Transplantation and survival of mouse inner ear progenitor/stem cells in the organ of Corti after cochleostomy of hearing-impaired guinea pigs: preliminary results

In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.

Modified titration intratympanic gentamicin injection for unilateral intractable Ménière's disease / 华中科技大学学报（医学）（英德文版）

This study looked into the efficacy of a modified titration protocol of intratympanic gentamicin injection (ITG) in the patients with unilateral intractable Ménière's disease (MD). Modified titration protocol of ITG at a low dose (20 mg/mL) was administered to 10 patients with definite unilateral intractable MD. After initial first two fixed ITGs on weekly basis, the patients might or might not be given any more injections, depending on the appearance of unilateral vestibular loss (UVL). ITG was terminated if the patients satisfied the criteria of UVL. All patients were followed-up for at least two years. The effects of ITG on the vertigo attack, functional level scores and postural balance were evaluated. Of the 10 cases, 8 showed the sign of UVL after receiving initial two ITGs and were not given any more intratympanic injections, and the other 2 patients were administered three ITGs. A two-year follow-up revealed that complete and substantial vertigo control was achieved in 9 cases, and limited vertigo control in 1 patient. Hearing level was lowered in 2 patients. The posture stability and functional level scores were improved. Our study showed that the modified titration protocol of ITG at a low dose could effectively control vertigo in patients with unilateral intractable MD.

Roles of protein synthesis inhibitors in long-term potentiation and depotentiation in hippocampal CA1 region of adult rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the roles of protein synthesis inhibitors in long-term potentiation(LTP) and depotentiation(DP) in hippocampal CA1 region of adult rats.</p><p><b>METHODS</b>Standard extracellular recording technique was used to record field EPSP(fEPSP) evoked by Schaffer collateral stimulation from the CA1 subfield of adult rat hippocampal slices. Paired-pulse low-frequency stimulation(PP-LFS) or high-intensity paired-pulse low-frequency stimulation(HI-PP-LFS) was delivered to induce depotentiation 2 h after LTP induction induced by six theta-burst stimulations. Protein synthesis inhibitors were applied before and after LTP induction to study their roles in LTP and DP in hippocampal CA1 region of adult rats.</p><p><b>RESULTS</b>When HI-PP-LFS was applied at 2 h after LTP induction, the depotentiation was induced. The mean fEPSP slopes reduced from 346.2%±26.3% to 207.1%±21.6%. This depotentiation was named as partial LTP depotentiation and maintained at least for 30 min. The percentage of depotentiation was 59.81%. Application of protein synthesis inhibitors, anisomycin and cycloheximide prior to tetanus resulted in smaller LTP compared to control group, and almost complete depotentiation was induced by HI-PP-LFS. With application of protein synthesis inhibitors anisomycin and cycloheximide 90 min after LTP induction, HI-PP-LFS still induced partial LTP depotentiation. However, there was no significant difference in the percentage of depotentiation between this group and control group.</p><p><b>CONCLUSION</b>HI-PP-LFS partially reverses late phase LTP. When protein synthesis inhibitors are applied prior to tetanus, LTP amplitude is markedly reduced, and HI-PP-LFS completely reverses late-phase LTP. Application of protein synthesis inhibitors after LTP induction does not significantly affect either the amplitude or depotentiation of LTP.</p>

Effects of erythromycin on γ-glutamyl cysteine synthetase and interleukin-1β in hyperoxia-exposed lung tissue of premature newborn rats / Efeitos da eritromicina sobre a γ-glutamil-cisteína-sintetase e a interleucina-1β no tecido pulmonar exposto à hiperóxia de ratos recém-nascidos prematuros

