Anti-tumor Efficacy and Safety of Zeylenone on Acute T Lymphoblastic Leukemia / 中国实验方剂学杂志

Objective:To investigate the anti-tumor efficacy, mechanism and safety of zeylenone on acute T lymphocytic leukemia. Method:In vitro, Molt-4 cells were treated with various concentrations of zeylenone (0.2, 0.4, 0.8, 1.6, 3.2 μmol·L-1) for 48 h, and the cell viability was measured with cell counting kit-8 (CCK-8) assay. nonobese diabetic-severce combined immunodeficient mice（NOD/SCID） mice were randomly divided into six groups: normal group, model group, vincristine group (1 mg·kg-1), low-dose zeylenone group (12.5 mg·kg-1), medium-dose zeylenone group (25 mg·kg-1), high-dose zeylenone group (50 mg·kg-1). With the exception of normal group, mice were pre-irradiated with 60Co and inoculated subcutaneously with Molt-4 cells to establish the Molt-4 xenograft model. Then NOD/SCID mice were sacrificed after 13 days of administration. The tumor inhibition rates, relative tumor growth rates and organ indexes were calculated. Hematoxylin and eosin (HE) staining was used to observe the pathological changes of liver and spleen tissues in mice. The expressions of phosphorylation signal transducer and activator of transcription (p-STAT3), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and cysteine aspartate-specific protease-3 (Caspase-3) were detected in tumor tissues by Western blot. Result:In vitro, zeylenone had an obvious inhibitory effect on Molt-4 cells. IC50 values of zeylenone was 1.49 μmol·L-1. In vivo, compared with the model group, medium and high-dose zeylenone groups had significant tumor inhibition effects, with the inhibition rates of 50.24% and 60.75%, respectively (P<0.01). Additionally, liver and spleen injuries were slight in the above mentioned two groups compared with the vincristine group, indicating that zeylenone was safe. Western blot analysis showed that medium and high-dose zeylenone groups showed significant declines in proteins p-STAT3, Caspase-3 and Bcl-2, and marked increases in pro-apoptotic protein Bax compared with the model group (P<0.05, P<0.01). Conclusion:zeylenone could obviously inhibit the proliferation and induce the apoptosis of Molt-4 cells; and its mechanism may be related to the down-regulation of p-STAT3, Caspase-3, Bcl-2 and the up-regulation of Bax expressions. In addition, zeylenone had less damage to liver and spleen, and was safer than vincristine.

β-arrestin1 overexpression suppresses progression of human T-cell acute lymphatic leukemia Molt-4 cell xenograft in mice / 南方医科大学学报

OBJECTIVE@#To investigate the effect of β-arrestin1 overexpression on tumor progression in a NCG mouse model bearing T-cell acute lymphocytic leukemia (T-ALL) Molt-4 cell xenograft.@*METHODS@#Molt-4 cells were tagged with firefly-luciferase (F-Luc) by lentiviral infection, and fluorescence intensity of the cells was detected using a luminescence detector. Molt-4 cell lines with β-arrestin1 overexpression or knockdown were constructed by lentivirus infection and injected the tail vein in sub-lethal irradiated NCG mice. Body weight changes and survival time of the xenografted mice were observed, and the progression of T-ALL in the mice was evaluated using an fluorescence imaging system. Sixteen days after xenografting, the mice were euthanatized and tumor cell infiltration was observed in the slices of the liver and spleen.@*RESULTS@#We successfully tagged Molt-4 cells with F-Luc and overexpressed or knocked down β-arrestin1 in the tagged cells. Bioluminescent imaging showed obvious luminescence catalyzed by F-Luc in Molt-4 cells. After injection of Molt-4-Luc cells into irradiated NCG mice, a gradual enhancement of luminescence in the xenografted mice was observed over time, while the body weight of the mice decreased. Compared with the control mice, the mice xenografted with β-arrestin1-overexpressing Molt-4 cells had significantly prolonged survival time ( < 0.001), while the survival time of the mice xenografted with Molt-4 cells with β- arrestin1 knockdown was significantly shortened ( < 0.001). Histological examination revealed fewer infiltrating tumor cells in the liver and spleen of the mice xenografted with β-arrestin1-overexpressing Molt-4 cells in comparison with the mice bearing parental Molt-4 cell xenografts.@*CONCLUSIONS@#β-arrestin1 overexpression suppresses tumor progression in mice bearing Molt-4 cell xenograft.

