Effects of exogenous double-stranded RNA on the basonuclin gene expression in mouse oocytes

In plants and less-advanced animal species, such as C.elegans, introduction of exogenous double-stranded RNA (dsRNA) into cells would trigger degradation of the mRNA with homologous sequence and interfere with the endogenous gene expression. It might represent an ancient anti-virus response which could prevent the mutation in the genome that was caused by virus infection or mobile DNA elements insertion. This phenomenon was named RNA interference, or RNAi. In this study, RNAi was used to investigate the function of basonuclin gene during oogenesis. Microinjection of dsRNA directed towards basonuclin into mouse germinal-vesicle-intact (GV) oocytes brought down the abundance of the cognate mRNA effectively in a time- and concentration-dependent manner. This reduction effect was sequence-specific and showed no negative effect on other non-homologous gene expression in oocytes, which indicated that dsRNA can recognize and cause the degradation of the transcriptional products of endogenous basonuclin gene in a sequence-specific manner. Immunofluorescence results showed that RNAi could reduce the concentration of basonuclin protein to some extent, but the effect was less efficient than the dsRNA targeting towards tPA and cMos which was also expressed in oocytes. This result might be due to the long half life of basonuclin protein in oocytes and the short reaction time which was posed by the limited life span of GV oocytes cultured in vitro. In summary, dsRNA could inhibit the expression of the cognate gene in oocytes at both mRNA and protein levels. The effect was similar to Knock-out technique which was based on homologous recombination. Furthermore, hairpin-style dsRNA targeting basonuclin gene could be produced by transcription from a recombinant plasmid and worked efficiently to deplete the cognate mRNA in oocytes. This finding offered a new way to study the function of basonuclin in the early stage of oogenesis by infection of primordial oocytes with the plasmid expressing hairpin-style basonuclin dsRNA.

Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (greater than or equal to 1000 mum in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%)f 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P<0.05). The lowest (P<0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.

Serum-free culture of rhesus monkey embryonic stem cells

Previous studies have shown that the maintenance and proliferation of undifferentiated rhesus monkey embryonic stem (rES) cells requires medium supplemented with fetal bovine serum (FBS). Due to the uncharacterized composition and variation in serum nature, the present study aimed to replace the serum-containing medium with a serum-free medium in the rES cell culture. The results showed that after the initial 48-h culture in the routinely used serum-containing medium, rES cells can grow and proliferate for a prolonged period in the serum-free medium composed of DMEM supplemented with a cocktail of BSA, IGF-1, TGF-alpha, bFGF, aFGF, estradiol, and progesterone. rES cells cultured in the serum-free medium maintained high level of alkaline phosphatase activity and OCT4 level. There was no indication of differentiation as judged by the marker gene expression of all three embryonic germ layers and trophoblast. In addition, serum-free culture would not affect the passage capacity and differentiation potential of rES cells. This work will facilitate the future study of induced differentiation of rES cells and other applications.

The application of in vitro sperm competition test to evaluate the impact of ZP-derived peptides on fertilization capacity of cat sperm

The present study aimed to establish a sensitive in vitro assay to assess the binding capacity of cat spermatozoa. Cat oocytes and epididymal sperm cells were isolated from gonads and cultured for in vitro fertilization. Before fertilization, the sperm ce

Managing environmental impacts in aquaculture

The article presents the environmental impacts of aquaculture and the ways to manage them. The environmental impacts include issues of recreation and aesthetics, and the usage of resources and discharge of nutrients and organic matter into the environment. Likewise, the results are also discussed, such as reduced production, disease outbreak in cultured and wild populations, and an increase of regulatory restrictions being placed on aquaculture operations.

