Study on the activation mechanism of BCL-2 related ovarian killer (BOK)

BCL-2 family proteins are key regulators of the mitochondrial apoptotic machinery, controlling the mitochondrial outer membrane (MOM) permeabilization (MOMP). BCL-2 related Ovarian Killer (BOK) is a poorly understood pro-apoptotic member of this protein family. It has been reported that BOK localizes predominantly (although not exclusively) at membranes of the endoplasmic reticulum and of the Golgi apparatus. However, it is unclear whether BOK also operates at the MOM to promote apoptosis, as other pro-apoptotic BCL-2 family members do. Basing on the fact that the other two BAX-like pro-apoptotic members have been reported to oligomerize in order to induce MOMP, site-directed mutagenesis was used to generate two point mutations that predictably eliminated BOK’s oligomerization capacity. Then, the effect of such mutations on BOK’s membrane activity was examined using fluorescence spectroscopy.

Histological distribution and ultrastructure of exocrine pancreas in Indian major carp (Labeo rohita Ham.) and its alteration in aflatoxicosis

The distribution pattern of exocrine pancreas in Labeo rohita besides its general location along the course of intestinal mesentery was studied. It is evenly distributed within the liver around portal vessels and also within the spleen near a blood vessel. On ultrastructure, two cell types of different degrees of staining intensities containing abundant rough endoplasmic reticulum, mitochondria, pre-zymogen and zymogen granules were marked. During aflatoxicosis, the mesenteric pancreas and hepatic pancreas were mostly affected revealing necrotic changes to acini. The zymogen granular activities were markedly reduced. Ultra structurally, the rough endoplasmic reticulum was fully dilated and formed whorled pattern. The damage to the exocrine pancreas might be affecting digestive enzymes' secretion which may be one of the causes of aflatoxin-induced anorexia in fish.

An analysis of the cooperative mechano-sensitive feedback between intracellular signaling, focal adhesion development, and stress fiber contractility

Cells communicate with their external environment via focal adhesions and generate activation signals that in turn trigger the activity of the intracellular contractile machinery. These signals can be triggered by mechanical loading that gives rise to a cooperative feedback loop among signaling, focal adhesion formation, and cytoskeletal contractility, which in turn equilibrates with the applied mechanical loads. We devise a signaling model that couples stress fiber contractility and mechano-sensitive focal adhesion models to complete this above mentioned feedback loop. The signaling model is based on a biochemical pathway where IP3 molecules are generated when focal adhesions grow. These IP3 molecules diffuse through the cytosol leading to the opening of ion channels that disgorge Ca2+ from the endoplasmic reticulum leading to the activation of the actin/myosin contractile machinery. A simple numerical example is presented where a one-dimensional cell adhered to a rigid substrate is pulled at one end, and the evolution of the stress fiber activation signal, stress fiber concentrations, and focal adhesion distributions are investigated. We demonstrate that while it is sufficient to approximate the activation signal as spatially uniform due to the rapid diffusion of the IP3 through the cytosol, the level of the activation signal is sensitive to the rate of application of the mechanical loads. This suggests that ad hoc signaling models may not be able to capture the mechanical response of cells to a wide range of mechanical loading events. © 2011 American Society of Mechanical Engineers.

Survey of reproductive physiology on Epinephelus coioides in Khozestan province, Persian Gulf