In vitro Antiproliferative activity of Melissa officinalis L. (Lamiaceae) leaves essential oil / Actividad antiproliferativa in vitro de aceite esencial de hojas de Melissa officinalis L (Lamiaceae)

In the present study, we investigated the antiproliferative activity of essential oil from leaves of Melissa officinalis L. grown in Southern Bosnia and Herzegovina. In vitro evaluation of antiproliferative activity of the M. officinalis essential oil was carried out on three human tumor cell lines: MCF-7, NCI-H460 and MOLT-4 by MTT assay. M. officinalis essential oil was characterized by high percentage of monoterpenes (77,5%), followed by the sesquiterpene fraction (14,5%) and aliphatic compounds (2,2%). The main constituents of the essential oil of M. officinalis are citral (47,2%), caryophyllene oxide (10,2%), citronellal (5,4%), geraniol (6,6%), geranyl acetate (4,1%) and ß- caryophyllene (3,8%). The essential oil showed significant antiproliferative activity against three cancer cell lines, MOLT-4, MCF-7, and NCI-H460 cells, with GI50 values of <5, 6±2 and 31±17 µg/mL, respectively. The results revealed that M. officinalis L. essential oil has a potential as anticancer therapeutic agent.

En el presente estudio, investigamos la actividad antiproliferativa del aceite esencial de las hojas de Melissa officinalis L. cultivadas en el sur de Bosnia y Herzegovina. La evaluación in vitro de la actividad antiproliferativa del aceite esencial de M. officinalis se llevó a cabo en tres líneas celulares de tumores humanos: MCF-7, NCI-H460 y MOLT-4 utilizando el ensayo de MTT. El aceite esencial de M. officinalis se caracterizó por un alto porcentaje de monoterpenos (77,5%), seguido de la fracción sesquiterpénica (14,5%) y compuestos alifáticos (2,2%). Los principales constituyentes del aceite esencial de M. officinalis fueron citral (47,2%), óxido de cariofileno (10,2%), citronelal (5,4%), geraniol (6,6%), acetato de geranilo (4, 1%), y ß-cariofileno (3,8%). El aceite esencial mostró una actividad antiproliferativa significativa contra las líneas celulares de cáncer MOLT-4, MCF-7 y NCI-H460, con valores GI50 de <5, 6±2 y 31±17 µg/mL, respectivamente. Los resultados revelaron que el aceite esencial de M. officinalis L. tiene potencial como agente terapéutico contra el cáncer.

New Records and Range Extensions for Birds from the Western Slope of the Eastern Andes, Santander, Colombia / Nuevos registros y ampliación de distribución de aves en la vertiente occidental, cordillera oriental, santander, colombia

RESUMEN Las aves de la vertiente occidental de la Cordillera Oriental en Santander han sido poco estudiadas. La Serranía de los Yariguíes y zonas aledañas, al norte del municipio de El Peñón, han sido muestreados en la última década con nuevas subespecies descritas y ampliaciones de distribución. Estudiamos las aves de la parte alta del municipio de El Peñón entre los 2750 a 2850 m s.n.m. durante ocho días de campo en agosto de 2016, mediante captura con redes de niebla y observaciones visuales y auditivas en bosques de roble. Registramos 77 especies de aves pertenecientes a 13 órdenes y 29 familias. La familia mejor representada fue Tyrannidae (Atrapamoscas) con 12 especies, seguida por Trochilidae (Colibríes) con nueve y Thraupidae (Fruteros y afines) con ocho. Registramos la ampliación de distribución geográfica para la endémica Pyrrhyra calliptera para el flanco occidental de la Cordillera Oriental, la segunda localidad para Macroagelaius subalaris para este flanco y al sur de los registros conocidos de la Serranía de los Yariguíes, ampliaciones menores o confirmación de presencia para 18 especies y otras 11 poco conocidas para este flanco. Casi el 45 % de los individuos capturados presentaron indicios de actividad reproductiva (parches de incubación o gónadas desarrolladas). Así mismo, 50 % de los individuos se encontraban mudando el plumaje, donde 15 % presentaron solapamiento entre muda y reproducción. Aunque comparte especies con los bosques al norte y sur, los bosques de roble remanentes están en buen estado e inmersos entre ecosistemas cársticos poco conocidos.

ABSTRACT Birds from the western slope of the East Andean Cordillera at Santander have been little studied. Serranía de los Yariguies and nearby areas, just north of El Peñon, have been studied in the last decade with new subspecies described and new range extensions. We studied birds from oak forests from the upper part of the municipality of El Peñon between 2750 and 2850 m a.s.l during eight field days during August 2016. Surveys included vocal and visual observations and mist netting. We report 77 species belonging to 13 different Orders and 29 Families. The best-represented family was Tyrannidae (Flycatchers) with 12 species, followed by Trochilidae (Hummingbirds) with nine and Thraupidae (fruit eaters and allies) with eight. Mayor range extensions were made for the endemic Pyrrhura calliptera - Brown-breasted Parakeet from the eastern to the western flack, confirmed a second locality for Macroagelaius subalaris-Colombian Mountain Grackle south of previously known Serranía de los Yariguies, minor range extensions or confirmed presence records for 18 species and new information for 11 poorly known species for this flank. Almost 45 % of all captured individuals showed reproductive activity (brood patches or developed gonads). 50 % of individuals were molting feathers, where 15 % showed some overlap between reproduction and molt. Even though species are shared with other forests to the north and south, the remnant oak forests are in good condition and immersed in karst ecosystems poorly known.

