group. CRP (125 and 62.5 μg/mL) could significantly enhance the phagocytic capacity of RAW264.7 cells, and imprive the content of IL-1β content compared with control group. 250 μg/mL of CRP possessed the significant inhibitory effect on TGEV, which could significantly reduce the apoptosis compared to TGVE group and inhibit the decrease in mitochondrial membrane potential compared to TGVE group. The mRNA expression of TGEV N gene in CRP groups was significantly lower than TGEV group. PPI showed that the core targets of immune-enhancing were AKT1, MMP9, HSP90AA1, etc., and the core targets of TGE were CASP3, MMP9, EGFR, etc. Molecular docking show that CRP has binding potential with target. These results indicated that CRP possessed the better immune enhancement effect and anti-TGEV activity.

Effect of miR-17 on Polygonum Cillinerve polysaccharide against transmissible gastroenteritis virus.

Transmissible gastroenteritis virus (TGEV) could cause diarrhea, vomiting, dehydration and even death in piglets, miRNA played an important role in the interaction between virus and cell. The study aimed to investigate the impact of miR-17 on the polysaccharide of Polygonum Cillinerve (PCP) in combating TGEV. miR-17 was screened and transfection validation was performed by Real-time PCR. The function of miR-17 on PK15 cells infected with TGEV and treated with PCP was investigated by DCFH-DA loading probe, JC-1 staining and Hoechst fluorescence staining. Furthermore, the effect of miR-17 on PCP inhibiting TGEV replication and apoptosis signaling pathways during PCP against TGEV infection was measured through Real-time PCR and Western blot. The results showed that miR-17 mimic and inhibitor could be transferred into PK15 cells and the expression of miR-17 significantly increased and decreased respectively compared with miR-17 mimic and inhibitor (P < 0.05). A total 250 μg/mL of PCP could inhibit cells apoptosis after transfection with miR-17. PCP (250 μg/mL and 125 μg/mL) significantly inhibited the decrease in mitochondrial membrane potential induced by TGEV after transfection with miR-17 (P < 0.05). After transfection of miR-17 mimic, PCP at concentrations of 250 μg/mL and 125 μg/mL significantly promoted the mRNA expression of P53, cyt C and caspase 9 (P < 0.05). Compared with the control group, the replication of TGEV gRNA and gene N was significantly inhibited by PCP at concentrations of 250 μg/mL and 125 μg/mL after transfection of both miR-17 mimic and inhibitor (P < 0.05). PCP at 62.5 μg/mL significantly inhibited the replication of gene S following transfection with miR-17 inhibitor (P < 0.05). These results suggested that PCP could inhibit the replication of TGEV and apoptosis induced by TGEV by regulating miR-17.

LINC01936 inhibits the proliferation and metastasis of lung squamous cell carcinoma probably by EMT signaling and immune infiltration

Purpose To discover the biological function and potential mechanism of LINC01936 in the development of lung squamous cell carcinoma (LUSC). Methods Transcriptome data of LUSC from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to analyze the differentially expressed lncRNAs in LUSC and normal tissues by R “DEseq2”, “edgeR” and “limma” packages. The subcellular localization of LINC01936 was predicted by lncLocator. Cell proliferation and apoptosis were measured by CCK-8, MTT assay and Hoechst fluorescence staining. The migration and invasion were detected by Transwell assay. The function and pathway enrichment analysis were performed by Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene set variation analysis (GSVA). The downstream targets of LINC01936 were predicted using RNA-Protein Interaction Prediction (RPISeq) program. The effect of LINC01936 on tumor immune infiltration was analyzed using Pearson Correlation Analysis using R “ggpubr” package. Results Based on the gene expression data of LUSC from TCGA database, 1,603, 1,702 and 529 upregulated and 536, 436 and 630 downregulated lncRNAs were obtained by DEseq2, edgeR and limma programs, respectively. For GSE88862 dataset, we acquired 341 differentially expressed lncRNAs (206 upregulated and 135 downregulated). Venn plot for the intersection of above differential expressed lncRNAs showed that there were 29 upregulated and 23 downregulated genes. LINC01936 was one of downregulated lncRNAs in LUSC tissues. The biological analysis showed that the overexpression of LINC01936 significantly reduced proliferation, migration and invasion of LUSC cells, and promoted cell apoptosis. The knockdown of LINC01936 promoted cell proliferation and metastasis. Pathway and GSVA analysis indicated that LINC01936 might participated in DNA repair, complement, cell adhesion and EMT, etc. LINC01936 was predicted to interact with TCF21, AOC3, RASL12, MEOX2 or HSPB7, which are involved in EMT and PI3K-AKT-MTOR pathway, etc. The expression of LINC01936 was also positively correlated with the infiltrating immune cells in LUSC. Conclusions LINC01936 is downregulated in LUSC. LINC01936 affected proliferation, migration and invasion of LUSC cells probably by EMT and immune infiltration, which might serve as a new target for the treatment of LUSC.