Epinephelus coioides (family serranidae) is protogynous. This species is one of the most important fishes in food chain of marine proteins of persian Gulf. Therefore knowing about the reproductive biology and physiology of this species is an important role on aquaculture procedures. Monthly samples of Epinephelus coioides were obtained in khozestan Bahrekan province from 2001 to 2002 for annual variation of base line of reproductive hormone. The hormones such as: 17-B estradiol, Testosteron, Progesterone, Gonadotropin I ,II GTHI, II) and cortisol have assayed and also different stages of gonads from the histological point of view were studied by light and electron microscope. Aditional to morphometric and fecundity measurements, the important factors such as : Gonadosomatic index (GSI) Hepatosomotic index (HSI) and Condition factor (KF) were also studied. Environmental factors such as temperature, salinity, photoperiod and pH were analyzed for the determination of effective factors responsible for the changes of reproductive cycles. The flactmation of estroid hormones and gonadotropines show a significant variation in different stages of maturation, e.g 17-B estradiol's concentration in the third stages, GTH II in fourth stages of sexual maturation or final oocyte maturation, plasma Testosteron in post ovulation and Progesterone during maturation indicates the highest levels of above mentioned hormones. The total calcium concentration was high in all year. calcium concentration was correlated with GTH II synthesis and increases with GTH II in June. 17-B estradiol concentration was also correlated with GSI. The high concentration of cortisol throughout the year was an index of stress and development of ovary maturational processes. This species was protogynous synchronous hermaphrodites , and belongs to annual spawning species, being monandric. The sexual transition was found to occure in individuals of 51.2- 105 cm in length. GSI and HSI level confirms the time of spawning period is in April- June. Electrone microscopic studies of gonad tissues showed some changes in mitochondria and endoplasmic reticulum in the post ovulation, maturation and post spawning periods. During the monthly sampling the biochemistry of tissues variations indicated decrease in protein and lipid content, but an increase in water content of spawning fishes which was correlated to the maturation of Epinephelus coioides . sex ratio indicative of higher frequences of females to males during monthly sampling periods. The females were smaller than males in sizes, therefore the females lived in 8-15m depth, but males were living in upper limits of depth. The results indicated that the temperature was the most effective parameter in reproductive cycle of Epinephelus coioides and the mean 24°c was a convenient temperature for spawning. Photoperiod was the second effective. factor on the reproductive cycle for this species. It seemed that the increase in the photoperiod between January to May caused a development of the oocyte. Regarding to the results of this research, it seems that the period of spawning in Epinephelus coioides is in May- June and the aquaculture procedure of Epinephelus coioides could be performed in the above mentioned periods.

Study on the reproductive biology of Pinctada fucata (Gould) (Mollusca: Pteriidae)

Annual cycle of gonad development and spawning in pearl oyster, Pinctada ficata (Gould) in Nakhiloo, Northeast Persian Gulf, was investigated over two years from August 1994 to June 1996. Gonadal condition was assessed by staging criteria to describe gametogenic development from histological preparations of randomly collected individuals of all sizes. A bimodal gametogenic pattern with summer and autumn spawning periods was evident throughout the study. Gametogensis commenced in November-December which proceeded by major gonadal maturation during February-April. Summer spawning was observed from April to July with major spawning at the latter end. During spawning peak in July, low level of gametogensis was noticed. Gametogenic activity was picked up again in August-September which proceeded by autumn spawning from September to December. Towards the end of spawning season, incidence of gonadal inactivation increased. Minimum level of gonadal activity was observed in November. Temperature regime appears to have influential role in regulation of gametogenic and spawning processes. Gonadal development and spawning trends were similar in both sexes. P. radiaata was found to be protandrous hermaphrodite which matured as a male at shell height greater than 20 mm. Biseivality was uncommon and the sex ratio was about 1:1. Ultrastructure of gametes were investigated in the Pictada fucata (Gould). "Auxiliary cells" closely accociated with developing oocytes were observed. Each oocyte seems to be associated with only one secretory cell. which is characterized by an abundant rough endoplasmic reticulum at the onset of vitellogenesis. Contact between this cell and a developing oocytes is maintained by a desmosome-like junction which can be observed when the vitelline coat is formed. these "auxiliary or nursing cells" seem to play a tropic role in vitellogenesis, and may be involved in the formation of the vitelline coat of the oocytes. Oocytic degeneration is observed in this species, it is a continuous phenomenon of varing intensity throughout the year. The ultrastructural changes resulting in lysis of the oocyte are described. Mature spermatozoa consist of a broad, cap-shaped acrosomal vesicle, subacrosomal material, a round nucleus, two triplet substructure centrioles surrounded by four spherical mitochondria, and a flagellum anchored to the distal centriole and plasma membrane. Spermatozoa of Plucata closley resemble to those of other investigated Pteriidae. Changes in proximate composition of soft tissue and gonadal cycle of Pinctada fucata was studied. Mobilization and utilization of stored reserves are apparent during gametogenesis and gonadal maturation. Protein reserves are utilized during spermatogenesis while reserved carbohydrates form the main energy donor in oogenesis. The role of lipid as am.: energy reserve is second to that of carbohydrate.