Effects of toxins induced by phytoplankton bloom on physiological function of indicator animals in Anzali Lagoon

The present overview summarizes data from one year's study during the period of 1379 to 1380 in the regions by "Anzali" lagoon called "Abkenar" and "Hendkhale". Specimens from this lagoon obtained weekly during mordad and shahrivar mounths (July 21 to september 21). The study included 67 types of 5 phytoplanktonal phylum. In "Abkenar" region Cyanobacters with maximum of 97% and minimum of 64.5% of total combination of phytoplanktones made the dominating combination during the period of study , while in "hendkhale" chyrsophyta with maximum of 89% and minimum of 38.7% of total phytoplanktonal was the dominating figure at the same period of time. Researches on ecological parameteres showed that the avarage dissolved oxygen in -Abkenar" and "hendlchale" regions was 10.7 and 8.0 mg/lit respectivly, also total rate of Phosphat in these regions was 0.085 and 0.15 mg/lit respectivly. This study showed that the rates of Nitrat and Amonium in "Abkenar" region was 0.043 and 0.79 mg / lit while for the same substances in "Ilendkhale" measured 0.08 and 0.7 mg I lit respective. Also the avarage rate of chlorophyll a in these two areas measured 58,38 and 40.45 j.un /ht respectivly. Depending on results of correlation cofficient in "Abkenar" region we had Cyanobacters , water and air temperature , Chlorophyll a and total amount of Phosphat as a poitive correlation with transparency while Amonium and Nitrat showed , a negative correlation , EC and finally dissolved oxygen showed a very low rate of correlation coffiocient. To perform this research 5 genus of Cyanobacteres horn "Anzali" lagoon have been isolated and cultured in a laboratorial conditions Later by using Mouse Bioassay method one of these genus identified as a toxic algae. Levels of LD50 with intra peritoneal injection of toxin on mouse in 24 hours was 660 mg/kg and Levels of LC50 by using Bioassay method on Artemia and Daphnia has been shown 618 and 1000 mg kg respectively. Also the physiological effects of were investigated. algae on two types of Cypronides family called Cyprinus carpi° and Hypophrthabnichthys Resultes of blood analyses of Cypronides who were feeded by toxic algae showed a significant decline (P < 0.05) in white and red blood cells and their hematocrites. Levels of LDH , SGOT and SGPT in their blood serum had a significant increase in porportion to control group (P < 0.05) but there was no evidance a differences in Total Protein levels. Pathological studies show damage and destruction of hepato pancreas and kidney of these fishes. Signs and symptoms of intoxication caused by Cyanobacter called Planktohrix agardlltii in mouse and fish show heptotoxic character. This toxin belongs to cyclicpeptides of microcystines group.

Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons

The characterization of acid-sensing ion channel (ASIC)-like currents has been reported in hippocampal neurons in primary culture. However, it is suggested that the profile of expression of ASICs changes in culture. In this study, we investigated the properties of proton-activated current and its modulation by extracellular Ca2+ and Zn2+ in neurons acutely dissociated from the rat hippocampal CA1 using conventional whole-cell patch-clamp recording. A rapidly decaying inward current and membrane depolarization was induced by exogenous application of acidic solution. The current was sensitive to the extracellular proton with a response threshold of pH 7.0-6.8 and the pH(50) Of 6.1, the reversal potential close to the Na+ equilibrium potential. It had a characteristic of acid-sensing ion channels (ASICs) as demonstrated by its sensitivity to amiloride (IC50 = 19.6 +/- 2.1 muM). Either low [Ca2+](0) or high [Zn2+](0) increased the amplitude of the current. All these characteristics are consistent with a current mediated through a mixture of homomeric ASIC1a and heteromeric ASIC1a + 2a channels and closely replicate many of the characteristics that have been previously reported for hippocampal neurons cultured for a week or more, indicating that culture artifacts do not necessarily flaw the properties of ASICs. Interestingly, we found that high [Zn2+] (>10(-4) M) slowed the decay time constant of the ASIC-like current significantly in both acutely dissociated and cultured hippocampal neurons. In addition, the facilitating effects of low [Ca2+](0) and high [Zn2+](0) on the ASIC-like current were not additive. Since tissue acidosis, extracellular Zn elevation and/or Ca2+ reduction occur concurrently under some physiological and/or pathological conditions, the present observations suggest that hippocampal ASICs may offer a novel pharmacological target for therapeutic invention. (C) 2004 Elsevier B.V. All rights reserved.