Comparison of hemocytes of V-instar nymphs of Rhodniusprolixus (Stål) and Rhodnius robustus (Larousse 1927), before and after molting / Comparación de los hemocitos de las ninfas de V instar de Rhodnius prolixus (Stål) and Rhodnius robustus, antes y después de la muda (Larousse 1927)

Background. The immune response of insects involves humoral factors and cellular elements known as hemocytes. There are different reports that explore the response of hemocytes to infections, but the effect that molting has on this response has not been explored so far. We hypothesized that there would be a change in the percentage of hemocytes as a response to the molting process. Objective. The aim of this work was to compare the hemogram (CBC), the formula, and the differential count of hemocytes in IV instar before molting and in V instar nymphs 24 hours after molting in two species: Rhodnius prolixus and Rhodnius robustus. Materials and methods. We assayed different staining methodologies including Giemsa, Alcian Blue pH 2.0, Alcian Blue pH 2.6, Gomori substrate, PAS (Schiff), Sudan Black and Papanicolau with positive controls for each one. In the Gomori staining, we observed lysosomes in the granulocytes and plasmatocytes, but the differentiation was better detected using Giemsa staining. Results. There were no statistically significant differences between the two species studied in plasmatocytes (p=0,053) or even in granulocytes (p=0,5). However, differences were significant in the prohemocytes (p=0,001) during the molting process in both Rhodnius prolixus and Rhodnius robustus. Conclusions. Significant differences in prohemocytes between nymphs of IV and V instar were detected. No significant differences in the amount of cells were observed between the two species and the two stages. These findings may be explained due to their role as precursor cell of prohemocytes.

Antecedentes. La respuesta inmune de los insectos involucra factores humorales y elementos celulares llamados hemocitos. Existen varios trabajos explorando la respuesta de los hemocitos frente a infecciones, pero no como respuesta al proceso de muda. Objetivo. Comparar el hemograma: la fórmula y el recuento diferencial de hemocitos de Rhodnius prolixus y Rhodnius robustus en ninfas de IV estadio antes de la muda y ninfas de V estadio 24 horas después de la muda. Materiales y métodos. Se ensayaron las coloraciones Giemsa, Alcian Blue pH 2.0, Alcian Blue pH 2.6, sustrato Gomori, PAS (Schiff), Sudán Negro, Papanicolau, con controles positivos para cada una. Con la coloración de Gomori se observaron lisosomas en los granulocitos y en plasmatocitos, pero la diferenciación se logró mejor con la coloración de Giemsa. Resultados. Los plasmatocitos fueron más abundantes en las ninfas de IV estadio de Rhodnius robustus y en las de V estadio de las dos especies, pero en las ninfas de V estadio de R. prolixus, el recuento de prohemocitos fue mayor; para los oenocitoides, esferulocitos y adipohemocitos el recuento no alcanzó al 1 %. En las dos especies no se encontraron diferencias estadísticamente significativas en plasmatocitos (p=0,05319), ni en granulocitos (p=0,5), entre las ninfas de IV y V estadio, pero fue significativa en prohemocitos (p=0,001). Conclusiones. Se detectaron diferencias significativas en prohemocitos entre las ninfas de IV y V estadio. No hubo un aumento significativo en el conteo de células en los diferentes estadios.

Supplementation with humic substances in laying hens during molt / Suplementação com substâncias húmicas em galinhas poedeiras durante a fase após muda / Suplementación con sustancias húmicas en gallinas ponedoras durante la fase posmuda

Humic substances (HS) have been used in the last two decades as a supplement in animal feed to improve performance and physiological processes that are reflected in the quality of products. 120 Hy Line Brown hens were supplemented with HS during the first 60 days of the molting stage. The experimental design was completely randomized. The birds were divided into four groups, each with 30 hens. The first and second groups were supplemented with 0.1 and 0.2% HS, respectively. The third group was supplemented with 0.25 mg/kg levamisole hydrochloride. The fourth group received no supplementation. The effect of humic substances on production parameters and egg quality (laying percentage, daily mortality, feed conversion ratio, shell thickness, albumen thickness, and yolk color) was evaluated. Mortality was not related to the experiment. HS supplementation resulted in no significant differences on hen weight, laying percentage, egg weight, or feed conversion ratio (p> 0.05). However, HS supplementation improved egg quality in terms of increased shell thickness and albumin at days 30 and 60 post-molt.