Objective: To explore the effect of erythromycin on hyperoxia-induced lung injury. Methods: One-day-old preterm offspring Sprague-Dawley (SD) rats were randomly divided into four groups: group 1, air + sodium chloride; group 2, air + erythromycin;group 3, hyperoxia + sodium chloride; and group 4, hyperoxia + erythromycin. At one, seven, and 14 days of exposure, glutathione (GSH) and interleukin-1 beta (IL-1 beta) were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and bicinchoninic acid (BCA) was used to detect GSH protein. γ-glutamine-cysteine synthetase (γ-GCS) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results: Compared with group 1, expressions of GSH and γ-GCS mRNA in group 3 were significantly increased at one and seven days of exposure (p < 0.05), but expression of γ-GCS mRNA was significantly reduced at 14 days; expression of IL-1 beta in group 3 was significantly increased at seven days of exposure (p < 0.05), and was significantly reduced at 14 days. Compared with group 3, expressions of GSH and γ-GCS mRNA in group 4 were significantly increased at one, seven, and 14 days of exposure (p < 0.05), but expressions of GSH showed a downward trend at 14 days; expression of IL-1 beta in group 4 was significantly reduced at one and seven days of exposure (p < 0.05). Conclusions: Changes in oxidant-mediated IL-1 beta and GSH are involved in the development of hyperoxia-induced lung injury. Erythromycin may up-regulate the activity of γ-GCS, increasing the expression of GSH, inhibiting the levels of oxidant-mediated IL-1 beta and alleviating hyperoxia-induced lung injury via an antioxidant effect. .

Objetivo: Explorar o efeito da eritromicina sobre lesões pulmonares induzidas por hiperóxia. Métodos: Uma prole de ratos Sprague-Dawley (SD) prematuros com um dia de vida foi dividida aleatoriamente em quatro grupos: grupo 1 ar + cloreto de sódio, grupo 2 ar + eritromicina, grupo 3 hiperóxia + cloreto de sódio e grupo 4 hiperóxia + eritromicina. Com um, sete e 14 dias de exposição, foram detectadas Glutationa (GSH) e Interleucina-1 beta (IL-1 beta) pelo ensaio imunossorvente ligado à enzima (ELISA), e o ácido bicinconinico (BCA) foi utilizado para detectar a proteína GSH. O mRNA da γ-glutamil-cisteina-sintetase (γ-GCS) foi detectado por reação em cadeia da polimerase via transcriptase reversa (RT-PCR). Resultados: Comparadas ao grupo 1, as expressões do mRNA da GSH e da γ-GCS no grupo 3 aumentaram significativamente com um e sete dias de exposição (p < 0,05), porém a expressão de mRNA da γ-GCS diminuiu significativamente aos 14 dias; a expressão de IL-1 beta no grupo 3 aumentou significativamente aos 7 dias de exposição (p < 0,05) e diminuiu significativamente aos 14 dias. Comparadas ao grupo 3, as expressões do mRNA da GSH e da γ-GCS no grupo 4 aumentaram significativamente com um, sete e 14 dias de exposição (p < 0,05), porém as expressões de GSH mostraram uma tendência de queda aos 14 dias; a expressão de IL-1 beta no grupo 4 foi reduzida significativamente com um e sete dias de exposição (p < 0,05). Conclusões: As variações de IL-1 beta e GSH mediadas por oxidantes estão envolvidas no desenvolvimento de lesão pulmonar induzida por hiperóxia. A eritromicina poderá regular positivamente a atividade da γ-GCS, aumentando a expressão de GSH, inibindo os níveis de interleucina-1beta mediada por ...

Methanolic extract of Momordica cymbalaria enhances glucose uptake in L6 myotubes in vitro by up-regulating PPAR-γ and GLUT-4 / 中国天然药物

The present study was undertaken to evaluate the influence of the methanolic fruit extract of Momordica cymbalaria (MFMC) on PPARγ (Peroxisome Proliferator Activated Receptor gamma) and GLUT-4 (Glucose transporter-4) with respect to glucose transport. Various concentrations of MFMC ranging from 62.5 to 500 μg·mL(-1) were evaluated for glucose uptake activity in vitro using L6 myotubes, rosiglitazone was used as a reference standard. The MFMC showed significant and dose-dependent increase in glucose uptake at the tested concentrations, further, the glucose uptake activity of MFMC (500 μg·mL(-1)) was comparable with rosigilitazone. Furthermore, MFMC has shown up-regulation of GLUT-4 and PPARγ gene expressions in L6 myotubes. In addition, the MFMC when incubated along with cycloheximide (CHX), which is a protein synthesis inhibitor, has shown complete blockade of glucose uptake. This indicates that new protein synthesis is required for increased GLUT-4 translocation. In conclusion, these findings suggest that MFMC is enhancing the glucose uptake significantly and dose dependently through the enhanced expression of PPARγ and GLUT-4 in vitro.

