Ad-ING4-IL-24 双基因共表达对人肺腺癌化疗的增敏效应

研究ING4 (肿瘤生长抑制因子4) 和IL-24 (人白细胞介素24) 双基因共表达腺病毒载体 (Ad-ING4-IL-24) 对肺腺癌的化疗增敏效应及分子机制。将Ad-ING4-IL-24感染A549肺癌细胞及联合顺铂 (DDP) 化疗药物作用,RT-PCR和Western blotting检测ING4和IL-24基因在A549肺癌细胞中的转录和表达,MTT法、流式细胞仪 (FCM) 和 Hoechst 染色法检测Ad-ING4-IL-24联合DDP (联合组) 对A549肺癌细胞的生长抑制和凋亡效应。采用A549细胞株建立人肺腺癌裸鼠模型,然后瘤体局部注射干预用药,动态测量肿瘤体积,并计算瘤重抑瘤率,免疫组化检测ING4、IL-24、bax、bcl-2、VEGF等基因的表达。结果表明,Ad-ING4-IL-24感染A549肺癌细胞后可获得成功转录和表达,体外联合组能明显抑制A549肺癌细胞生长和诱导细胞凋亡,呈现出典型细胞凋亡形态学变化。体内联合组同样能显著抑制肿瘤生长,瘤重抑瘤率达52.81％,免疫组化结果显示联合组能上调bax基因表达,同时下调bcl-2、VEGF等基因的表达。以上结果表明Ad-ING4-IL-24具有化疗增敏的作用,该作用机制可能与促进肿瘤细胞凋亡和抑制血管形成有关。     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

Activities in vitro and in vivo of enzymes of benzodiazepine alkaloid biosynthesis during development of Penicillium cyclopium

In the hyphae of Penicillium cyclopium the in vitro measurable activities of 3 enzymes of alkaloid biosynthesis are induced endogenously during     

Measurements of mitochondrial calcium in vivo

Mitochondria play a pivotal role in intracellular Ca²⁺ signalling by taking up and releasing the ion upon specific conditions. In order to do so, mitochondria depend on a number of factors, such as the mitochondrial membrane potential and spatio-temporal constraints. Whereas most of the basic principles underlying mitochondrial Ca²⁺ handling have been successfully deciphered over the last 50 years using assays based on in vitro preparations of mitochondria or cultured cells, we have only just started to understand the actual physiological relevance of these processes in the whole animal. Recent advancements in imaging and genetically encoded sensor technologies have allowed us to visualise mitochondrial Ca²⁺ transients in live mice. These studies used either two-photon microscopy or bioluminescence imaging of cameleon or aequorin-GFP Ca²⁺ sensors, respectively. Both methods revealed a consistent picture of Ca²⁺ uptake into mitochondria under physiological conditions even during very short-lasting elevations of cytosolic Ca²⁺ levels. The big future challenge is to understand the functional impact of such Ca²⁺ signals on the physiology of the observed tissue as well as of the whole organism. To that end, the development of multiparametric in vivo approaches will be mandatory.     

Low immunogenicity of allogeneic human umbilical cord blood-derived mesenchymal stem cells in vitro and in vivo

Evaluation of the immunogenicity of human mesenchymal stem cells (MSCs) in an allogeneic setting during therapy has been hampered by lack of suitable models due to technical and ethical limitations. Here, we show that allogeneic human umbilical cord blood derived-MSCs (hUCB-MSCs) maintained low immunogenicity even after immune challenge in vitro. To confirm these properties in vivo, a humanized mouse model was established by injecting isolated hUCB-derived CD34+ cells intravenously into immunocompromised NOD/SCID IL2γnull (NSG) mice. After repeated intravenous injection of human peripheral blood mononuclear cells (hPBMCs) or MRC5 cells into these mice, immunological alterations including T cell proliferation and increased IFN-γ, TNF-α, and human IgG levels, were observed. In contrast, hUCB-MSC injection did not elicit these responses. While lymphocyte infiltration in the lung and small intestine and reduced survival rates were observed after hPBMC or MRC5 transplantation, no adverse events were observed following hUCB-MSC introduction. In conclusion, our data suggest that allogeneic hUCB-MSCs have low immunogenicity in vitro and in vivo, and are therefore “immunologically safe” for use in allogeneic clinical applications.     

Effects of freezing on biological membranes in vivo and in vitro

Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.     

Piperazine derivatives inhibit PrP/PrP propagation in vitro and in vivo

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc^C into a disease-associated isoform PrP^Sc. PrP^Sc accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP^Sc species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP^Sc formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge).     

Enhancement of bone formation ex vivo and in vivo by a helioxanthin-derivative

To effectively treat serious bone defects using bone-regenerative medicine, a small chemical compound that potently induces bone formation must be developed. We previously reported on the osteogenic effect of 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin-derivative, in vitro. Here, we report on TH’s osteogenic effects ex vivo and in vivo. TH-induced new bone formation in both calvarial and metatarsal organ cultures. A novel monitoring system of osteoblastic differentiation using MC3T3-E1 cells revealed that TH was released from α-TCP bone cement and this release continued for more than one month. Lastly, the implantation of the α-TCP carrier containing TH into defects in mouse skull resulted in increased new bone areas within the defects after 4 weeks. A TH-containing scaffold may help establish a more efficient bone regeneration system.     

