Cloning and expression analysis of a heat shock protein 90 ß isoform gene from the gills of Wuchang bream (Megalobrama amblycephala Yih) subjected to nitrite stress.

Heat shock protein 90 (HSP90), a highly conserved and multi-functional molecular chaperone, plays an essential role in cellular metabolism and stress response. In this study, HSP90 cDNA named MaHSP90 was cloned from Wuchang bream (Megalobrama amblycephala) gills by using rapid amplification of cDNA ends. The full-length MaHSP90 cDNA is 2674 bp and consists of a 3',5'-untranslated region and a 2250-bp open reading frame encoding a 750-amino acid long protein. Identity analysis revealed that the amino acid sequence of MaHSP90 is highly conserved. Homology analysis and structure comparison further indicated that MaHSP90 should be the ß isoform member of the HSP90 family. MaHSP90 mRNA was ubiquitously expressed in the liver, heart, muscle, gill, intestine, kidney, and brain. The MaHSP90 mRNA levels under nitrite stress were analyzed using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR); the mRNA levels significantly increased at 3, 6, and 12 h after nitrite exposure in the gills and then stabilized between 24 and 48 h. Furthermore, a similar relationship between mRNA expression (qRT-PCR) and HSP90 protein levels (densitometric band analysis) was found. Transcriptional analysis of caspase-8 and caspase-9 expression in the gills of juvenile M. amblycephala after a 48-h exposure to nitrite suggested that MaHSP90 expression is related positively with nitrite-induced apoptosis. Fish exposed to nitrite also showed gill damage. Our results suggest that MaHSP90 mRNA is constitutively expressed in various tissues and inducible in the gills under nitrite stress, suggesting its important role in nitrite stress response.

Characterization of the male-specific lethal 3 gene in the oriental river prawn, Macrobrachium nipponense.

In this study, male-specific lethal 3 homolog (Mnmsl3) was cloned and characterized from the freshwater prawn Macrobrachium nipponense (Crustacea: Decapoda: Palaemonidae) by rapid amplification of cDNA ends. The deduced amino acid sequences of Mnmsl3 showed high-sequence homology to the insect Msl3 and contained a conserved chromatin organization modifier domain and an MORF4-related gene domain. Real-time quantitative reverse transcription-polymerase chain reaction showed that the Mnmsl3 gene was expressed in all the investigated tissues, with the highest level of expression in the testis. The expression level of Mnmsl3 between males and females was different in the gonad (testis or ovary), abdominal ganglion, and heart. The results revealed that the Mnmsl3 gene might play roles in regulating chromatin and in dosage compensation of M. nipponense. Real-time quantitative reverse transcription-polymerase chain reaction also revealed that Mnmsl3 mRNA expression was significantly increased in both 5 and 20 days post-larvae after metamorphosis, suggesting that Mnmsl3 plays complex and important roles in the early embryonic development and sex differentiation of M. nipponense.

Effect of High Dietary Carbohydrate on the Growth Performance and Physiological Responses of Juvenile Wuchang Bream, Megalobrama amblycephala.

An optimum dietary carbohydrate content is important for maximum fish growth. In this study, we fed Wuchang bream (Megalobrama amblycephala) with either control diet (30.42%) or high carbohydrate diet (52.92%) for 90 d. Fish were fed to apparent satiation three times daily in an aquarium with automatic temperature control and circulated water. Growth performance, plasma biochemical parameters, hepatic morphology and enzyme activities were determined. It was shown that compared to fish fed control diet, fish fed high carbohydrate diet had higher plasma triglyceride and cortisol levels for d 90, and lower alkaline phosphatase level for d 45, lower hepatic superoxide dismutase and total antioxidative capacity for d 90, higher malondialdehyde for d 45 and glycogen content for d 45 and 90 (p<0.05). Histological and transmission electron microscopy studies showed that hepatocytes of fish fed high carbohydrate diet contained large lipid droplets, causing displacement of cellular organelles to periphery of hepatocytes. The relative level of hepatic heat shock protein 70 (HSP70) mRNA of Wuchang bream fed high carbohydrate diet was significantly higher than that of fish fed the control diet for 90 d (p<0.05). These changes led to decreased specific growth rate and increased feed conversion ratio (p<0.05). Upon hypoxia challenge, fish fed high carbohydrate diet had higher cumulative mortality than those fed the control diet (p<0.05). These results suggested that high dietary carbohydrate (52.92%) was detrimental to the growth performance and health of Wuchang bream.