Successful treatment of recurrent follicular B-cell lymphoma with clarithromycin, prednisolone, and cyclophosphamide

No abstract available.

Zebrafish model for the study on drug ototoxicity of aminoglycoside antibiotics / 药学学报

Aminoglycoside antibiotics, due to their strong antibacterial effects and broad antimicrobial spectra, have been very commonly used in clinical practice in the past half century. However, aminoglycoside antibiotics manifest severe ototoxicity and nephrotoxicity, and are one of top factors in hearing loss. In this study, three members of the aminoglycoside antibiotics family, gentamycin, neomycin and streptomycin, were chosen as the representatives to be investigated for their toxicity to the embryonic development and the larva hair cells in zebrafish, and also to their target genes associated with hearing-related genes. The results showed that: (1) the lethal effect of all three drugs demonstrated a significant dependence on concentration, and the severity order of the lethal effect was streptomycin > neomycin > gentamycin; (2) all the three drugs caused the larva trunk bending in resting state at 5 dpf (day past fertilization), probably due to their ototoxicity in the physical imbalance and postural abnormalities; (3) impairment and reducing of the hair cells were observed in all three cases of drug treatment; (4) four genes, eya1, val, otx2 and dlx6a, which play an important role in the development of hearing organs, showed differential and significant decrease of gene expression in a drug concentration-dependent manner. This study for the first time reports the relevance between the expression of hearing genes and the three ototoxic antibiotics and also proved the feasibility of establishing a simple, accurate, intuitive and fast model with zebrafish for the detection of drug ototoxicity.

[ effect of chloramphenicol on atropine and scopolamine contents in datura stramonium L]

In regard to the importance of atropine and scopolamine as medicines with natural source, this study was aimed to investigate the effect of chloramphenicol as an inhibitor of protein synthesis on the alkaloids content of Datura stramonium. After initial preparation of medicinal garden seeds, they were planted 50 cm apart in four different sections. Three different concentrations of chloramphenicol [100, 200 and 400 ppm] were sprayed on the plant once a week for a period of 18 weeks. Water was applied for the control group. Replicate samples were taken randomly every two weeks from each section. After extraction and separation of the samples, the quantitative analyses were carried out. The levels of atropine and scopolamine were determined using UV spectroscopy [¹³CNMR, UV, IR, Mass]. The maximum levels of alkaloids were observed in young stems of the plants. The best result was obtained following applying the concentration of 200 ppm, which caused a significant increase in atropine and scopolamine levels in all parts of plant by 100% and 110% respectively in comparison with the control group [p< 0.005]. Increase in the availability of amino acids may lead to an increase of alkaloids production following protein inhibition synthesis

Primary amebic meningoencephalitis.

We report an 8 month-old infant with primary amebic meningoencephalitis (PAME) due to Naegleria fowleri. The child was treated with amphotericin B, chloramphenicol and rifampicin for 3 weeks. PAME is an almost universally fatal condition with very few survivors till date. Our patient was one of the rare survivors who recovered after treatment and was discharged without any residual neurological deficit.

Rosette formation by macrophages with adhered T lymphocytes is precluded by inhibitors of antigen processing and presentation

We had previously found in autologous human leukocyte cultures, in which dead neutrophils phagocytosis by macrophages occur, macrophages and T CD4 lymphocytes perform a selective cell-cell interaction showing many figures of either one, two or several T- lymphocytes adhering to a central macrophage were seen. Considering that antigen presentation would be necessary for the formation of these immune synapses, we attempted to block rosette formation (i.e., the formation of macrophage associations with at least three lymphocytes) by interfering with both antigen processing and presentation. Culture samples of autologous leukocytes from 7 healthy donors were subjected to either brefeldin A, chloroquine or to an anti-HLA DR antibody. Rosette formation was significantly inhibited in the treated samples (either with brefeldin A, chloroquine or the anti- HLA DR; ANOVA, p<0.001, as compared with the untreated controls). It is concluded that interference with antigen processing and presentation precludes the formation of these cell-cell interactions.