En las últimas dos décadas, las sustancias húmicas (SH) han sido utilizadas como suplemento en la alimentación animal con el propósito de mejorar los parámetros productivos y algunos procesos fisiológicos que se reflejan en la calidad de los productos. En el presente estudio se suplementaron 120 gallinas Hy Line Brown con SH durante los primeros 60 días de la etapa posmuda, en un diseño experimental completamente aleatorizado. Las aves fueron divididas en cuatro grupos, cada uno con 30 gallinas. El primer y segundo grupo fueron suplementados con 0,1 y 0,2% de SH respectivamente, el tercer grupo fue suplementado con 0,25 mg/kg de clorhidrato de levamisol y el cuarto grupo no tuvo suplementación. Se evaluó el efecto de las sustancias húmicas sobre parámetros de producción y de calidad de huevo (porcentaje de producción, mortalidad diaria, tasa de conversión alimenticia, grosor de la cáscara, grosor de la albúmina y color de la yema respectivamente). La mortalidad registrada no se relacionó con el experimento. La suplementación con SH no presentó diferencia significativa sobre el peso de las gallinas, el porcentaje de producción, el peso del huevo y la tasa de conversión alimenticia (p>0,05). No obstante, cuando se suplementó con SH la calidad de los huevos mejoró en términos de aumento en el grosor de la cáscara y de la albúmina en el día 30 y 60 posmuda.

Nas últimas duas décadas, as substancias húmicas ( SH) tem sido utilizadas como suplemento na alimentação animal com o propósito de melhorar os parâmetros produtivos e alguns processos fisiológicos que se refletem na qualidade dos produtos. No presente estudo se suplementaram 120 galinhas Hy Line Brown com SH durante os primeiros 60 dias da etapa após muda, se fez um desenho completamente ao acaso. As aves foram divididas em quatro grupos, cada um com 30 galinhas. O primeiro e o segundo grupo foram suplementados com 0,1 e 0,2% de SH respectivamente, o terceiro grupo foi suplementado com 0,25 mg/kg de cloridrato de levamisol e o quarto grupo não teve suplementação. Avaliou-se o efeito das substancias húmicas sobre os parâmetros de produção e da qualidade do ovo (percentagem de produção, mortalidade diária, taxa de conversão alimentar, grosso da casca, grosso da albumina e coloração da gema respectivamente). A mortalidade registrada não se relaciono com o experimento. A suplementação com SH não apresentou diferença significativa sobre o peso das galinhas, a porcentagem de produção, o peso do ovo e a taxa de conversão alimentar (p>0,05). No entanto, quando se suplementou com SH a qualidade dos ovos melhorou em termos de aumentar o grosso da casca e da albumina no dia 30 e 60 após muda.

Binding pattern of 125Iodine thyroxine and tri-iodothyronine in skin and liver tissues of spotted munia, Lonchura punctulata: Co-relation to seasonal cycles of breeding and molting.

Prevalent notion about thyroid hormones is that thyroxine (T4) is a mere precursor and physiological effects of thyroid hormones are elicited by tri-iodothyronine (T3) after mono-deiodination of T4. Earlier studies on feather regeneration and molt done on spotted munia L. punctulata suggest that T4 (mono-deiodination suppressed by iopanoic acid and thyroidectomized birds) is more effective than T3 in inducing feather regeneration. The binding pattern of 125I labeled T4 and T3 has been investigated in the nuclei prepared from skin and liver tissues (samples obtained during different months) of spotted munia using scatchard plot analysis. The results show that binding capacity (Bmax – pmole/80 µgm DNA) of 125I-T3 to nuclei of skin was significantly higher in November as compared to April and June, whereas the binding affinity (Kd-10-9M-1) was significantly lower in November as compared to April and June. During November, Bmax for binding of T3 and T4 did not vary in liver and skin nuclei but Kd varied significantly. Binding capacity of 125I- T3 to skin and liver did not vary but binding affinity of 125I- T4 to skin was approximately 7 times higher than that of liver. The results suggest that T4 does show a variation in binding pattern that co-relates to the molting pattern of spotted munia. These variations might play important role in different physiological phenomenon in this tropical bird. The experiments do point towards the possibility of independent role of T4 as a hormone, however, further experiments need to be done to ascertain the role of T4 in this model and work out the exact molecular mechanism of action.

Pharmacophore based approach to design inhibitors in Crustaceans: An insight into the molt inhibition response to the receptor guanylyl cyclase.

The first set of competitive inhibitors of molt inhibiting hormone (MIH) has been developed using the effective approaches such as Hip-Hop, virtual screening and manual alterations. Moreover, the conserved residues at 71 and 72 positions in the molt inhibiting hormone is known to be significant for selective inhibition of ecdysteroidogenesis; thus, the information from mutation and solution structure were used to generate common pharmacophore features. The geometry of the final six-feature pharmacophore was also found to be consistent with the homology-modeled MIH structures from various other decapod crustaceans. The Hypo-1, comprising six features hypothesis was carefully selected as a best pharmacophore model for virtual screening created on the basis of rank score and cluster processes. The hypothesis was validated and the database was virtually screened using this 3D query and the compounds were then manually altered to enhance the fit value. The hits obtained were further filtered for drug-likeness, which is expressed as physicochemical properties that contribute to favorable ADME/Tox profiles to eliminate the molecules exhibit toxicity and poor pharmacokinetics. In conclusion, the higher fit values of CI-1 (4.6), CI-4 (4.9) and CI-7 (4.2) in conjunction with better pharmacokinetic profile made these molecules practically helpful tool to increase production by accelerating molt in crustaceans. The use of feeding sub-therapeutic dosages of these growth enhancers can be very effectively implemented and certainly turn out to be a vital part of emerging nutritional strategies for economically important crustacean livestock.