Effects of atrazine herbicide on physiological, biochemical and histological biomarkers of developmental stages of larvae and fry in Caspian kutum, Rutilus frisii kutum

To investigation of the toxic effects of atrazine on newly hatched larvae and releasing age fry of the Caspian Kutum, Rutilus frisii kutum, the 96h LC50 was determined as 18.53 ppm and 24.95 ppm, respectively. Newly hatched larvae were exposed to three sublethal concentrations of atrazine (1/2LC50, 1/4LC50 and 1/8LC50) for 7 days. Different histopathological alterations were observed in fins and integument, gills, Kidney, digestive system, liver and the brain of the exposed larvae. Fry’s were exposed to one sublethal concentration of atrazine (1/2LC50) for four days, and like the larvae’s, many histopathological alterations were observed in fins and integument, gills, Kidney, digestive system, liver and the brain of the exposed fry’s, too. Also, measurements of the body ions: Na+, K+, Ca2+, Mg2+ and Cl- in atrazine exposed larvae and fry’s compare to control groups showed that atrazine is changed the body ions composition. No significant differences were found in length growth rate, weight growth rate and the condition factor of the atrazine exposed larvae and fry. Immunohistochemical localization of the Na+, K+-ATPase in integumentary and gill ionocytes, showed no differences in dispersion pattern of the ionocytes in atrazine exposed larvae and fry, compare to control group. Measuring the dimensions of the ionocytes and counting the ionocytes showed that atrazine is affecting on ionocytes by mild increasing in size and mild decreasing in number. Ultrastructural studies, using SEM and TEM, showed that atrazine have significant effects on cellular and subcellular properties. It caused necrosis in surface of the pavement cells in branchial epithelium, necrosis in endoplasmic reticulum of the ionocytes and changed the shape of the mitochondria in these cells. Results showed that sublethal concentrations of atrazine were very toxic to larvae and fry of the Rutilus frisii kutum, and at these levels can made some serious histopathological alterations in their tissues. Related to the severe histopathological alterations in osmoregulatory organs, like gill, kidney and digestive system, and the alterations in the body ion composition, it could be concluded that atrazine could interfere with the osmoregulation process of the Rutilus frisii kutum at the early stages of the life history.

Ultrastructure of the bloodstream forms of the fish trypanosome Trypanosoma pseudobagri dogiel et achmerov, 1959

The ultrastructure of the bloodstream forms of Trypanosoma pseudobagri from its natural host, yellow catfish (Pseudobagrus fulvidraco), a freshwater fish, is described in the present work. The pellicle, consisting of a unit membrane with a superimposed surface coat, the structure and attachment of the flagellum and the subpellicular microtubules show the usual structural and organizational features. Cell organelles and cytoplasmic inclusions such as kinetoplast, mitochondria, nucleus and vacuoles, which occur in trypanosomidae, are observed and described in detail. The ultrastructure of T. pseudobagri has been compared with that of bloodstream forms of other species and culture forms of fish trypanosomes, and similarities and divergences are discussed. The Golgi-complex and endoplasmic reticulum could not be observed and need further investigation.

First report of aphantoxins in China - waterblooms of toxigenic Aphanizomenon flos-aquae in Lake Dianchi

The oligohaline cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs (A. flos-aquae) has been reported in several countries to produce paralytic shellfish poisons (PSPs) or protracted toxic effects. In the past years, A. flos-aquae blooms have occurred annually in the eutrophic Lake Dianchi (300 km(2) in area, located in southwestern China). Material from natural blooms dominated by A. flosaquae was collected and lyophilized. Acute toxicity testing was performed by mouse bioassay using extracts from the lyophilized material. Clear symptoms of PSPs, intoxications were observed. To confirm the production of PSPs, a strain of A. flos-aquae (DC-1) was isolated and maintained in culture. Histopathological effects were studied by examining the organ damages using transmission electron microscopy (TEM). Slight hepatocytic damage with swollen mitochondria was found. The ultrastructural pulmonary lesions were characterized by distortied nuclei and indenting of karyotheca, together with degeneration and tumefaction of mitochondria and endoplasmic reticulum. Control animals injected with acetic acid did not exhibit histopathological damage in any organ. Toxic effects of cultured algal cells on enzymatic systems in the mouse were studied using sublethal doses of extracts. Significant glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) increases, together with decrease of the glutathione (GSH) level, were measured. These results indicated a potential role of PSPs intoxicating and metabolizing in the test animals. HPLC-FLD and LC/MS analysis of extracts from cultured material demonstrated the PSP toxins produced by A. flos-aquae bloom. To the best of our knowledge, this is the first study reporting chemically and toxicologically confirmed PSP toxins related to A. flosaquae in China. (c) 2005 Elsevier Inc. All rights reserved.