Preparation and characterization of fenugreek leaf flavonoids and their protective effects against oxidative damage to hepatocytes

The present study investigated the main components of fenugreek(Trigonella foenum-graecum L.) leaf flavonoids(FLFs) and their antioxidant activity. FLFs were prepared and enriched by solvent extraction, and the flavonoids were characterized by high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS). The protective effect of FLFs against H_2O_2-induced stress damage to L02 hepatocytes was also investigated. Firstly, the cell viability was measured by MTT assay. The oxidative stress injury model was induced by H_2O_2 in L02 cells. The release of lactate dehydrogenase(LDH), the content of reduced glutathione(GSH) and malondialdehyde(MDA), and the activities of superoxide dismutase(SOD) and catalase(CAT) were measured by assay kits. Hoechst fluorescence staining was performed to observe the cell apoptosis. The expression levels of c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase 1/2(ERK1/2), nuclear factor erythroid-2 related factor 2(Nrf2), heme oxygenase 1(HO-1), and their phosphorylated proteins were detected by Western blot. Based on the MS fragment ion information and data in databases, FLFs contained eight flavonoids with quercetin and kaempferol as the main aglycons. The cell viabi-lity assay revealed that as compared with the conditions in the H_2O_2 treatment group, 3.125-25 μg·mL~(-1) FLFs could increase the viability of L02 cells, reduce LDH release and MDA content in a dose-dependent manner, potentiate the activities of SOD, CAT, and GSH, decrease the phosphorylation of JNK and ERK1/2 proteins, and up-regulate the expression of Nrf2 and HO-1. The results of fluorescence staining showed that the nucleus of the H_2O_2 treatment group showed concentrated and dense strong blue fluorescence, while the blue fluorescence intensity of the FLFs group decreased significantly. FLFs showed a protective effect against H_2O_2-induced oxidative damage in L02 cells, and the underlying mechanism is associated with the enhancement of cell capability in clearing oxygen free radicals and the inhibition of apoptosis by the activation of the MAPKs/Nrf2/HO-1 signaling pathway. The antioxidant effect of fenugreek leaf is related to its rich flavonoids.

Schisandrin Protects against Norepinephrine-Induced Myocardial Hypertrophic Injury by Inhibiting the JAK2/STAT3 Signaling Pathway.