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca2+ permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASICla-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca2+ elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.

The technology of milkfish hatchery in Indonesia

The culture of milkfish (Chanos chanos), which is the oldest fish cultured in Indonesia, has spread to almost all the provinces in the country. In order to answer the demand of milkfish fry, without decreasing production and wild fry supply, artificial fry production in big and small-scale hatcheries is being practiced. Details are given of the milkfish hatchery system, listing also the major criteria to be taken into account for site selection. Operation of the hatchery is described, outlining the following activities: broodstock rearing; hormone implantation; broodstock maintenance; egg production and harvest; and, larval rearing.

Milkfish grow-out culture in ponds

Milkfish farming is regarded as the backbone of Philippine aquaculture. In 2001, nearly 50% of the Philippines' aquaculture production was milkfish cultured from brackishwater ponds. It has 3 major culture systems: extensive; semi-intensive and intensive. A brief outline is given of the stocking and feeding procedures, providing also some details as to investment costs and returns.

Catfish culture in Southeast Asia

Catfish rank fifth in the world in terms of fresh and brackishwater fish culture. In Asia and the Pacific, the Clariidae family dominates production, representing nearly 80% of the total catfish production. Among the most cultured species are Clarias batrachus, C. macrocephalus, C. gariepinus. The domestic market generally absorbs catfish produce in Asia, although high-producing countries like Thailand and Vietnam engage in export. There are two basic markets for catfish: live fish and processed fish. Particular details are given of catfish production in Thailand, Indonesia and Malaysia.

胎牛血清促进色素上皮衍生因子在皮肤角质形成细胞和成纤维细胞中的表达

下载PDF阅读器目的:探讨表皮角质形成细胞和真皮成纤维细胞中色素上皮衍生因子(PEDF) 表达的影响因素.方法:体外培养角质形成细胞和成纤维细胞,并以10%FBS(胎牛血清)刺激,通过免疫荧光和Western blot检测PEDF的表达.结果:PEDF大多位于细胞浆中,但在细胞核中也有少量表达.细胞浆中PEDF并非均质型分布,而是呈细颗粒状集聚.10%FBS促进角质形成细胞和成纤维细胞中PEDF的集聚和表达,而且这一分布形式不受组胺和佛波肉豆蔻醋酸(PMA) 的影响.结论:10% FBS促进角质形成细胞和成纤维细胞中PEDF的集聚和表达.

The Philippine seaweed industry

A brief outline is given of the Philippine seaweed industry, which involves mainly the red seaweed Kappaphycus alvarezii, produced as a source of the semi-refined or Philippine natural grade (PNG) carrageenan. Other species cultured and marketed include: Gracilaria changii, G. firma, G. heteroclada, G. manilaensis and G. tenuistipitata. The Philippines has the largest carrageenan refinery in Asia. However, production of seaweeds is rather erratic, which is attributed to the following factors: 1) indiscriminate harvesting, seasonal abundance of different species in different areas and lack of proper management; 2) shortage and deteriorating quality of seedlings, especially for Kappaphycus; and 3) natural calamities.

Sustainability in aquaculture

Sustainable aquaculture in Asia is discussed, considering in particular the loss of mangroves associated with the rapid increase in production of cultured shrimp. Southeast Asia's development and population growth brought non-sustainable exploitation of its coastline; the destruction has increased through the years as development proceeded and the international markets entered the scenario. An outline is given of the current status of mangroves in some Asian countries. Some worldwide remedies for sustainable aquaculture are described.

The Sargeant fish and the eel

The culture of sargeant fish, or cobia (Rachycentron canadum) and eel (Anguilla spp) in the Philippines is discussed. Market potentials for the cultured products are examined.