Nutritional requirements of protoplast-derived,haploid tobacco cells grown at low cell densities in liquid medium

Preliminary attempts to define a completely synthetic medium able to support divisions of haploid tobacco mesophyll protoplasts at low initial densities have failed. High protoplast concentrations together with large amounts of naphtaleneacetic acid in the medium (3 mg l^-1 NAA) were required for maximal induction of protoplast division. However, cell suspensions derived from haploid protoplasts after four days of preculture at high initial cell densities could be diluted to densities as low as 1–4 cells ml^-1, provided the concentration of NAA in the medium was lowered to below 0.3 mg l^-1. The optimal NAA supply for low cell density growth was affected by the nature of the nitrogen source.A simple minimal medium which supports the growth of these haploid cells with a plating efficiency of 30–40%, independent of the cell density in the range of 1–4 to 3·10⁴ cells ml^-1, has been established. In this medium inositol was the only vitamin stringently required for growth.Growth of cells at low densities was also possible in a medium initially containing 3 mg l^-1 NAA, provided it was conditioned by the growth of protoplasts at high densities. Preliminary experiments with [¹⁴C]NAA showed that the amount of free NAA remaining in the medium after preincubation at high densities was drastically reduced. Simultaneously, NAA conjugates accumulated in the medium. The implications of these results are discussed.Abbreviations BA 6-benzyladenine - EDTA ethylene diaminetetraacetic acid - NAA naphtaleneacetic acid     

A method for the microinjection and culture of protoplasts at very low densities

A method has been developed which allows the recovery of calli from a high proportion of individual, injected, mesophyll protoplasts of Nicotiana tabacum c.v. Xanthi. A small drop of low melting point agarose is used both to hold protoplasts during microinjection and for their subsequent culture in feeder dishes. The feeder dishes consist of "beads" of protoplasts at a high density set in agarose to "feed" the infected protoplasts across a liquid medium.The method has been used successfully both with normal protoplasts and protoplasts from which the vacuole has been removed.Abbreviations NT medium Nagata-Takebe medium (Nagata and Takebe, 1971) - MS medium Murashige-Skoog medium (Murashige and Skoog, 1962) - NAA 1-Naphthaleneacetic acid - BAP 6-Benzylaminopurine - LMP agarose low melting point agarose     

Organization of microtubules in dividing and elongating cells of Vicia hajastana Grossh. in suspension culture

Cells of Vicia hajastana Grossh. cultured with 2,4-D showed coupled division and growth and formed multicellular files of small isodiametric cells. In GA without added 2,4-D, the cells stopped dividing and continued elongating for several days. Total growth was the same in both hormone conditions. An immunofluorescent technique was developed to study microtubule (MT) distribution. Cells in GA showed parallel MT arrays oriented transversely to the axis of elongation. In some cells the number of MT per unit length was maintained during growth while other elongating cells showed reduced frequency of MT. Microtubules often appeared as thickened, branched strands, probably as a result of lateral aggregation. In cells grown in 2,4-D some pre-prophase bands of MT were observed. Cells in mitosis lacked cortical MT, and all organized staining was in spindles or phragmoplasts. Interphase cells in 2,4-D showed variable organization of cortical MT ranging from disordered to transversely ordered. Cells in early interphase had disordered MT while larger cells showed order. These observations indicate that MT in cycling cells are continually changing organization, probably accounting for the different distributions observed in interphase cells. On cessation of the mitotic cycle, reorganization of MT stops and transverse arrays of cortical MT are maintained as cells elongate. These processes are similar to those observed in organized tissues; however, cultured cells offer distinct advantages for experimental manipulation and microscopic observation of cytoskeleton.     

Nutritional requirements and media development for Lactococcus lactis IL1403

Lactic acid bacteria are extensively used in food technology and for the production of various compounds, but they are fastidious in nutrient requirements. In order to elucidate the role of each component precisely, defined multicomponent media are required. This study focuses on determining nutrient auxotrophies and minimizing media components (amino acids, vitamins, metal ions, buffers and additional compounds) for the cultivation of Lactococcus lactis subsp. lactis IL1403, using microtitre plates and test tubes. It was shown that glutamine and asparagine were the most important media components for achieving higher biomass yields while the branched-chain amino acids were necessary to increase specific growth rate. The amino acid and glucose ratio was reduced to achieve minimal residual concentration of amino acids in the medium after the growth of cells, whereas the specific growth rate and biomass yield of cells were not considerably affected. As the percentage of each consumed amino acid compared to initial amount is larger than measurement error, these optimized media are important for achieving more precise data about amino acid utilization and metabolism.     