Aims. Heart failure is closely associated with norepinephrine-(NE-) induced cardiomyocyte hypertrophy. Schisandrin is derived from the traditional Chinese medicine Schisandra; it has a variety of pharmacological activities, and the mechanism of schisandrin-mediated protection of the cardiovascular system is not clear. Main Methods. NE was used to establish a cardiomyocyte hypertrophy model to explore the mechanism of action of schisandrin. An MTT assay was used for cell viability; Hoechst fluorescence staining was used to observe the cell morphology and calculate the apoptosis rate. The cell surface area was measured and the protein to DNA ratio was calculated, changes in mitochondrial membrane potential were detected, and the degree of hypertrophic cell damage was evaluated. WB, QRT-PCR, and immunofluorescence were used to qualitatively, quantitatively, and quantitatively detect apoptotic proteins in the JAK2/STAT3 signaling pathway. Key Findings. In the NE-induced model, schisandrin treatment reduced the apoptosis rate of cardiomyocytes, increased the ratio of the cell surface area to cardiomyocyte protein/DNA, and also, increased the membrane potential of the mitochondria. The expression of both JAK2 and STAT3 was downregulated, and the BAX/Bcl-2 ratio was significantly reduced. In conclusion, schisandrin may protect against NE-induced cardiomyocyte hypertrophy by inhibiting the JAK2/STAT3 signaling pathway and reducing cardiomyocyte apoptosis.

Luteolin has a significant protective effect against cadmium-induced injury in lung epithelial Beas-2B cells

To investigate the protective effect of luteolin against cadmium (Cd)-induced injury in human lung epithelial Beas-2B cells. Beas-2B cells were treated with different concentrations of luteolin (0-160 μmol/L) or Cd (0-40 μmol/L) for 24 h, and the cell viability was examined using MTT assay. After treatment with luteolin (0.25, 0.5 and 0.75 μmol/L) with or without Cd (5 μmol/L) for 24 h, the cells were examined for viability, lactate dehydrogenase (LDH) activity and morphological changes of the cell nuclei using Hoechst fluorescent staining. The levels of ROS, SOD, GSH and MDA in the treated cells were detected, and the expression levels of Akt, p-Akt and nuclear factor E2-related factor 2 (Nrf2) proteins were determined using Western blotting. Luteolin within the concentration range of 0-80 μmol/L did not significantly affect the survival rate of Beas-2B cells (P>0.05), but Cd at 5 μmol/L significantly decreased the cell viability (P < 0.05) with an IC50 of 24.6 μmol/L. In Cd-treated cells, treatment with luteolin significantly mitigated the decrease of cell viability, reduced LDH release and cell apoptosis, enhanced SOD activity and GSH content, and inhibited the production of MDA and ROS (all P < 0.05). Luteolin also significantly up-regulated the expression levels of p-Akt and Nrf2 protein in Cd-treated Beas-2B cells (P < 0.05). Luteolin has a significant protective effect against Cd-induced injury in Beas-2B cells, and the effects are probably mediated, at least in part, by promoting the activation of Akt and Nrf2.

Jianpi Yangwei decoction promotes apoptosis and suppresses proliferation of 5-fluorouracil resistant gastric cancer cells in vitro and in vivo

BackgroundThe exploration of new therapeutic agents targeting 5-Fu resistance may open a new opportunity to gastric cancer treatment. The objective is to establish a 5-Fu resistant gastric cancer cell line and observe the effect of Jianpi Yangwei decoction (JPYW) on its apoptosis and drug-resistance related proteins.MethodsMTT assay was used to measure the effect of JPYW on the BGC823 cells proliferation, and the apoptosis was observed by flow cytometry and Hoechst fluorescence staining. The BGC823 xenograft tumor nude mice models were established, the apoptosis was detected by Tunel method. BGC-823/5-Fu was established by repeated low-dose 5-Fu shocks, the drug resistance index and proliferation were detected by the MTT assay; MDR1 mRNA was detected by real-time RT-PCR; Western blot was used to detect the ratio of p-AKT to AKT; The BGC823/5-Fu xenograft tumor nude mice models were established and apoptosis was measured. The expressions of MRP1, MDR1, ABCG2, AKT, p-AKT, caspase-3 and bcl-2 were detected by immunohistochemistry and the AKT mRNA expression was detected by real-time RT-PCR.ResultsJPYW induced apoptosis in BGC823 cells; Drug-resistant cell line BGC-823/5-Fu was sucessfully established; JPYW induced apoptosis of BGC823/5-Fu cells, down-regulated the expression of MRP1, MDR1 and ABCG2 in vitro and in vivo, and further decreased MDR1 expression when combined with pathway inhibitor LY294002 (P < 0.05); JPYW down-regulated the ratio of p-AKT to AKT in vitro in a dose-dependent manner, the same as after the combination with LY294002 (P < 0.05).ConclusionJPYW can induce apoptosis of BGC823 and BGC823/5-Fu cells, and down-regulate the expression of MDR1, MRP1, ABCG2 in vitro and in vivo. Its in vitro effect is related to the PI3K/AKT signaling pathway.

