HPLC analysis of fusaric acid, 9,10-dehydrofusaric acid and their methyl esters,toxic metabolites from weed pathogenic Fusarium species

A simple and rapid HPLC method, using a high-density C18 column, has been developed for the quantitative analysis of fusaric and dehydrofusaric acids and their methyl esters in the methanol extract of lyophilised culture filtrates of species of Fusarium. The method has been used to determine the content of these metabolites in two strains of Fusarium oxysporum and in strains of F. nygamai and F. udum. Fusaric acid has been isolated and identified from a strain of F. udum for the first time.     

Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina

Over the last 20 years, the zebrafish has become an important model organism for research on retinal function and development. Many retinal diseases do not become apparent until the later stages of life. This means that it is important to be able to analyze (gene) function in the mature retina. To meet this need, we have established an organotypic culture system of mature wild-type zebrafish retinas in order to observe changes in retinal morphology. Furthermore, cell survival during culture has been monitored by determining apoptosis in the tissue. The viability and excitability of ganglion cells have been tested at various time points in vitro by patch-clamp recordings, and retinal functionality has been assessed by measuring light-triggered potentials at the ganglion cell site. Since neurogenesis is persistent in adult zebrafish retinas, we have also monitored proliferating cells during culture by tracking their bromodeoxyuridine uptake. Reverse genetic approaches for probing the function of adult zebrafish retinas are not yet available. We have therefore established a rapid and convenient protocol for delivering plasmid DNA or oligonucleotides by electroporation to the retinal tissue in vitro. The organotypic culture of adult zebrafish retinas presented here provides a reproducible and convenient method for investigating the function of drugs and genes in the retina under well-defined conditions in vitro.     

Optimization of porcine urothelial cell cultures: Best practices,recommendations, and threats

Many experimental approaches have been conducted in order to isolate urothelial cells from bladder tissue biopsies, but each method described has utilized different protocols and sources of bladder tissue. In this study, we compared the different methods of urothelial cell isolation available in literature together with standardized methods in order to obtain more unified results. Five methods for primary porcine urothelial culture establishment were compared: tissue explants and four enzymatic methods utilizing collagenase II, dispase II, combination of dispase II and trypsin, and trypsin alone. The average number of isolated cells, cell morphology, success of established culture, average number of cells from the first passage, expression of p63 and pancytokeratin and the characterization of urothelial cell growth, and aging were analyzed during the in vitro culture. The method utilizing dispase II was the most efficient and reproducible method for the isolation and culture of porcine urothelial cells when compared to the other tested methods. Urothelial cells obtained by this method grew considerably well and the cultures were established with high efficiency, which enabled us in obtaining a large quantity of cells with normal morphology. Contamination with fibroblasts in this method was the lowest. The utilization of a proper method for urothelial cell isolation is a critical step in the urinary tract regeneration when using tissue engineering techniques. In summary, this study demonstrated that by utilizing the described method with dispase II, a suitable number of cells was achieved, proving the method useful for tissue regeneration.     

A RAPID, HIGHLY EFFICIENT METHOD FOR COLLECTING, FIXING, AND EMBEDDING PLANKTONIC AND OTHER SMALL CELLS FOR ELECTRON MICROSCOPY

A rapid, highly efficient method is described for fixation, dehydration, and embedding of small (e.g. planktonic) cells dispersed in large volumes of culture medium. The entire protocol, based on continuous filtration, can be completed within about an hour, and the yield of cells is very high. Fixation quality has been excellent with several different types of samples.     

Hepatocytes from newborn and weanling rats in monolayer culture: isolation by perfusion, fibronectin-mediated adhesion, spreading, and functional activities

The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 2 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and alpha-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.     

Selection of the medium for the simultaneous synthesis of an antibiotic, protease and pigment by a Nocardia fructiferi culture

Using method of mathematical planning of experiment the culture medium ensuring the simultaneous intensive production of ristomycin, protease and violet pigment by Nocardia fructiferi has been worked out.     