Potentiation of a novel palladium (II) complex lethality with bee venom on the human T-cell acute lymphoblastic leukemia cell line (MOLT-4)

Background : Although honeybee venom (BV) has been reported to induce apoptosis in different types of cancerous cells, its synergistic effects with customary anti-cancer drugs remain largely unknown. In the present study, we evaluated the cytotoxic effect of BV alone (as a natural product) and the synergistic cytological effects of this component in combination with [Pd (bpy) (Pi-Pydtc)]NO3 - a novel palladium complex on human T-cell lymphoblastic leukemia cells. To investigate the cytotoxic effect of the BV alone and in combination with palladium complex on MOLT-4 cells MTT assay was performed. In order to determine the apoptotic effects of BV separately and in combination with Pd (II) complex on these cells and its ability to induce apoptosis, morphological examination, flowcytometric analysis and caspase-3 colorimetric assay were done. Results : We found that BV induced morphological changes, namely nuclear shrinkage, and inhibited MOLT-4 cell proliferation; both effects were dose- and time-dependent. Flow cytometry by Annexin-V antibody demonstrated that BV induced apoptosis in MOLT-4 cells. Furthermore, BV induced apoptosis independently of caspase-3 in these cells. In addition, we proved a clear synergistic effect of BV on [Pd (bpy) (Pi-Pydtc)]NO3. The apoptotic pathway activated by BV in combination with Pd complex was caspase-3-dependent. Conclusions : These observations provide an explanation for the anti-proliferative properties of BV, and suggest that this agent may be useful for treating lymphoblastic leukemia alone or in combination with chemotherapy drugs pending further investigations on animal models as preclinical tests.

Potentiation of a novel palladium (II) complex lethality with bee venom on the human T-cell acute lymphoblastic leukemia cell line (MOLT-4)

Background : Although honeybee venom (BV) has been reported to induce apoptosis in different types of cancerous cells, its synergistic effects with customary anti-cancer drugs remain largely unknown. In the present study, we evaluated the cytotoxic effect of BV alone (as a natural product) and the synergistic cytological effects of this component in combination with [Pd (bpy) (Pi-Pydtc)]NO3 - a novel palladium complex on human T-cell lymphoblastic leukemia cells. To investigate the cytotoxic effect of the BV alone and in combination with palladium complex on MOLT-4 cells MTT assay was performed. In order to determine the apoptotic effects of BV separately and in combination with Pd (II) complex on these cells and its ability to induce apoptosis, morphological examination, flowcytometric analysis and caspase-3 colorimetric assay were done. Results : We found that BV induced morphological changes, namely nuclear shrinkage, and inhibited MOLT-4 cell proliferation; both effects were dose- and time-dependent. Flow cytometry by Annexin-V antibody demonstrated that BV induced apoptosis in MOLT-4 cells. Furthermore, BV induced apoptosis independently of caspase-3 in these cells. In addition, we proved a clear synergistic effect of BV on [Pd (bpy) (Pi-Pydtc)]NO3. The apoptotic pathway activated by BV in combination with Pd complex was caspase-3-dependent. Conclusions : These observations provide an explanation for the anti-proliferative properties of BV, and suggest that this agent may be useful for treating lymphoblastic leukemia alone or in combination with chemotherapy drugs pending further investigations on animal models as preclinical tests.(AU)

Effect of bortezomib combined with doxorubicin on lymphoblastic lymphoma Molt-4 cells / 白血病·淋巴瘤

Objective To investigate the effect of bortezomib combined with doxorubicin on lymphoblastic lymphoma cell line Molt-4.Methods Molt-4 cells were cultured in the presence of bortezomib and doxorubicin,cell viability was monitored by CCK-8 and trypan-blue exclusion.Apoptosis was detected by flow cytometry and mitochondrial membrane potential,expression of Fas was also measured with flow cytometry.Results Molt-4 cell proliferation was substantially inhibited in concentration-dependent manners when treated with either bortezomib or doxorubicin.The combination of both drugs synergistically inhibited Molt-4 cell proliferation at 48 hours [(57.24±0.10) ％].Combination therapy further enhanced bortezomib and doxorubicin induced apoptosis [48 h (23.08±1.25) ％] (P ＜ 0.05).Detection of mitochondrial membrane potential showed that combination therapy could promote apoptosis (15.84 ％,5.38 ％,5.52 ％) but did not significantly change the level of Fas expression (P ＞ 0.05).Conclusion Combination therapy of bortezomib with doxorubicin efficiently inhibits proliferation and induces apoptosis of Molt-4 cells.Activation of mitochondrial and intrinsic apoptotic pathway may play important roles.