Long-term toxic impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the reproduction, sexual differentiation, and development of different life stages of Gobiocypris rarus and Daphnia magna

The sexual ratio of Gobiocypris rarus exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 17 beta -estradiol from embryo to sexually mature revealed feminization and overdevelopment of connective tissue in male fish gonad in 2-30 pg/L TCDD concentration range. Daphnia magna was not sensitive to the high dose of TCDD (0.1-1000 ng/ml), but the reproduction of D. magna treated with TCDD decreased after the 8th day. 7-Ethoxyresorufin-O-deethylase (EROD) activities in newly fertilized eggs of G. rarus exposed to TCDD dosage groups (1000-100,000 pg/L) were significantly induced and increased with TCDD concentrations at the early life stage, while no difference was found between low TCDD dosage groups (<100 pg/L), but a good relationship between the EROD activity and the TCDD concentration was observed during a long-term developmental stage. There was a pericardial edema formed in a 2-week yolk-sac at the concentration of 1000 pg/L TCDD. But in the exposure group (2 pg/L TCDD for 120 days), the cell nuclei of hepatocytes was far from the center and packed toward the cell membrane; the cristae of most mitochondria in the cell dropped and collapsed; the rough endoplasmic reticulum broke into fragments; and numerous lipid droplets formed in the cell. (C) 2001 Academic Press.

Cytological and biochemical alterations in Carassius auratus hepatocytes from exposure to sediment containing dioxins and related compounds

Cytological and biochemical alterations of crucial carp (Carassius auratus) hepatocytes were characterized after exposure to sediments from a lake contaminated with dioxins and other industrial chemicals. Carp were exposed in 20 L water containing 25, 50, or 100 g of contaminated sediment for 2 and 4 weeks. Ultrastructural changes in the liver were characterized by severe enlargement of hepatocytes. Alterations in the cell. included formation of condensed and irregular cell nucleus, polynuclei, dispersed heterochromatin, enlargement of the nucleolus, and degeneration of the nucleus. Mitochondrial numbers were reduced and cristae were deformed. Myelin figures and lysosomes were increased, and sometimes cell organelles and cell matrix were totally lost after 4 weeks of exposure. The ultrastructural alterations were correlated with exposure time and sediment concentrations. Hepatosometic index was significantly increased in experimental groups at 2 and 4 weeks as compared with the control group. EROD enzyme activities were strongly induced in liver. A trend from rough endoplasmic reticulum (RER) to SER was observed. Our results suggest that the dioxin-like compounds bound by sediment were bioavailable to C. auratus and cause sublethal effects.

Intercellular calcium wave propagation in linear and circuit-like bone cell networks

In the present study, the mechanism of intercellular calcium wave propagation in bone cell networks was identified. By using micro-contact printing and self-assembled monolayer technologies, two types of in vitro bone cell networks were constructed: open-ended linear chains and looped hexagonal networks with precisely controlled intercellular distances. Intracellular calcium responses of the cells were recorded and analysed when a single cell in the network was mechanically stimulated by nano-indentation. The looped cell network was shown to be more efficient than the linear pattern in transferring calcium signals from cell to cell. This phenomenon was further examined by pathway-inhibition studies. Intercellular calcium wave propagation was significantly impeded when extracellular adenosine triphosphate (ATP) in the medium was hydrolysed. Chemical uncoupling of gap junctions, however, did not significantly decrease the transferred distance of the calcium wave in the cell networks. Thus, it is extracellular ATP diffusion, rather than molecular transport through gap junctions, that dominantly mediates the transmission of mechanically elicited intercellular calcium waves in bone cells. The inhibition studies also demonstrated that the mechanical stimulation-induced calcium responses required extracellular calcium influx, whereas the ATP-elicited calcium wave relied on calcium release from the calcium store of the endoplasmic reticulum.

Functional gold nanoparticle-peptide complexes as cell-targeting agents

In this paper, we report a novel approach using peptide CALNN and its derivative CALNNGGRRRRRRRR (CALNNR(8)) to functionalize gold nanoparticles for intracellular component targeting. The translocation is effected by the nanoparticle diameter and CALNNR8 surface coverage. The intracellular distributions of the complexes are change from the cellular nucleus to the endoplasmic reticulum by increasing the density of CALNNR8 at a constant nanoparticle diameter. Additionally, increasing the nanoparticle diameter at a constant density of CALNNR8 leads to less cellular internalization.