Method for studying the adaptation of Escherichia coli to the acidification of the medium

Apparatus permitting to regulate the growth rate of turbidostat culture of microorganisms has been worked out. The process of adaptation of culture microorganisms to the medium acidity has been investigated by the stabilization of growth rate method. The correlation coefficients between adaptation time and changes of acid concentration in medium have been determined. The effect of aeration and density of microorganisms on the parameters of adaptation process has been considered. The conclusion concerning prospects of the growth rate stabilization method application for studying the regularities of culture microorganisms adaptation to the action of the stress has been made.     

Archaeology, Anthropology, and the Culture Concept

The culture concept has been central to anthropology since the formational period of the discipline. Yet for much of the discipline's history it was used without explicit definition. Recent attempts to define it have yielded a range of varied formulations in the subdisciplines of archaeology and sociocultural anthropology. Does this mean that the center of anthropology—shared belief in a unified culture concept—has been destroyed? Quite the opposite, the author concludes—the debate has yielded benefits.     

东方田鼠皮肤成纤维细胞的分离培养及生物学特性

目的探索和建立东方田鼠皮肤成纤维细胞体外分离、培养的技术方法并观察其生物学特性。方法采用含10%小牛血清的Dulbecco改良Eagle培养液(DMEM)和1640两种培养体系,运用组织块贴壁法和胰酶消化法,分别对出生后1、3 d和5 d的东方田鼠乳鼠皮肤成纤维细胞进行原代分离、培养。苏木素-伊红染色及倒置相差显微镜下观察成纤维细胞形态和生长特性。结果 0.25%胰酶消化分离出生后1 d和3 d东方田鼠乳鼠皮肤较出生后5 d组织可获得较多数量细胞,成纤维细胞在体外快速贴壁生长,一般6～7 d长满培养瓶,细胞纯度高,HE染色细胞呈长梭形,胞核明显;DMEM和1640两种培养液均可用于东方田鼠皮肤成纤维细胞的培养,但细胞传代后生长趋缓,只可传代2～3次。本实验运用组织块贴壁法未能培养出皮肤成纤维细胞。结论确定了有效分离东方田鼠皮肤成纤维细胞的日龄和方法,为进一步深入研究提供了技术方法和操作依据。     

Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep

Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.     

Hepatocytes from newborn and weanling rats in monolayer culture: Isolation by perfusion,fibronectin-mediated adhesion,spreading, and functional activities

Summary The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 3 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and α-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture. Part of this work was presented at the 30th Annual Meeting of the Tissue Culture Association, Seattle, June, 1979.     

A rapid and simple method for extracting intracellular metabolites from Escherichia coli bacteria

The article deals with the development of a new method for the extraction of intracellular glycolytic metabolites from bacterial cells. The study has been made on the culture of E. coli B/r CSH. In accordance with this method, the same bacterial filter is used for both filtration (the removal of the culture fluid) and the extraction of low-molecular components of the cells with perchloric acid. The advantage of this method is the absence of unnecessary operations due to the use of a filter installation designed by the author. Quantitatively, this method yields better and reproducible results. The filtration capacity of different types of filters has been analyzed. The optimal time for the extraction of low-molecular cell components has been determined. A change in the concentration of pyruvate in the process of the cellular cycle of E. coli synchronous culture grown in the presence of glucose has been shown to occur. The newly developed method of extraction can be used not only for E. coli, but also for cells of other types.     

丛枝菌根的双重培养方法及其菌丝际的建立*

成功地建立起丛枝菌根真菌Glomusintraradices与转移RiT-DNA胡萝卜根器官的双重培养体系,且在此基础上将根与菌丝分隔开来(两室系统),在人工培养条件下形成了无杂菌的菌丝际环境。菌丝际的建立为研究菌丝的生理、生化特性奠定了基础。     

The production of mycobacterial antigens by homogeneous culture in a fermentor

Mycobacterium bovis strain BCG, substrain 1173P2, has been grown in homogeneous culture in classical synthetic Sauton medium without supplementary ingredients. The culture conditions are described. The protein release in the culture medium and the tuberculin yield after 2% trichloroacetic acid precipitation were significantly improved. The antigenicity of the tuberculin has been successfully assayed on specifically sensitized guinea-pigs. It is concluded that homogeneous mycobacterium culture in a fermentor using synthetic medium is a suitable method for the large scale production of antigen.     

