南极乔治王岛冰锥洞微生物培养探索

【目的】南极洲具备独特的环境和相对的生物地理隔离,南极洲各类生境中蕴藏了大量尚未培养和难培养的微生物,也是新颖微生物物种的重要来源之一。本研究以南极冰锥洞这类特殊生境为研究对象,通过培养条件的多样化提升南极微生物的培养率和多样性,揭示南极冰锥洞可培养微生物类群多样性,为该环境可培养微生物功能研究奠定基础,也为南极极端环境未培养微生物的培养方法提供借鉴。【方法】通过采用不同培养基添加复苏促进因子(resuscitation promoting factor, Rpf)的方式,提高南极柯林斯冰盖冰锥洞生境中微生物的可培养率,探究该生境中微生物的多样性。采用4种不同营养水平的培养基,平行添加Rpf进行菌株培养,经分离纯化与16S rRNA基因鉴定,分析冰锥洞可培养微生物的多样性及培养条件对多样性的影响。【结果】本研究共分离培养细菌407株,涵盖5个门、18个科、29个属,其中：放线菌门(Actinomycetota)为优势门,占72.73%;微杆菌科(Microbacteriaceae)为优势科,占69.78%;Lacisediminihabitans属为优势属,占45.70%。从培养基效果...     

Lysozyme as a regulator of interleukin-2-activated lymphocyte proliferation

Summary Lysozyme at 1 to 100μg/ml of exposure levels augmented or inhibited proliferative response of human peripheral blood lymphocytes stimulated with interleukin-2 (IL-2). This contradictory effect of lysozyme depended on IL-2 concentration, activating state of lymphocytes, addition time of lysozyme, and serum existence. Lymphocytes increased their IL-2-mediated proliferating ability in response to lysozyme when stimulated with less than suboptimal concentration of IL-2. Lymphocyte activation with anti-CD3 antibody changed the augmented proliferative response into the inhibited response by lysozyme addition whereas elimination of MHC class II molecule-expressing cells augmented the response. Addition of lysozyme within 1 h after IL-2 exposure was most effective in promoting the proliferation whereas additions after 16 to 24 h were ineffective or inhibitory. Addition after longer than 24 h inversely restored the proliferative response. Serum seemed to retard lysozyme action because either sequential serum addition 1 h after exposure of IL-2 and lysozyme to cells or exposure of IL-2 and serum after pretreatment of cells with lysozyme changed the proliferative responsiveness from inhibition into augmentation. Thus lysozyme may regulate lymphocyte proliferation responding to a magnitude of antigenic stimuli and to the progression of cellular events that periodically occur.     

Cultures of bovine tracheal epithelium with differentiated ultrastructure and ion transport

Summary Tracheal epithelial cells were grown on Nuclepore filters coated with human placental collagen. When grown immersed in medium containing fetal bovine serum, cells displayed an undifferentiated ultrastructure (no cilia and a cell height of ∼ 10 μm). Short-circuit current (I_sc) was approximately 1/10 that of the native epithelium. By contrast, when grown in hormonally defined, serum-free medium with an air interface, cells showed I_sc equal to or greater than the original tissue, possessed cilia, and had a cell height of ∼ 50 μm. Responses in I_sc to mediators were similar to those of the original tissue, but differed from those of dog or human tracheal epithelium. Given the ready availability and low cost of the native tissues, bovine tracheal cultures grown in serum-free medium with an air interface should prove useful in studies of airway epithelial physiology.     

海南坡鹿围栏种群动态与食物资源关系的初步分析

1986年海南省大田国家级自然保护区在境内设立面积为95公顷的围栏,将86只海南坡鹿分隔成一个孤立的种群。经过3个繁殖年度,种群发展到158只。随着种群数量的增加,坡鹿采食植物的种类密度、高度和频率均明显减少,而坡鹿不食的植物种类则明显增加。种群出生率和年度增长率随种群数量增加而逐年降低,而种群死亡率却有增加趋势。种群密度过高产生的拥挤效应和食物资源不足是造成种群出生率和年度增长率下降的主要原因。本文还就保护区应采取的管理措施,提出了相应的建议。     

Population genetic structure,parasite infection and somatic condition of pumpkinseed Lepomis gibbosus (Actinopterygii: Centrarchidae) in the Oder river basin