Connexin 43 gap junction affects survival and drug resistance of multiple myeloma side population cells.

Drug resistance remains a major challenge for multiple myeloma (MM) treatment, and side population (SP) cells may play a key role in this resistance. The function of connexin 43 (Cx43)-mediated gap junction intercellular communication (GJ-IC) in MM cells is poorly understood. Bone marrow mesenchymal stem cells (BMSCs) from different sources were isolated and cultured. SP cells of MM cell line RPMI 8266 were separated by flow cytometry. Real-time reverse transcriptase-polymerase chain reaction and Western blot were used to detect Cx43 mRNA and protein expression in BMSCs, RPMI 8266 and SP cells from different sources. The effects of BMSCs from different sources on SP cell cycle, in vitro colony formation ability, stem cell-related gene expression and drug resistance, and the addition of 18α glycyrrhetinic acid (18αGA) as a pathway inhibitor were observed. Here, we demonstrate that MM cells expressed Cx43 and contained a high percentage of SP cells. We observed an increase in the survival and proliferative capacity of SP cells compared with RPMI 8226 cells, but treatment with 18αGA decreased SP cell survival and proliferation (all P<0.05). MM cells were sensitive to dexamethasone- and bortezomib-induced apoptosis; however, this sensitivity was significantly decreased when MM cells were co-cultured with BMSCs, and 18αGA partly recovered this cytotoxicity (all P<0.05). Collectively, our data suggest that GJ-IC between BMSCs and MM cells is one of the important regulatory mechanisms underlying MM cells survival, proliferation, and drug sensitivity.

Mass mortality of pond-reared Carassius gibelio caused by Myxobolus ampullicapsulatus in China.

From June to August 2009, allogynogenetic silver crucian carp Carassius gibelio (Bloch) pond-cultured at the Nanquan Experimental Station, China, were found to be heavily infected with myxosporeans, which caused mortalities ranging from 33% (13/40) to 90% (36/40) in the cages. The pharynxes of infected fish were swollen, nodular, and severely damaged. Based on morphological characters and 18S small subunit ribosomal DNA sequence similarity, the myxosporean was identified as Myxobolus ampullicapsulatus. This is the first report of M. ampullicapsulatus causing mass mortality of pond-reared C. gibelio.

[Clinical study of baoyuan dahuang decoction in the treatment of chronic renal failure].

UNLABELLED: The quality of life of 56 chronic renal failure (CRF) patients using two kinds of treatment has been observed. 36 of them were taken as Group A with BUN 21.62 +/- 5.96 mmol/L, Cr 528.63 +/- 176.3 mumol/L and Hb 65 +/- 13 g/L were treated with Baoyuan Dahuang Decoction consisted of Panax ginseng, Astragalus membranaceus, Cassia cinnamomi, Glycyrrhiza uralensis and Rheum palmatum. RESULT: Six symptoms were observed, that were: fatigue, lassitude in loin and legs, aversion of cold, anorexia, sexual dysfunction and mental depression. Five of them improved markedly, from 12.5 +/- 2.91 before treatment to 5.58 +/- 4.68 after treatment in terms of symptom scores, P < 0.001. Furthermore, dihydrothelin (E2), testosterone (T) and pregnendione (P) were regulated significantly, the elevated level of creatine phosphokinase (CPK) before treatment lowered to near normal. The other 20 patients, BUN 20.24 +/- 6.57 mmol/L, Cr 487.08 +/- 238.68 mumol/L and Hb 68 +/- 13g/L formed Group B using comprehensive treatment of Western medicine as control. The main drug was coated aldehyde oxystarch. After treatment, the symptoms didn't show marked improvement, symptom scores being 12.55 +/- 2.0 compared with 10.05 +/- 2.72 before treatment, P > 0.05. E2, T, P and CPK were of insignificant differences (P > 0.05); Hb remained almost unchanged, even though BUN and Cr lowered for 22% and 29% respectively. It is assumed that Baoyuan Dahuang decoction could improve CRF patients' quality of life, and the mechanism of which was not only to reduce the urea nitrogen, but also to adjust the anemia and regulate the membrane function.