[Antitumor effects of redox-responsive nanoparticles containing platinum（⠣）in ovarian cancer].

Objectives: To explore the antitumor effects of redox-responsive nanoparticles containing platinum(Ⅳ)-NP@Pt(Ⅳ) in ovarian cancer. Methods: Redox-responsive polymer carriers were synthesized. Polymer carriers and platinum(Ⅳ)-Pt(Ⅳ) can self-assemble into NP@Pt(Ⅳ). Inductively coupled plasma mass spectrometry was performed to detect the platinum release from NP@Pt(Ⅳ) in reducing environment and the platinum content in ovarian cancer cells ES2 treated with cisplatin, Pt(Ⅳ) and NP@Pt(Ⅳ). The proliferation ability of the ovarian cancer cells were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was assessed by flow cytometry. Collection of primary ovarian cancer tissues from patients with primary high-grade serous ovarian cancer who were surgically treated at the Cancer Hospital of the Chinese Academy of Medical Sciences from October to December 2022. The high-grade serous ovarian cancer patient-derived xenograft (PDX) mice were intravenously injected with Cy7.5 labeled NP@Pt(Ⅳ) followed by in vivo imaging system. Mice were treated with PBS, cisplatin and NP@Pt(Ⅳ). Tumor volume and weight were measured in each group. Necrosis, apoptosis and cell proliferation of tumor tissues were detected by hematoxylin-eosin (HE) staining, TUNEL fluorescence staining and Ki-67 immunohistochemistry staining. Body weight and HE staining of heart, liver, spleen, lung and kidney of mice in each group were measured. Results: The platinum release of NP@Pt(Ⅳ) after 48 hours in reducing environment was 76.29%, which was significantly higher than that of 26.82% in non-reducing environment (P<0.001). The platinum content in ES2 cells after 4 hours and 7 hours of treatment with NP@Pt(Ⅳ) (308.59, 553.15 ng/million cells) were significantly higher than those of Pt(Ⅳ) (100.21, 180.31 ng/million cells) and cisplatin (43.36, 50.36 ng/million cells, P＜0.05). The half inhibitory concentrations of NP@Pt(Ⅳ) in ovarian cancer cells ES2, A2780, A2780DDP were 1.39, 1.42 and 4.62 µmol/L, respectively, which were lower than those of Pt(IV) (2.89, 7.27, and 16.74 µmol/L) and cisplatin (5.21, 11.85, and 71.98 µmol/L). The apoptosis rate of ES2 cells treated with NP@Pt(Ⅳ) was (33.91±3.80)%, which was significantly higher than that of Pt(Ⅳ) [(16.28±2.41)%] and cisplatin [(15.01±1.17)%, P＜0.05]. In high-grade serous ovarian cancer PDX model, targeted accumulation of Cy7.5 labeled NP@Pt(Ⅳ) at tumor tissue could be observed. After the treatment, the tumor volume of mice in NP@Pt(IV) group was (130±98) mm3, which was significantly lower than those in control group [(1 349±161) mm3, P<0.001] and cisplatin group [(715±293) mm3, P=0.026]. The tumor weight of mice in NP@Pt(IV) group was (0.17±0.09)g, which was significantly lower than those in control group [(1.55±0.11)g, P<0.001] and cisplatin group [(0.82±0.38)g, P=0.029]. The areas of tumor necrosis and apoptosis in mice treated with NP@Pt(Ⅳ) were higher than those in mice treated with cisplatin. Immunohistochemical staining revealed that there were low expressions of Ki-67 at tumor tissues of mice treated with NP@Pt(Ⅳ) compared with cisplatin. The change in body weight of mice in NP@Pt(Ⅳ) group was not significantly different from that of the control group [(18.56±2.04)g vs.(20.87±0.79)g, P=0.063]. Moreover, the major organs of the heart, liver, spleen, lung, and kidney were also normal by HE staining. Conclusion: Redox-responsive NP@Pt(Ⅳ), produced in this study can enhance the accumulation of cisplatin in ovarian cancer cells and improve the efficacy of ovarian cancer chemotherapy.

Cloning and sequencing of the rDNA gene family of the water buffalo (Bubalus bubalis).

The rDNA genes coding for ribosomal RNA in animals are complicated repeat sequences with high GC content. We amplified water buffalo rDNA gene sequences with the long and accurate (LA) PCR method, using LA Taq DNA polymerase and GC buffer, based on bioinformatic analysis of related organisms. The rDNA genes were found to consist of 9016 nucleotides, including three rRNA genes and two internal transcribed spacers (ITS), which we named 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA. We tested and optimized conditions for cloning these complicated rDNA sequences, including specific rules of primer design, improvements in the reaction system, and selection of the DNA polymerase.

Identification of a locus for disseminated superficial actinic porokeratosis at chromosome 12q23.2-24.1.

Disseminated superficial actinic porokeratosis is an autosomal dominant cutaneous disorder characterized by many uniformly small, minimal, annular, anhidrotic, and keratotic lesions. The genetic basis for this disease is unknown. Using a genomewide search in a large Chinese family, we identified a locus at chromosome 12q23.2-24. 1 responsible for disseminated superficial actinic porokeratosis. The fine mapping study indicates that the disseminated superficial actinic porokeratosis gene is located within a 9.6 cM region between markers D12S1727 and D12S1605, with a maximum two-point LOD score of 20.53 (theta = 0.00) at D12S78. This is the first locus identified for a genetic disease where the major phenotype is porokeratosis. The study provides a map location for isolation of a gene causing disseminated superficial actinic porokeratosis.

Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (<12%) had condensed chromatin and fragmented nuclei, the morphological features of apoptosis. The predominant alterations in acutely infected cells were distended endoplasmic reticulum and abnormal mitochondria; these ultrastructural changes are consistent with necrosis, although some infected cells simultaneously displayed features of both necrosis and apoptosis. Viability of cells persistently infected by HIV-1 was only minimally reduced from that of uninfected cells. This reduction was accounted for by an increased propensity of the persistently infected cells to die by apoptosis. Alterations in [Ca2+]i and deltapsi(m) occurred in both acutely and persistently infected cells. CONCLUSION: Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

Costimulatory pathways mediate monocyte-dependent lymphocyte apoptosis in HIV.

Examination of annexin V binding, an indicator of early apoptosis, on lymphocytes from HIV+ people immediately after isolation showed that both CD4(+) and CD8(+) T cells were apoptotic, whereas B cell apoptosis was induced mainly after incubation. CD8(+) T cell apoptosis correlated with fewer CD4(+) T cells, but not the level of viremia. To determine potential mechanisms for apoptosis, we examined FasL expression, which was dramatically elevated on CD14(+) monocytes; however, antibody to FasL did not reproducibly inhibit apoptosis. Rather, CD8(+) T cell apoptosis was caused by antigen-presenting cells because removal of monocytes or addition of antibodies to CD80 and CD86 reduced apoptosis. B cell apoptosis also involved costimulatory signals delivered by T cells but not monocytes. A unique CD8(bright)CD28(dim) T cell population died after costimulation by monocytes. Because this population was increased in patients with undetectable viremia, abnormal antigen-presenting cells may contribute to continued CD8(+) T cell exhaustion by inducing apoptosis.

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.

Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells.

Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

Anergy and apoptosis in CD8+ T cells from HIV-infected persons.

CD8+T cells from HIV-infected persons increase early in infection, display increased levels of activation Ags, and abnormal MHC-restricted, HIV-specific and nonspecific cytotoxicity abilities. Paradoxically, these cells are also unresponsive to T cell signaling in vitro and have decreased in vitro cloning potential. HIV-specific CTL precursors also are lost late in infection. A quantitative Southern blotting technique showed that CD8+ T cells from asymptomatic, HIV-infected persons have increased DNA fragmentation after overnight incubation. DNA fragmentation was reduced by an endonuclease inhibitor but not by cycloheximide, suggesting that a pre-apoptotic state exists in vivo. Partial inhibition of DNA fragmentation also could be induced by IL-2 addition. No consistent difference in fragmentation was observed among CD8+ subpopulations from HIV-infected individuals, although only CD8+ T cells that did not express activation Ags (DR-, CD28+, CD57- phenotype) showed reduced fragmentation when incubated in IL-2. A dramatic increase in CD8+, CD28- cells was observed in asymptomatic HIV-infected people. A subset of CD8+, CD28- cells in both controls and HIV-infected people do not proliferate to T cell signals, and these cells from controls demonstrate increased DNA fragmentation in vitro after 3 days of incubation, regardless of stimulation conditions. This suggests that the cells are end-stage cells. Taken together, the data suggest an increase in anergic or apoptotic CD8+ T cells in HIV-infected persons. Eventual depletion of HIV-specific CD8+ T cells may occur through a process of proliferation, anergy induction, and apoptosis.

Sex pheromone components of female smaller tea tortrix moth,Adoxophyes sp. (Lepidoptera: Tortricidae) in Taiwan.

(Z)-11-Teiradecenyl acetate (Z11-14: Ac) and (Z)-9-tetrade-cenyl acetate (Z9-14: Ac) were isolated as major sex pheromone components from the female tips of the smaller tea tortrix moth,Adoxophyes sp., in Taiwan. The average amount ofZ11- andZ9-14: Ac in each female gland was 24.9 and 14.1 ng, in a ratio of 64∶36, respectively. When compared to a closely related Japanese strain, which used theZ9-14: Ac,Z11-14∶ Ac,E11-14:Ac, 10-Me-12:Ac four-component system (in a ratio of 63∶31∶4∶2), the Taiwan formulation of two components caught significantly more Taiwan males than the Japanese formulation of four components.

Alarm pheromone of pentatomid bug,Erthesina fullo Thunberg (Hemiptera: Pentatomidae).

In the pentatomid bug,Erthesina fullo Thunberg, the odor of male metathoracic scent gland elicits an alarm response, making the male individuals of the same species alert and disperse; the alarm response of males is more obvious than that of females. Chemical composition of the glandular secretion was identified by gas chromatography and mass spectrometry data in comparison with authentic compounds. No sexual dimorphism exists in the glandular composition in this species. A total of 9 compounds [(E)-2-hexenal, (E)-4-keto-2-hexenal, (E)-2-hexenyl acetate,n-undecane,n-dodecane, (E)-2-decenal,n-tridecane, (E)-2-decenyl acetate, andn-pentadecane] are identified, among whichn-tridecane and (E)-4-keto-2-hexenal comprised nearly 70% of the total secretion in both females and males.

Simultaneous determinations by theophylline and its major metabolites in urine by reversed-phase ion-pair high-performance liquid chromatography.

A new, highly slective high-performance liquid-chromatographic (HPLC) assay for theophylline and its major metabolites in urine is described. The method utilizes an ion-pair extraction followed by separation and quantitation by reversed-phase ion-pair gradient-elution HPLC. Comparison with several other methods showed that interferences were present in too many blank urine samples to allow for the accurate quantitation of the metabolites of theophylline by direct injection--isocratic HPLC assays. Sample processing involving ion-pair complexing and extraction together with gradient-elution systems is recommended for accurate pharmacokinetic studies.