Cloning and expression of antiviral/ribosome-inactivating protein from Bougainvillea xbuttiana.

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).

[ role of intrahippocampal microinjections of anisomycin on dexamethasone-induced modulation of memory consolidation in rats]

Evidence indicates that hippocampus and activation of glucocorticoid receptors in this area are necessary for emotional learning and memory processes; also some studies suggest that glucocorticoid's effects probably involve with processes of protein synthysis in the hippocmapus. The aim of this study was to determine the role of intrahippocampal microinjections of anisomycin [[AIMS] as a protein synthysis inhibitor]] on dexamethasone-induced modulation of memory consolidation in the passive avoidance learning [PAL] task in rats. In this study, 90 male Wistar rats [250-300 gr] were surgically implanted bilaterally with cannulae aimed at the dorsal hippocampus [DH] were trained in PAL task. In experiment 1. Dexamethasone [0.1, 0.5, 1 and 3 mg/kg IP] was injected immediately after training and vehicle injected into DH. In experiment 2. Anisomycin [0.5,1 micro g/ micro l/side] or vehicle were injected bilaterally into the DH followed immediately by IP injection of Dexamethasone [1 mg/kg] or vehicle. Two days after training, retention tests were done and step-through latency [STL] and total time spent in light chamber [TLC] of apparatus were recorded during 10 min and compared with controls. Data indicated that injection of Dexamethasone immediatly after training enhanced memory consolidation [P<0.01] and this effect was blocked by injection of ANS in to the DH [P<0.01]. The findings above showed that glucocorticoids play on important role in consolidation of emotional learning and probably in processes of protein synthesis in the hippocampus may play an important role in mediating these effects

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor gamma

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor gamma (PPAR gamma) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPAR gamma activity or knockdown of PPAR gamma expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPAR gamma through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPAR gamma siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress- induced gene expression by suppressing translation via activation of PPAR gamma in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPAR gamma.

Orbital rhabdomyosarcoma: a case series.

Orbital rhabdomyosarcoma is the most common orbital malignancy of childhood with the common presentation of rapidly evolving unilateral proptosis. We studied six patients who were diagnosed and treated for rhabdomyosarcoma between January 1999 and June 2004. The age of the patients ranged from 4 to 29 years. Four patients presented with acute onset proptosis associated with signs of inflammation, mimicking orbital cellulitis. One patient presented with lid mass. Another patient presented with a soft, blind eye that was pushed superotemporally by a large inflammed, vascularised mass. Embryonal rhabdomyosarcoma was the commonest histopathological type in our series found in five patients. One patient was completely cured with chemotherapy alone whereas two patients were treated with a combination of chemotherapy and radiotherapy. Three patients in our series required exenteration.

Chloramphenicol: screening and review to evaluate its potential beneficial effects in leukaemia.

Chloramphenicol is an antimicrobial agent having a very broad-spectrum of activity including Gram-positive bacteria, Gram-negative bacteria and anaerobes. However the use of chloramphenicol has reduced over a period of time due to the adverse effects of causing bone marrow depression or in some cases severe aplastic anaemia. As the effects are seen on the bone marrow cell, it was intended to find out if these adverse effects could be used for the benefits in leukaemia patients, using in-vitro study on leukaemic cell lines. The study showed inhibition of growth of the leukaemia cells by chloramphenicol which was comparable to or better than daunorubicin in some cell lines. The article also discusses the other adverse effect profile of chloramphenicol compared with anticancer drugs and its potential benefit in leukaemia and in neutropenic fever.