Production of BMAA and DAB by diatoms (Phaeodactylum tricornutum,Chaetoceros sp., Chaetoceros calcitrans and,Thalassiosira pseudonana) and bacteria isolated from a diatom culture

Microalgae have previously been reported to contain β-N-methylamino-l-alanine (BMAA), and the global presence of these primary producers has been associated with the widespread occurrence of BMAA in marine organisms. It has been repeatedly shown that filter-feeding bivalves accumulate phytoplankton species and their toxins. In this study, the concentrations of total soluble BMAA and DAB as a function of growth phase were observed for four non-axenic diatom species (i.e. Phaeodactylum tricornutum, Chaetoceros sp., Chaetoceros calcitrans and Thalassiosira pseudonana). These strains had previously been shown to contain BMAA using a highly selective HILIC-MS/MS method. BMAA cell quota appeared to be species-specific, however, highest BMAA concentrations were always obtained during the stationary growth phase, for all four species, suggesting that BMAA is a secondary metabolite. While DAB was detected in a bacterial culture isolated from a culture of P. tricornutum, the presence or absence of a bacterial population did not influence production of BMAA and DAB by P. tricornutum, i.e. no significant difference was noted for BMAA and DAB production between axenic and non-axenic cultures. The presence of DAB in bacteria had previously been shown, and raised the question as to whether DAB observed in many species of microalgae may arise from the non-axenic culture conditions or from the microalgae themselves.     

The Continuous Culture of Luminous Bacteria: a Luminostat

A method has been designed for the continuous culture of luminous bacteria. The control system for the culture uses a combination of luminescence and optical density as a light signal received by a photomultiplier. This combined signal operates pumps which exchange the growth medium. Using this method, a culture of brightly luminescing bacteria was maintained for periods up to 3 weeks.     

Method of radiocapacity factor assessment in study of cross adaptations of plants

Adaptation of plants to the action of gamma-irradiation and cadmium chloride has been studied in the experiments on water culture of maize plants. A new method of registration of the biological object state at the conditions of the stress-factor action - "the method of radiocapacity factor" based on the measurement of the culture medium radio-activity during the incubation process of vegetative objects has been developed. The method offered has allowed us to estimate in dynamics an integrated condition of the root system exposed to stimulating and inhibiting doses of acute gamma-irradiation. Application of the pattern of radio-adaptive response has allowed us to reveal it using both a conventional technique - by registering the values of growing parameters, and the radiocapacity factor method.     

Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan

A method for isolating purified populations of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells suitable for culture, using density gradient centrifugation on the polysaccharide material Stractan is described. A nonparenchymal cell digest of liver from either normal rats or rats treated with modest doses of vitamin A is layered on a discontinuous gradient of 6, 8, 12, ind 20% Stractan; lipocytes are separated efficiently from other nonparenchymal cells and are removed from the top of the gradient. Kupffer cells and sinusoidal endothelial cells, which migrate to denser interfaces in the gradient, are further purified by differential plating and selective trypsinization, respectively. Isolated highly viable lipocytes free of contaminants adhere and spread progressively over several days in primary culture and display both intrinsic vitamin A fluorescence and positive immunostaining for desmin. Lipocytes survive for prolonged periods on plain plastic, and collagen synthesis by these cells remains relatively constant for at least 28 days. Based on serial assay of DNA content, lipocytes in primary culture proliferate, beginning 7 days after plating. Kupffer cells and sinusoidal endothelial cells isolated by Stractan density centrifugation likewise retain their typical morphologic and functional characteristics in culture; the purity of these cell isolates has been confirmed by using specific fluorescent markers. This investigation demonstrates that Stractan density gradient centrifugation is an efficient, sensitive, and reproducible method for isolating pure populations of hepatic nonparenchymal cells.