RAj-Tspin, a novel recombinant peptide from Apostichopus japonicus, suppresses the proliferation, migration, and invasion of BEL-7402 cells via a mechanism associated with the ITGB1-FAK-AKT pathway.

Purpose Our study aimed to investigate the antitumor effects of rAj-Tspin on BEL-7402 hepatocellular carcinoma cells and to explore the underlying mechanism. Method For the in vivo experiment, BEL-7402 cells were inoculated subcutaneously into the axilla of nude mice to generate a BEL-7402 cell-bearing model, and model mice were then treated with different doses of rAj-Tspin. A CCK-8 assay was used to evaluate the in vitro viability of BEL-7402 and LO2 cells after treatment with different concentrations of rAj-Tspin. The effects of rAj-Tspin on BEL-7402 cell apoptosis, migration and invasion were evaluated by AO/EB and Hoechst fluorescent staining and by scratch and Transwell assays, and the tumor-suppressive mechanism of rAj-Tspin was explored by Western blotting. Results rAj-Tspin suppressed the proliferation of BEL-7402 cells with an IC50 of 0.89μM. The results of both microscopic analysis and Western blotting suggested that rAj-Tspin induced the apoptosis of BEL-7402 cells through a mitochondria-dependent pathway. Furthermore, rAj-Tspin disrupted EMT; this disruption ultimately caused BEL-7402 cells to lose their shape and decreased their migration and invasion. Moreover, rAj-Tspin might inhibit the proliferation and metastasis of BEL-7402 cells through the ITGB1-FAK-AKT pathway. Conclusion rAj-Tspin exerts an antitumor effect through the ITGB1-FAK-Akt signaling pathway and exhibits low toxicity at an effective dose.

Erratum to: Mesenchymal stem cells attenuate hydrogen peroxide-induced oxidative stress and enhance neuroprotective effects in retinal ganglion cells.

The apoptosis of retinal ganglion cells leads to visual impairment and blindness in ocular neurodegenerative diseases, especially in diabetic retinopathy (DR). Mounting evidence suggests that oxidative stress contributes to the pathogenesis of DR. In the present study, we investigated whether bone mesenchymal stem cells (BMSCs) have protective ability to relieve hydrogen peroxide (H2O2)-induced injury on retinal ganglion cells in vitro. An immortalized retinal ganglion cells, RGC-5 cells, were exposed to an indicated concentration of H2O2 for 24 h. Cell viability was analyzed by CCK-8 assay to find out a certain concentration to build H2O2 oxidative damage model. Morphological changes in RGC-5 cells were observed under optical microscope, and cell apoptosis was detected with Hoechst fluorescence staining. Then, BMSCs were co-cultured with RGC-5 cells in a transwell culture system for 24 h and 48 h. Flow cytometry was performed to qualify the apoptosis rate of RGC-5 cells. Conditioned medium was collected for evaluation the inflammatory cytokines by ELISA. The content of intracellular malondialdehyde (MDA) and superoxide dismutase (SOD) was assayed by thiobarbituric acid and xanthine oxidase method, respectively. qRT-PCR and ELISA were conducted for analysis of the expression changes in brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), respectively. After H2O2 exposure, the morphological varieties were observed as cytoplasm shrinking and paramorphia together with nuclear gathering. Meanwhile, the apoptotic cells had hyperfluorescence with Hoechst 33258 staining. Co-culture with BMSCs significantly inhibited retinal cell death. It was found that BMSCs reduced H2O2-induced inflammatory factors IL-1β and TNF-α, down-regulated intracellular oxidant factor MDA, up-regulated intracellular antioxidant factor SOD, and increased neurotrophins BDNF and CNTF expression. BMSCs may enhance protective effect of RGC-5 cells in H2O2-induced damage through improving antioxidant capacity, inhibiting pro-inflammatory cytokine secretion, and promoting neurotrophin expression.