Cloning and expression of glucose regulated protein 78 (GRP78) in Fenneropenaeus chinensis

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35A degrees C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25A degrees C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.

Molecular characteristics and expression analysis of calreticulin in Chinese shrimp Fenneropenaeus chinensis

Calreticulin (CRT), as an endoplasmic reticulum luminal resident protein, plays important roles in Ca2+ homeostasis and molecular chaperoning. CRT on the surface of the cell can modulate cell adhesion, phagocytosis and integrin-dependent Ca2+ signaling. The full length cDNA of calreticulin (FcCRT) was cloned from Chinese shrimp Fenneropenaeus chinensis. It consists of 1672 by with an open reading frame of 1221 bp, encoding 406 amino acids. This is the first reported cDNA sequence of calreticulin in Crustacea. The deduced amino acid sequence of FcCRT showed high identity with those of Bombyx mori (88%), Drosophila melanogaster (83%), Mus musculus (82%) and Homo sapiens (82%). Highest expression of FcCRT was detected in ovary by Northern blot and in situ hybridization. Different mRNA levels of FcCRT were detected at various molting stages. Expression of FcCRT was induced significantly after 3 h of heat shock treatment, reached the maximum at 4 h and dropped after that. Differential expression profiles of FcCRT were observed in hepatopancreas and haemocytes when shrimp were challenged by white spot syndrome virus (WSSV). From the above results, we inferred that FcCRT might play important roles in Ca2+ homeostasis, chaperoning and immune function in shrimp. (c) 2007 Elsevier Inc. All rights reserved.

SNAP-23蛋白在鲈鱼巨噬细胞白细胞介素1β分泌过程中的功能

SNARE蛋白家族是所有真核细胞胞吐及分泌作用中的关键因子，由其介导的运输囊泡膜与靶膜的锚靠、融合为胞内蛋白的运出提供了一条重要途径。体外试验表明，Syntaxin6-Syntaxin7-Vti1b，SNAP-23-Syntaxin4等SNARE核心蛋白之间精确的相互作用是哺乳动物巨噬细胞肿瘤坏死因子α (TNF-α)运输和分泌的必备条件，在机体非特异性免疫应答反应过程中起重要作用。 本研究受上述启示，旨在揭示SNARE蛋白在海洋鱼类免疫细胞内重要细胞因子白细胞介素1β (IL-1β)的分泌过程中的作用。参照Percoll密度梯度离心技术，从鲈鱼头肾组织分离纯化巨噬细胞进行稳定培养；利用RT-PCR方法克隆出鲈鱼t-SNARE蛋白SNAP-23和Syntaxin3的部分cDNA序列，再结合先前克隆的VAMP2和已知的鲈鱼IL-1β，TNF-α和IL-8的基因序列，设计特异性引物。利用Real-time PCR技术在mRNA水平上精确测定鲈鱼巨噬细胞中上述6种基因在革兰氏阴性菌脂多糖(LPS)分子刺激下的表达变化，发现SNAP-23基因与三种细胞因子基因的表达正相关；通过免疫印迹检测SNAP-23蛋白表达变化，利用酶联免疫吸附试验(ELISA)检测IL-1β的分泌水平，在蛋白水平上验证了SNAP-23表达与IL-1β分泌的正相关性；利用5`RACE和3`RACE技术克隆出鲈鱼SNAP-23全长基因，结合定点突变策略和靶向PCR克隆手段，构建鲈鱼SNAP-23野生型融合质粒pEGFP-SNAP-23wt，Cys缺失突变融合质粒pEGFP-SNAP-23ΔCys和模拟E型肉毒神经毒素(BoNT/E)切割突变融合质粒pEGFP-SNAP-23ΔBoNT/E，以及鲈鱼IL-1β野生型融合表达质粒IL-1β-pEGFP和IL-1β-pEYFP。所有融合蛋白均在鲈鱼巨噬细胞内成功表达，结合ELISA实验结果发现，SNAP-23野生型的表达对IL-1β的分泌有促进作用，而Cys缺失突变体的表达则抑制IL-1β向胞外分泌。首次证实了鱼类巨噬细胞内SNAP-23蛋白在IL-1β分泌过程中的重要作用。此外通过与GFP共表达，定位了IL-1β分子在巨噬细胞内的分布，发现新合成的IL-1β分子很可能像TNFα一样经“内质网－胞质－伪足－胞外” 的分泌路径运出胞外。