In Poland, distribution of non-native pumpkinseed Lepomis gibbosus (Centrarchidae) is strictly limited to the Oder river basin, where it was introduced in the early 20th century. Recently, several populations have been found in waterbodies adjacent to the Oder, particularly in its lower reaches. In this study, we compare the genetic relatedness of populations in the Oder basin with other European populations using nuclear (microsatellite) and mitochondrial (partial cytochrome c oxidase subunit I; cox1) markers. Microsatellite analysis indicated that four populations in the lower Oder form a separate cluster, while one in the middle Oder clustered with Danubian populations, from where probably having been introduced. Microsatellite data suggested that the lower Oder populations differ from other non-native European populations, making it impossible to estimate the source of introduction. Nevertheless, analysis of cox1 indicated that Oder pumpkinseeds belong to the same haplotype as the vast majority of European populations. Parasitological examination confirmed the presence of two North American species, the monogenean Onchocleidus dispar and trematode Posthodiplostomum centrarchi, in the lower Oder, both previously unknown in the region. Fifteen other parasite species were acquired, including glochidia of invasive Sinanodonta woodiana. In the middle Oder, parasite infection was more limited. Fish from the Gryfino Canal, considered one of the most invasive populations in Europe, showed the highest parasite abundance and diversity, and the highest somatic condition and growth rate due to warm water released from the Dolna Odra power plant. Our results highlight significant differences in somatic condition and parasite infection in long-established non-native pumpkinseed populations in the same river system, reflecting mainly environmental conditions.     

Floral sex ratio variation in hermaphrodites of gynodioeciousChionographis japonica var.kurohimensis Ajima et Satomi (Liliaceae)

Intrapopulation and interpopulation variations in floral sex ratio in hermaphrodites of gynodioeciousChionographis japonica var.kurohimensis (Liliaceae) were examined. The relative ratio of male flowers to total flowers (male and perfect flowers) decreased with plant size, suggesting size-dependent gender modification. The relative ratio of male flowers per population-basis is negatively correlated with the mean number of perfect flowers. Since the number of perfect flowers proportionally increased with plant size, populations showing low maleness consist of relatively bigger plants and are considered to be in high-quality environment. On the other hand, the relative ratio of male flowers per population basis is independent of female frequency in the population. Plasticity in gender expression probably plays an important role of maintenance of gynodioecy inC. japonica var.kurohimensis.     

Growth of cells in a new defined protein-free medium

The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum.Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR.     

Substitution of transferrin by FeCl3 in the development of a low foetal calf serum concentration medium for KB-26.5 hybridoma cell line

KB-26.5, a murine hybridoma cell line producing an IgG₃ monoclonal antibody used in blood type determination, primarily adapted to grow at 5% foetal calf serum (FCS) concentration has been adapted to grow at 0.5% FCS, maintaining its ability to produce antibodies at the same level. In the final step of adaptation, the addition of insulin, transferrin, ethanolamine and selenium to the media formulation was studied, using factorial assay techniques to check the effect of the different compounds and to optimize their required level for satisfactory growth and antibody secretion. KB-26.5 cells required only 20 g/ml of transferrin to adapt to 0.5% FCS medium. Furthermore, transferrin could be substituted by FeCl₃, at a relatively low level of 2 g/ml. Maximum cell density decreased by 31.5% in spinner flask test, but the antibody titer was maintained, thus the specific productivity increased. However, inoculum size had to be increased three-fold with 0.5% FCS medium in order to assure cell growth.     

Invasion of tissue culture cells by diarrhoeagenic strains of Escherichia coli which lack the enteroinvasive inv gene

Abstract Invasive Escherichia coli strains of certain serotypes invade by the same mechanism as the Shigella sp. It has been proposed that invasion of epithelial cells by EPEC strains may also occur; this is a previously overlooked property. In the present study E. coli strains isolated from patients with diarrhoea or ulcerative colitis, lacking the inv plasmid mediating classical invasion, but hybridizing with probes for different adhesins, were analyzed for their ability to invade HeLa and Caco-2 cells. The majority of strains invaded Caco-2 cells to a higher extent than HeLa cells. Adhesion to Caco-2 cells was a prerequisite for subsequent invasion of the cells but EAF, eae , EAgg and other known virulence factors were not sufficient to mediate invasion. In 8/9 E. coli strains invasion was enhanced after growth under iron restriction. Growth during anaerobic conditions did not influence subsequent invasion by E. coli strains whereas 6/9 strains had their invasive ability significantly decreased after growth in the presence of 1% glucose. The invasive process was inhibited by mannose but not by lactose, fucose or galactose. Our data indicate that strains of E. coli may invade Caco-2 cells by novel mechanisms which require adhesion to the cells but which differ from those of Salmonella sp., Yersinia sp., Shigella sp. and classical enteroinvasive E. coli .     

Normal rat kidney proximal tubule cells in primary and multiple subcultures

Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.