Brugia pahangi: Feeding and nutrient uptake in vitro and in vivo

Incubation in vitro of adult Brugia pahangi in an apparatus which permitted the separate exposure of the anterior, middle, or posterior region of the worms to medium-containing radioactively labeled d-glucose, l-leucine, and adenosine has provided evidence that these materials are taken up in physiologically significant amounts by a transcuticular route. No evidence for an oral ingestion of materials has been obtained from worms in vitro, but in vivo an oral uptake of Trypan blue has been demonstrated. The ultrastructure and cytochemical staining reactions for nonspecific esterase, acid phosphatase (EC-3.1.3.2), and leucine naphthylamidase of the gut and body wall are described.     

Inhibition of c-Jun N-terminal kinase increases apoE expression in vitro and in vivo

Apolipoprotein E (apoE), a ligand for the low-density lipoprotein receptor family, has been implicated in modulating glial inflammatory responses and the risk of neurodegeneration associated with Alzheimer’s disease. Glial cells activated by lipopolysaccharide (LPS) have decreased apoE levels and we recently showed that apoE itself can modulate the inflammatory response by reducing c-Jun N-terminal kinase (JNK) activation. Reduced JNK phosphorylation is vital to overcome the LPS-induced decrease in apoE expression, suggesting that JNK inhibition may be an effective way to increase apoE protein and protract its anti-inflammatory properties. This study investigates the impact of JNK inhibition on apoE production using two JNK inhibitors. Our work in primary glia and in vivo in mice injected with JNK inhibitor demonstrates that inhibition of JNK may be an effective way to increase apoE expression.     

Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.     

Knockdown of Akt isoforms by RNA silencing suppresses the growth of human prostate cancer cells in vitro and in vivo

The serine/threonine kinase Akt has three highly homologous isoforms in mammals: Akt1, Akt2, and Akt3. Recent studies indicate that Akt is often constitutively active in many types of human malignancy. Here we investigated the expression and function of Akt isoforms in human prostatic carcinoma cells. Initially, we used Western blotting to examine Akt expression in four human prostate cancer cell lines. Next, small-interfering RNAs (siRNAs) specific for Akt isoforms were used to elucidate their role on the in vitro and in vivo growth of prostate cancer cells. Expression of Akt1 and Akt2 was detected in all cells tested, but Akt3 was expressed only in cancer cells that did not express androgen receptors. All synthetic siRNAs against Akt isoforms suppressed their expression and inhibited the growth of cancer cells in vitro. Furthermore, atelocollagen-mediated systemic administration of siRNAs significantly reduced the growth of tumors that had been subcutaneously xenografted. These results suggest that targeting Akt isoforms could be an effective treatment for prostate cancers.     

Anticonvulsant, antidepressant-like activity of Abelmoschus manihot ethanol extract and its potential active components in vivo

Depression is the most common psychiatric comorbidity in patients with epilepsy. Searching for antiepileptic (anticonvulsant) and antidepressant-like medicines from natural products is very important for the treatment of this disease. The flower of Abelmoschus manihot (Linn.) Medicus has been reported to have neuroprotective effect against cerebral ischemia injury. In order to further explore the activity of Abelmoschus manihot on the central nervous system, the anticonvulsant and antidepressant-like effects of Abelmoschus manihot ethanol extract (AMEE) as well as its potential active components in vivo was investigated in the present study. It was found that AMEE could protect mice against PTZ-induced clonic convulsions and mortality. AMEE could also decrease immobility time in the FST in mice. Furthermore, the potential active components of AMEE in rat brain were identified by ultra performance liquid chromatography-mass spectrometer (UPLC-MS). Five parent components including isoquercitrin, hyperoside, hibifolin, quercetin-3′-O-glucoside, quercetin and three metabolites were detected in rat brain after administration of AMEE. In conclusion, eight flavonoids were identified in rat brain after administration of AMEE; meanwhile, these flavonoids might represent the potential bioactive components of AMEE and contribute to its anticonvulsant and antidepressant-like activity in vivo.     

Non-invasive in vivo measurement of the shear modulus of human vocal fold tissue

Voice is the essential part of singing and speech communication. Voice disorders significantly affect the quality of life. The viscoelastic mechanical properties of the vocal fold mucosa determine the characteristics of the vocal folds oscillations, and thereby voice quality. In the present study, a non-invasive method was developed to determine the shear modulus of human vocal fold tissue in vivo via measurements of the mucosal wave propagation speed during phonation. Images of four human subjects' vocal folds were captured using high speed digital imaging (HSDI) and magnetic resonance imaging (MRI) for different phonation pitches, specifically fundamental frequencies between 110 and 440 Hz. The MRI images were used to obtain the morphometric dimensions of each subject's vocal folds in order to determine the pixel size in the high-speed images. The mucosal wave propagation speed was determined for each subject and at each pitch value using an automated image processing algorithm. The transverse shear modulus of the vocal fold mucosa was then calculated from a surface (Rayleigh) wave propagation dispersion equation using the measured wave speeds. It was found that the mucosal wave propagation speed and therefore the shear modulus of the vocal fold tissue were generally greater at higher pitches. The results were in good agreement with those from other studies obtained via in vitro measurements, thereby supporting the validity of the proposed measurement method. This method offers the potential for in vivo clinical assessments of vocal folds viscoelasticity from HSDI.     

Temperature dependence of absorption and fluorescence spectra of bacteriochlorophylls in vivo and in vitro

The “short wave” far-red absorption bands (795–825 nm) of bacteriochlorophyll in photosynthetic red bacteria are sharpened but not shifted upon cooling, the “long wave” far-red bands (840–890 nm) are sharpened less but shifted appreciably towards longer wavelengths. The fluorescence bands are shifted about as much as the corresponding “long wave” absorption bands. Warming results in changes in the opposite direction. The temperature effects are reversible.