Mesenchymal stem cells attenuate hydrogen peroxide-induced oxidative stress and enhance neuroprotective effects in retinal ganglion cells

The apoptosis of retinal ganglion cells leads to visual impairment and blindness in ocular neurodegenerative diseases, especially in diabetic retinopathy (DR). Mounting evidence suggests that oxidative stress contributes to the pathogenesis of DR. In the present study, we investigated whether bone mesenchymal stem cells (BMSCs) have protective ability to relieve hydrogen peroxide (H2O2)-induced injury on retinal ganglion cells in vitro. An immortalized retinal ganglion cells, RGC-5 cells, were exposed to anindicated concentration of H2O2 for 24h. Cell viability was analyzed by CCK-8 assay to find out a certain concentration to build H2O2 oxidative damage model. Morphological changes in RGC-5 cells were observed under optical microscope, and cell apoptosis was detected with Hoechst fluorescence staining. Then, BMSCs were co-cultured with RGC-5 cells in a transwell culture system for 24 h and 48h. Flow cytometry was performed to qualify the apoptosis rate of RGC-5 cells. Conditioned medium was collected for evaluation the inflammatory cytokines by ELISA. The content of intracellular malondialdehyde (MDA) and superoxide dismutase (SOD) was assayed by thiobarbituric acid and xanthine oxidase method, respectively. qRT-PCR and ELISA were conducted for analysis of the expression changes in brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), respectively. After H2O2 exposure, the morphological varieties were observed as cytoplasm shrinking and paramorphia together with nuclear gathering. Meanwhile, the apoptotic cells had hyperfluorescence with Hoechst 33258 staining. Co-culture with BMSCs significantly inhibited retinal cell death. It was found that BMSCs reduced H2O2-induced inflammatory factors IL-1β and TNF-α, down-regulated intracellular oxidant factor MDA, up-regulated intracellular antioxidant factor SOD, and increased neurotrophins BDNF and CNTF expression. BMSCs may enhance protective effect of RGC-5 cells in H2O2-induced damage through improving antioxidant capacity, inhibiting pro-inflammatory cytokine secretion, and promoting neurotrophin expression.

Flavonoids Isolated from KoreaCitrus aurantiumL. Induce G2/M Phase Arrest and Apoptosis in Human Gastric Cancer AGS Cells

Aim of the Study. Citrus species is used in traditional medicine as medicinal herb in several Asian countries including Korea. Flavonioids became known as various properties, such as anti-oxidants, anti-inflammation and anti-cancer, and so forth. The present study, the anti-cancer effect of flavonioids isolated from Citrus aurantium L. in human gastric cancer AGS cells has been investigated. Materials and Methods. The anti-proliferative activity was assayed using MTT assay. Cell cycle analysis was done using flow cytometry and apoptosis detection was done using by hoechst fluorescent staining and Annexin V-propidium iodide double staining. Western blot was used to detect the expression of protein related with cell cycle and apoptosis. Results. Flavonoids isolated from Citrus aurantium L. have the effect of anti proliferation on AGS cells with IC50 value of 99 μg/mL. Flavonoids inhibited cell cycle progression in the G2/M phase and decrease expression level of cyclin B1, cdc 2, cdc 25c. Flavonoids induced apoptosis through activate caspase and inactivate PARP. Conclusions. Flavonoids isolated from Citrus aurantium L. induced G2/M phase arrest through the modulation of cell cycle related proteins and apoptosis through activation caspase. These finding suggest flavonoids isolated from Citrus aurantium L. were useful agent for the chemoprevention of gastric cancer.