Different pathways in the larval development of the crab Ucides cordatus (Decapoda, Ocypodidae) and their relation with high mortality rates by the end of massive larvicultures / Diferentes rotas de desenvolvimento larval do caranguejo Ucides cordatus (Decapoda, Ocypodidae) e sua relação com altas taxas de mortalidade no fim de larviculturas massivas

One of the most limiting factors affecting the larval rearing of Ucides cordatus in the laboratory is a period of high mortality, which usually occurs late in the course of the larviculture during the metamorphosis from the zoeal to the megalopal phase. The objective of the present research was to analyze the post-embryonic development of U. cordatus on an individual basis and, in particular, to search for patterns linking disturbances in the molting process to the high larval death rates observed in massive larvicultures. A total of 50 larvae were individually reared from hatching to metamorphosis into the megalopal phase under controlled conditions, fed a combination of microalgae and rotifers. The survivorship rate was 70% until zoea V. The 35 surviving zoea V larvae followed two different pathways. Eleven underwent metamorphosis directly to megalopa, eighteen molted to zoea VI and six died as zoea V. In the last molting event, only two zoea VI larvae reached the megalopal stage, while the remaining sixteen died. In further observation under microscope, 13 of the dead zoea VI showed characteristics of the pre-molt stage and pereiopods disproportionably large in relation to the carapace. The observed pattern resembles the Molt Death Syndrome (MDS) described for other decapod species, in which larvae die in the late pre-molt phase of the molting cycle. We suggest that U. cordatus larvae develop disturbances in the molting process similar to the MDS described for other species and that these disturbances are related to a more complex pathway involving the emergence of larval stage zoea VI.

Um dos fatores mais limitantes que afetam o cultivo larval de Ucides cordatus (Linnaeus, 1763) em laboratório é um período de alta mortalidade, o qual usualmente ocorre no fim do curso da larvicultura durante a metamorfose da fase de zoea para a de megalopa. O objetivo da presente pesquisa foi analisar o desenvolvimento pósembrionário de U. cordatus em uma base individual e, em particular, procurar por padrões ligando distúrbios no processo de muda com as altas taxas de mortalidade observadas nas larviculturas massivas. Um total de 50 larvas foram cultivadas individualmente da eclosão até a fase de megalopa em condições controladas, alimentadas com uma combinação de microalgas e rotíferos. A taxa de sobrevivência foi de 70% até zoea V. As 35 larvas zoea V sobreviventes seguiram dois diferentes caminhos. Onze realizaram a metamorfose diretamente para megalopa, dezoito mudaram para zoea VI e seis morreram como zoea V. No último evento de muda, apenas duas larvas zoea VI atingiram a fase de megalopa, enquanto as dezesseis restantes morreram. Em investigação posterior sob microscópio, 13 das zoea VI mortas mostravam características de estágio de prémuda e pereiópodes desproporcionalmente grandes em relação à carapaça. O padrão observado lembra a Síndrome da Morte na Muda (MDS) descrita para outras espécies de decápodes, na qual larvas morrem no fim da fase de prémuda do ciclo de mudas. Nós sugerimos que larvas de U. cordatus desenvolvem distúrbios no processo de muda similares à MDS descrita para outras espécies e que esses distúrbios são relacionados a um caminho mais complexo que envolve a ocorrência do estágio larval zoea VI.

Detection of p15 methylation and deletion status in acute lymphoblastic leukemia using hn-MSP / 白血病·淋巴瘤

Objective To explore the characteristics of hemi-nested methylation specific polymerase chain reaction (hn-MSP) and to find out the possible relationship between patterns of methylation or deletion and the developmet of adult acute lymphoblastic leukemia(ALL).Methods hn-MSP and bisulfit-sequencing PCR (BSP) were designed and adopted to analyze p15 gene methylation or deletion patterns in 25 adult ALL patients,malignant hematopathy cell lines and normal lymphocytes. hn-MSP and BSP products were cloned and sequenced.The sensitivity and specificity of hn-MSP were also analized.Results The sequencing results of hn-MSP and BSP products were consistent, and the sensitivity of detection of p15 methylation was up to 1.0×10-5.17 adult ALL patients (68 ％) were p15 gene hypermethylation and 3 patients were with deletion of p15 gene exon 1.There were no hypermethylation or deletion in the 10 controls.Conclusions The detection rate of p15 methylation in many tumors,especially in adult ALL,is frequent high.hn-MSP is highly sensitive and specific in analyzing p15 methylation.