Parâmetros da degradação protéica ruminal de diferentes alimentos e rações estimados por técnica in vitro / Parameters of ruminal protein degradation of different feeds and diets estimated by an in vitro method

Foram realizados três experimentos para estudar os parâmetros de degradação protéica ruminal. No primeiro, foram incubadas, em líquido ruminal de bovinos, dietas isoprotéicas contendo capim-elefante, fubá de milho e farelo de soja, em cinco níveis de concentrado (0:100, 25:75, 50:50, 75:25 e 100:0), adicionado ou não de monensina (5µM). Houve efeito linear decrescente do nível de concentrado sobre a concentração de amônia e degradabilidade da proteína bruta (DPB), e efeito cúbico sobre a concentração de proteína solúvel, com máximo valor em dieta com 25 por cento de concentrado. A monensina diminuiu a DPB e a concentração de proteína solúvel, sem afetar a produção de amônia. No segundo experimento, foram incubados cinco diferentes volumosos (silagens de milho - Zea mays L. e de capim-elefante - Pennisetum purpureum, silagem pré-secada de braquiária -Brachiaria decumbens, feno de Tifton 85 -Cynodon sp. amonizado e feno de Tifton 85). A silagem pré-secada de capim-braquiária e o feno de Tifton 85 amonizado apresentaram as maiores concentrações de amônia (8,7 e 5,3mg/dl) e proteína solúvel (5,4 e 7,0mg/dl), devido aos seus maiores teores de PB, seguidos da silagem de capim-elefante e feno de Tifton 85. A DPB variou de 29,6 a 80,6 por cento, para a silagem pré-secada de braquiária e para o feno de Tifton 85, e a degradabilidade potencial da matéria seca de 40,1 a 64,3 por cento, para a silagem de capim-elefante e silagem pré-secada de braquiária, respectivamente. A degradabilidade efetiva da proteína bruta apresentou baixos valores devido à baixa taxa de degradação da fração insolúvel. No terceiro experimento, foram incubados diferentes tipos de camas de frango (casca de café, capim-elefante seco picado, sabugo de milho ou cepilho), contendo ou não monensina (5µM). Não houve diferença nas concentrações de amônia entre as diferentes camas de frango, na ausência de monensina. Entretanto, com monensina, a cama de capim-elefante apresentou o...

Three experiments were carried out in order to study the parameters of ruminal protein degradation. In the first, isoproteic diets, constituted of elephant grass, ground corn and soybean meal, at five concentrate levels (0:100, 25:75, 50:50, 75:25 e 100:0), with or without monensin (5µM), were incubated in ruminal fluid of bovines. There was a decreasing linear effect of the concentrate level on ammonia concentration and degradability of crude protein, and cubic effect in soluble protein concentration, with the largest value in the diet with 25 percent concentrate. Monensin decreased degradability of crude protein and soluble protein concentration with no effect on ammonia production. In the second, five different roughages (corn and elephant grass silage - Pennisetum purpureum, Brachiaria haylage - Brachiaria decumbens, ammoniated Tifton 85 hay - Cynodon sp. e Tifton 85 hay). The were incubated Brachiaria haylage and the ammoniated Tifton 85 hay showed the greatest concentrations of ammonia (8.7 and 5.3mg/dl) and soluble protein (5.4 and 7.0mg/dl), due to their higher crude protein content, followed by elephant grass silage and Tifton 85 hay. The degradability of crude protein ranged from 29.6 to 80.6 percent for Brachiaria haylage and Tifton 85 hay, and the degradability of dry matter ranged from 40.1 to 64.3 percent for elephant grass silage and Brachiaria haylage, respectively. The effective degradability of crude protein showed low values due to low degradation rate of the insoluble fraction. In the third, four different poultry litter (hulls coffee, shopped dry elephant grass, corn cobs and wood shavings) were incubated, with or without monensin (5µM). No difference in ammonia concentration among the poultry litter samples, was observed in the absence of monensin. However, when monensin was present, the grass poultry litter showed the lowest ammonia level and wood poultry litter the highest. The poultry litter influenced the soluble...