AICAR inhibits proliferation and induced S-phase arrest, and promotes apoptosis in CaSki cells

The aim of the present study was to determine the effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) on proliferation, cell cycle, and apoptosis in the human epithelial cervical cancer cell line CaSki cells. Cell count and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were used to determine cell proliferation and viability. Hoechst 33258 staining was conducted to distinguish the apoptotic cells. Cell cycle and Annexin-V/propidium iodide staining were analyzed by fluorescence-activated cell sorting (FACS). A Western blot assay was used to evaluate the expression of AKT (also known as protein kinase B), mammalian target of rapamycin (mTOR), p53, and extracellular signal-regulated kinase (ERK). AICAR (500 micromol/L) significantly inhibited the proliferation of CaSki cells treated for 24, 48, and 72 h as determined by cell count. The cells at the G1 and G2 phases were dramatically decreased while cells at the S phase were increased in response to AICAR treatment for 24, 48, and 72 h. The MTT assay showed less viable cells and Hoechst fluorescent staining showed more apoptotic cells upon AICAR stimulation. The results of the Annexin-V staining demonstrated a time-dependent increase of apoptosis in cells treated with AICAR for 24, 36, and 48 h. Furthermore, AICAR activated caspase-3 in a time-dependent manner. It was also found that AICAR inhibited the phosphorylation of AKT and mTOR, which are important kinases regulating cell growth and survival. AICAR stimulation obviously increased the expression of the tumor suppressor p53 and the phosphorylation of ERK. AICAR inhibited proliferation and induced S phase arrest and promoted apoptosis in CaSki cells, which might be mediated by the downregulation of the AKT/mTOR pathway and the upregulation of the p53/ERK pathway.

Strain-dependent induction of enterocyte apoptosis by Giardia lamblia disrupts epithelial barrier function in a caspase-3-dependent manner.

We recently demonstrated that Giardia lamblia rearranges cytoskeletal proteins and reduces transepithelial electrical resistance. The effect of G. lamblia on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and altered epithelial permeability has yet to be established. The aim of this study was to assess whether G. lamblia induces enterocyte apoptosis in duodenal epithelial monolayers and whether this effect increases epithelial permeability. Monolayers of nontransformed human duodenal epithelial cells were incubated with sonicated or live G. lamblia trophozoites (NF, S2, WB, or PB strains) for 8, 24, and 48 h. Cell cultures were assessed for apoptosis by Hoechst fluorescence staining, enzyme-linked immunosorbent assay for apoptotic nucleosomes, and electron microscopy. In separate experiments, monolayers were pretreated with or without 120 microM caspase-3 inhibitor (Z-DEVD-FMK) for 1 h and were assessed for production of apoptotic nucleosomes, tight junctional integrity (with fluorescent ZO-1 staining followed by confocal laser microscopy), and transepithelial permeability for fluorescein isothiocyanate-dextran. G. lamblia strains NF and S2, but not strains WB or PB, induced enterocyte apoptosis within the monolayers, and this effect was inhibited by Z-DEVD-FMK pretreatment. Using the G. lamblia NF isolate, additional experiments investigated the possible link between enterocyte apoptosis and altered epithelial permeability. G. lamblia NF disrupted tight junctional ZO-1 and increased epithelial permeability, but these effects were also prevented by pretreatment with the caspase-3 inhibitor. These findings indicate that strain-dependent induction of enterocyte apoptosis may contribute to the pathogenesis of giardiasis. This effect is responsible for a loss of epithelial barrier function by disrupting tight junctional ZO-1 and increasing permeability in a caspase-3-dependent manner.

Neural transplantation of cells expressing the anti-apoptotic gene bcl-2.