Apoptosis of leukemia Molt-4 cells induced by resveratrol and effect of WAVE1 gene expression / 白血病·淋巴瘤

Objective To investigate the possible mechanism of apoptosis effect induced by resveratrol in human acute T lymphoblast leukemia Molt-4 cells. Methods MTT method was used to analyze the inhibition rate. The cell cycle and apoptosis percentage of Molt-4 cells treated with resveratrol were detected by flow cytometry. The expression of WAVE1 mRNA was assessed by semi-quantitative RT-PCR. Results After treated with 12.5, 25.0, 50.0, 100.0, 200.0μmol/L resveratrol for 24, 48 and 72 hours, the inhibitory effects were at 29.32 %, 36.11 %, 53.92 %, 62.50 %, and 74.98 %, respectively. Resveratrol was able to inhibit the proliferation of Molt-4 cells in a time-dependent and dose-dependent manner (F =33.614, P <0.05).Compared with control group, after treated with 50.0, 100.0 μmol/L resveratrol for 48 h, the cell number in S phase of Molt-4 cells was 68.6 % and 78.1 %, respectively, and the cell cycle was arrested at S phase (F = 19.453, P < 0.01) after treated with 50.0, 100.0 μmol/L resveratrol for 48 h, the ratio of WAVE1/GAPDH was 0.356±0.03, 0.382±0.05. Compared with control group 0.586±0.06, the ratio of WAVE1/GAPDH was decreased (F =8.950, P <0.01). Conclusion Resveratrol could induce the cells to undergo apoptosis, which was probably related to downregulation the expression of WAVE1.

Microarray Study of Mechanism of Trichostatin A Inducing Apoptosis of Molt-4 Cells / 华中科技大学学报（医学）（英德文版）

to the changes of pro-proliferation genes and anti-apoptosis genes.

Influência do comportamento gregário nos processos de muda de Rhipicephalus sanguineus (Latreille, 1806) (acari: ixodidae), em condições laboratoriais / Influence of aggregation on the molting process of Rhipicephalus sanguineus, (Latreille, 1806) (acari: ixodidae) under laboratory conditions

Objetivou-se com o experimento verificar a influência de diferentes agregações sobre os parâmetros relacionados ao processo de ecdise ninfal e de adulto de Rhipicephalus sanguineus (Latreille, 1806). Foram utilizados como hospedeiros 10 coelhos domésticos (Oryctolagus cuniculus) primos-infestados. Foram analisados os períodos de pré-ecdise, de ecdise e de muda, o percentual e o ritmo de ecdise para todos os estágios estudados. Durante toda a fase não-parasitária, os carrapatos foram mantidos em condições controladas de umidade (80±5 por cento) e temperatura (27±1ºC). Um total de 1560 larvas ingurgitadas e de 1560 ninfas ingurgitadas foi utilizado. Os carrapatos foram divididos em seis grupos contendo um, cinco, dez, 20, 40 e 80 espécimes cada. Para cada um desses grupos foram realizadas dez repetições. Os processos de ecdise ninfal e de adulto foram acompanhados diariamente. Após a análise dos resultados, verificou-se que não houve relação entre as diferentes agregações e o percentual de ecdise em nenhuma etapa experimental. Em todos os outros parâmetros biológicos estudados, verificaram-se diferenças significativas (p<0,05). Os resultados obtidos sugerem que em elevadas agregações o processo de ecdise tende a ser mais rápido.

The objective of this experiment was to evaluate the influence of different aggregations on the molting process of Rhipicephalus sanguineus (Latreille, 1806). Tick-naïve domestic rabbits (Oryctolagus cuniculus) were used as experimental hosts for the parasitic phase. A total of 10 rabbits were used on the experiment. All tick free-living phases were kept in incubator at 27±1ºC, 80±5 percent relative humidity and scotophase. The periods of pre-molt, molt, percentage of ecdisis and molt rhythm of larvae and nymphs were studied. A total of 1560 engorged larvae and 1560 nymphs of R. sanguineus were divided in six groups of aggregations: 1, 5, 10, 20, 40 and 80 ticks with ten repetitions each one. The ecdises of nymphs and adults were daily observed. The percentage of ecdysis was the only parameter with no aggregations effects. All others parameters were statistically different (p<0,05). The results suggested that highest aggregations accelerate the molting process.

Molt and reproduction of the European green crab Carcinus maenas (Decapoda: Portunidae) in Patagonia, Argentina

The green crab Carcinus maenas, a decapod crustacean native to the northeastern Atlantic, has beeninvading distant areas, mainly for the last 25 years. This species is currently distributed along the coasts of thenortheastern Pacific, South Africa, Japan, South Australia, Tasmania, and western and eastern North America,among others. Here we provide information on the biology of the green crab occurring in the central area of SanJorge Gulf, Argentina, where it has been established since 1999-2000. Crabs of both sexes were hand-collectedbetween January 2004 and May 2005 from the intertidal zone and the upper sublittoral fringe. Sex, carapacewidth and molting stage were recorded. The reproductive status of males was based on the presence of sperm andspermatophores in testes and deferent ducts, and that of females on ovarian development and presence-absenceof eggs. Stages of embryonic development for ovigerous females were also recorded. The most importantphysiological events taking place during the annual cycle of the adult population were as follows: (1) male moltoccurred mainly in November and female molt between January and the beginning of March; (2) the reproductiveseason started in January, after a courtship in which the male, larger in size, holds the female until the molt,and spermatophores are ejaculated once the old exoskeleton is cast off; (3) females left the intertidal zone earlyand moved to lower littoral levels during fall and winter; (4) larvae appeared in September.