Determination of micronucleus frequency by acridine orange fluorescent staining in peripheral blood reticulocytes of mice treated topically with different lubricant oils and cyclophosphamide

To ascertain whether used and re-refined lubricant oil absorbed through the skin can produce a genotoxic effect or cytotoxicity in mouse bone marrow cells, we examined the induction of micronucleated erythrocytes of peripheral blood after cutaneous application. Both re-refined and used lubricant oils showed a weak but significant induction of micronucleated polychromatic erythrocytes compared with control, while virgin oil did not show micronucleus induction. Cyclophosphamide (CP) was used not only as positive control but also to compare the sensitivity between intraperitoneal and dermal routes of administration of the test compounds in mice. The efficacy of intraperitoneal injection of CP is well known. On the other hand, dermal exposure is not so common and when CP was diluted in glycerin statistically significant values (P = 0.0036) of micronuclei were also found. Topically applied lubricant oils (virgin, re-refined and used) have the capacity to interfere with mouse bone marrow hematopoiesis evidenced by a statistically significant decrease in the proportion of polychromatic erythrocytes in the peripheral blood. Physical and chemical analysis revealed that used oil is more viscous than other lubricants, suggesting the presence of insoluble compounds, oxidized products and water as well as aromatic hydrocarbons. Used oil differs from other lubricant oils in metal and polyaromatic hydrocarbon content. Re-refined oil revealed a neutral value typical of pure mineral oil. This assay is an important tool to evaluate environmental pollutants that cause genotoxicity and/or cytotoxicity through skin exposure.

Effect of N-tosyl-L-phenylalnylchloromethyl ketone and dexamethasone on expression of nuclear transcription factor-kappaB in childhood acute lymphoblastic leukemia and its significance / 中国实验血液学杂志

In order to investigate the effect of N-tosyl-L-phenylalnylchloromethyl ketone (TPCK) and dexamethasone (Dex) on expression of nuclear transcription factor-kappaB (NF-kappaB) in childhood acute lymphoblastic leukemia (ALL) and its significance, so as to provide the experimental basis for corresponding clinical treatment of ALL, in which NF-kappaB is taken as a target. The biotin-streptavidin method was used to detect the expression of NF-kappaB P65 protein and the effects of TPCK and Dex at clinically relevant dosage on activity of NF-kappaB P65 protein in 20 childhood ALL patients. The results indicated that the expression of NF-kappaB P65 protein was strongly diminished and reached to negative level at 2 hours by treatment with 40 micromol/L TPCK, the positive expression of NF-kappaB P65 protein was (2.5 +/- 1.6)%. TPCK had a time-dependent inhibitory effect on ALL cells cultured in vitro. The expression of NF-kappaB P65 protein in ALL cells was strongly inhibited by clinically relevant concentration of dexamethasone 5.0 microg/ml for 24 hours in vitro. The positive expression was (25.0 +/- 3.0)%, there was significant difference, as compared with untreated ALL cells (T=55, P<0.01). It is concluded that TPCK and Dex can inhibit NF-kappaB activity. Inhibition of NF-kappaB activity may be one of the effect mechanism of dexamethasone on ALL cells. Inhibition of NF-kappaB conduction pathway may have a significant value in childhood ALL treatment.

Subcellular localization and identification of hydrogenase isolated from the marine green alga Platymonas subcordiformis using immunoprecipitation and MALDI-TOF MS / 生物工程学报

A marine unicellular green alga, Platymonas subcordiformis, was demonstrated to photobiologically produce hydrogen gas from seawater. The objective of this study was to localize and identify the hydrogenase isolated from P. subcordiformis. Adaptation in the presence of inhibitors of protein biosynthesis indicated that the hydrogenase was much more inhibited by cycloheximide than that by chloramphenicol. The result suggested that the hydrogenase isolated from P. subcordiformis is probably synthesized in cytoplasmic ribosomes. Both Western blot analysis and immunogold electron microscopy demonstrate that the P. subcordiformis hydrogenase is mainly located in the chloroplast stroma. The proteins that reacted specifically with the antibodies against the iron hydrogenase isolated from Chlamydomonas reinhardtii were concentrated by immunoprecipitation. The separated protein bands were cut out of the SDS-PAGE gel, in-gel digested by trypsin, and analyzed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Mascot was employed for analysis of the MALDI data using the public databases NCBInr. The hydrogenase isolated from P. subcordiformis was identified to be the Fe-hydrogenase.