Long-term survival of grafted neural cells is a major goal of neural transplantation, but typical survival rates of grafted fetal neurons are in the range of 5-10%. Whether the death of transplanted neural cells is apoptotic or necrotic is unknown. The expression of the proto-oncogene bcl-2 inhibits both apoptotic and necrotic neural cell death. In a 6-OHDA induced rat model of Parkinson's disease, Hoechst 33258 prelabelled conditionally immortalized nigral cells engineered to express bcl-2 were stereotactically transplanted into the striatum ipsilaterally to the lesioned nigrostriatal pathway. Sixteen rats received bcl-2 transfected cells, 15 received cells transfected with vector alone, and 12 received either a nondopaminergic cell line or were sham transplanted as controls. Four wk following transplantation, the rats with grafts containing bcl-2 expressing cells showed an approximately 43% decrease in apomorphine-induced rotational behavior. In contrast, 12% improvement occurred in the rats with transplanted cells transfected with vector alone (p < 0.05), and no improvement occurred in sham-operated animals (p < 0.05). Histological examination showed no tumor formation. Despite the difference in behavioral effect, no clear difference in Hoechst fluorescent staining or staining for TH, GFAP was noted; therefore, it is unknown at present whether the observed effect was due to a difference in survival or to increased efficacy per surviving transplanted neural cell, or both.

Neural transplantation of cells expressing the anti-apoptotic gene bcl-2

Long-term survival of grafted neural cells is a major goal of neural transplantation, but typical survival rates of grafted fetal neurons are in the range of 5–10%. Whether the death of transplanted neural cells is apoptotic or necrotic is unknown. The expression of the proto-oncogene bcl-2 inhibits both apoptotic and necrotic neural cell death. In a 6-OHDA induced rat model of Parkinson's disease, Hoechst 33258 prelabelled conditionally immortalized nigral cells engineered to express bcl-2 were stereotactically transplanted into the striatum ipsilaterally to the lesioned nigrostriatal pathway. Sixteen rats received bcl-2 transfected cells, 15 received cells transfected with vector alone, and 12 received either a nondopaminergic cell line or were sham transplanted as controls. Four wk following transplantation, the rats with grafts containing bcl-2 expressing cells showed an approximately 43% decrease in apomorphine-induced rotational behavior. In contrast, 12% improvement occurred in the rats with transplanted cells transfected with vector alone ( p < 0.05), and no improvement occurred in sham-operated animals ( p < 0.05). Histological examination showed no tumor formation. Despite the difference in behavioral effect, no clear difference in Hoechst fluorescent staining or staining for TH, GFAP was noted; therefore, it is unknown at present whether the observed effect was due to a difference in survival or to increased efficacy per surviving transplanted neural cell, or both.

BrdU-dye characterization of late replication and meiotic recombination in Armenian hamster germ cells.

In vivo BrdU-dye methodology is used to reveal patterns of replication kinetics and meiotic recombination in male germ cells of the Armenian hamster. Analogue substitution over a fraction of the DNA synthesis period results in distinctive 33258 Hoechst fluorescence staining of late replicating chromatin, detectable at spermatogonial or spermatocyte I and II stages. Spermatogonial cells which are extensively substituted with BrdU over the penultimate synthesis period reveal sister chromatid differentiation in all chromosomes of primary or secondary spermatocytes. In chiasmatic regions, exchanges between unlike-stained non-sister chromatids are indicated by isolatelled segments, while those occurring between like-stained non-sister chromatids are not directly detected. In sex chromosomes from young animals, these alternative images occur in a ratio of 1:1, which supports the concept that homologue non-sister chromatid regions are randomly broken and re-united in the process of chiasma formation. Deviations from randomness appeared to occur in older animals. Sex bivalent chiasmata are either coincident with points of visible exchange, or they appear to have variable degrees of terminalization. Secondary spermatocytes display sharp chromatid contrast, and aid in mapping the positions and frequencies of homologue exchanges.