El cangrejo verde europeo es un crustáceo decápodo originario del Océano Atlántico nororiental, el cual principalmente en los últimos 25 años ha invadido, entre otros, el Pacífico nororiental, Sudáfrica, Japón, sur de Australia, Tasmania y ambas costas de América del Norte. Aquí resumimos las observaciones biológicas en el área central del Golfo San Jorge, Argentina, donde este cangrejo se ha instalado a partir de 1999 ó 2000. Recolectamos manualmente muestras de ambos sexos entre enero de 2004 y mayo de 2005 en el intermareal y la franja superior del sublitoral. La muda de los machos ocurre principalmente en noviembre y la de las hembras, entre enero y principios de marzo. La época reproductiva se inicia en enero, con un cortejo previo donde el macho, de mayor talla que la hembra, la retiene hasta la muda y, una vez desprendido el viejo exoesqueleto, se produce la eyaculación de los espermatóforos. Las hembras abandonan tempranamente el intermareal y migran a niveles inferiores del litoral durante el otoño e invierno, eclosionando las larvas en setiembre.

Dual effect of Molt-4 cell lines onset with Roscovitine / 肿瘤研究与临床

Objective To investigate the effect of Mlot-4 cells onset with Roscovitine (ROSC)as some Cyclin-dependent kinases(CDK),inhibitor.Methods The logarithmic growth phase Molt-4 cells treated with ROSC at a final concentration ranging between 1～20 μmol/L and harvested in different time point,DNA assay of single cells by flow cytometry was used to detect the effect of cell cycle arrest and Annexin-V/FITC assay was used to detect the effect of cell apoptosis. Results It showed that ROSC exerted strong inhibitory effect on proliferation and cell cycle progression of Molt-4 Accumulation of G2/M arrested cells starting 6 h after onset of 10 μmol/L and 20 μmol/L ROSC;at the same time,the cell apoptosis of Molt-4 would be detected,According with the time and concentration changing,the cell apoptosis rate would rise.Conclusion It is concluded that Roscovitine(ROSC)as some Cyclin-dependent kinases(CDK),inhibitor,It has dual effects to Molt-4 cells,not only the effect of cell cycle arrest but also the effect of cell apoptosis.

Effect of Rapamycin on the Cell Cycle Arrest of T Lymphocytes

PURPOSE: Rapamycin (RPM) and its analogues are known for their potent immunosuppressant and anti-proliferative properties, which stem from their ability to modulate the signal transduction pathways involved in cell cycle progression from the G1 to S phase. Thus, RPM has been shown to inhibit the proliferation of a number of non-immune cell types, including hepatocytes, vascular smooth cells and fibroblasts. In addition to its effects on proliferation, RPM may also play a role in the regulation of apoptosis under certain circumstances. METHODS: The effects of RPM on the activation, proliferation and expression of cytotoxic effector molecules were examined on Molt-4 human T-lymphocyte by determining its effects on apoptosis, cell viability, reactive oxygen species (ROS) production and mitochondrial dysfunction. Cells were cultured in the presence or absence of RPM, and then analyzed by Flow cytometry after staining with PI (propidium iodide). RESULTS: The viability of Molt-4 T cells dose- and time-dependently decreased on the addition of RPM. CONCLUSION: RPM induced cytotoxicity was characterized by G2/M phase cell cycle arrest. In addition, a pharmacological scavenging study of ROS, including H2O2, revealed the cytotoxicity was mainly induced by the generation of ROS, which might modulate the expression of Bak protein and mitochondrial dysfunction.

Effects of Trichostatin A on HDAC8 Expression, Proliferation and Cell Cycle of Molt-4 Cells / 华中科技大学学报（医学）（英德文版）

The effects of Trichostatin A (TSA) on histone deacetylase 8 (HDAC8) expression, proliferation and cell cycle arrest in T-lymphoblastic leukemia cell line Molt-4 cells in vitro were investigated. The effect of TSA on the growth of Molt-4 cells was studied by MTT assay. Flow cytometry was used to examine the cell cycle. The expression of HDAC8 was detected by using immunocytochemistry and Western blot. The results showed that proliferation of Molt-4 cells was inhibited in TSA-treated group in a time- and dose-dependent manner. The IC50 of TSA exposures for 24 h and 36 h were 254.3236 and 199.257 μg/L respectively. The cell cycle analysis revealed that Molt-4 was mostly in G0/G1 phase, and after treatment with TSA from 50 to 400 μg/L for 24 h, the percents of G0/G1 cells were decreased and cells were arrested in G2/M phase. Treatment of TSA for 24 h could significantly inhibit the expression of HDAC8 protein in Molt-4 cells (P＜0.01). It was concluded that TSA could decrease the expression of HDAC8 in Molt-4 cells, which contributed to the inhibition of proliferation and induction of cell cycle arrest in Molt-4 cells.