Nutritional requirements and growth characteristics of a biosurfactant-producingRhodococcus bacterium

The nutritional requirements and growth characteristics of a biosurfactant-producingRhodococcus bacterium isolated from Kuwaiti soil were determined. Maximum cell yields (6.6 g/l) and biosurfactant production were achieved with a medium containing 2% (v/v)n-paraffin as a carbon and energy source, 0.2% lactose broth, optimal concentrations of nitrogen (nitrate), phosphorus, iron, magnesium and sodium sources, and minimal concentrations of potassium and trace element sources. The optimal pH was 6.8 for surfactant production and optimal temperature was 37°C. The biosurfactant produced after 16 to 33 h growth in a 7 I fermenter decreased both surface tension and interfacial tension of culture broth to below 27 and 1.8 mN/m, respectively, and was effective at critical micelle dilutions of 10^–3. Data on biosurfactant biosynthesis suggest that the product is produced as a primary metabolite and, therefore, could be produced effectively under continuous fermentation conditions.A.S. Abu-Ruwaida, S. Haditirto and A. Khamis are with the Kuwait Institute for Scientific Research, Biotechnology Department, P.O. Box 24885, 13109, Safat, Kuwait. I.M. Banat is now in Londonderry, Northern Ireland but was at the Kuwait Institute for Scientific Research at the time this paper was written. A.S. Abu-Ruwaida is the corresponding author.In view of the annexation of Kuwait by Iraq in August 1990, this paper has been accepted without return to the author for attention to minor details. The Editor-in-Chief therefore assumes full responsibility for any errors or omissions.     

Nutritional requirements for growth of Helicobacter pylori.

A chemically defined medium consisting of a buffered mineral base supplemented with amino acids, a purine, and thiamine supported growth of 23 clinical isolates and the type strain of Helicobacter pylori. The growth of four strains was inhibited by the presence of certain amino acids. All but one strain required alanine for growth. The amino acids leucine, valine, phenylalanine, methionine, arginine, and histidine were generally required. Isoleucine either was required or stimulated growth. Strains could be differentiated into groups on the basis of a requirement for one or more of the amino acids cysteine, serine, and proline. Only one strain however, demonstrated a requirement for all three of these amino acids.     

Evidence for the presence of low density and very low density lipoproteins in human amniotic fluid

Previous analysis of amniotic fluid (AF) noted only the presence of high density lipoprotein (HDL). In this study AF lipoprotein profile was examined using gel filtration column chromatography and Ouchterlony gel diffusion. Unlike previous studies which showed only the presence of HDL, we found significant amounts of low density lipoprotein (LDL) and very low density lipoprotein (VLDL). AF-LDL and AF-VLDL were identified by reactions with anti-h-apolipoprotein AI and AII antiserum and anti-h-apolipoprotein B-antiserum, respectively. Furthermore, bulk of the cholesterol mass was carried in VLDL (53.6 +/- 7.7%) and LDL (32.5 +/- 4.3%) with minor amounts (13.9 +/- 1.3%) in HDL fraction. It is concluded that human AF contains all three lipoproteins with most of the cholesterol being carried in very low density lipoprotein fraction.     

Immersible probe for continual monitoring of the population density of microorganisms grown in liquid media

A common technique of measuring population density of microorganisms grown in liquid media is to withdraw a sample of the suspension and measure its apparent optical density with a spectrophotometer. The device we describe is capable of continually and automatically monitoring the population density of microorganisms grown in suspension.     

Human fibroblast conditioned media contains growth-promoting activities for low density cells

Normal diploid human fibroblasts, cultured at high density (1–2 × 10⁵ cells per cm²) release two growth promoting activities into the culture medium. The fibroblast proliferation activity-conditioned medium facilitates the attachment of low density cells to the substrate. That activity resides in a non-dialyzable material that is sensitive to proteolytic inactivation. A second activity is dialyzable and can be recovered in the dialysate. In the presence of serum it stimulates cell growth. After 168 hours of incubation conditioned medium cultures contain five times more cells than are present in comparable cultures without conditioned medium. A reproducible biological assay for each activity is described.     

Is hypertriglyceridemic very low density lipoprotein a precursor of normal low density lipoprotein?

The precursor-product relationship of very low density (VLDL) and low density lipoproteins (LDL) was studied. VLDL obtained from normal (NTG) and hypertriglyceridemic (HTG) subjects was fractionated by zonal ultracentrifugation and subjected to in vitro lipolysis. The individual subfractions and their isolated lipolysis products, as well as IDL and LDL, were rigorously characterized. A striking difference in the contribution of cholesteryl ester to VLDL is noted. In NTG subfractions, the cholesteryl ester to protein ratio increases with decreasing density (VLDL-I----VLDL-III). This is the expected result of triglyceride loss through lipolysis and cholesteryl ester gain through core-lipid transfer protein action. In HTG subfractions there is an abnormal enrichment of cholesteryl esters that is most marked in VLDL-I and nearly absent in VLDL-III. Thus, the trend of the cholesteryl ester to protein ratios is reversed, being highest in HTG-VLDL-I and lowest in VLDL-III. This is incompatible with the precursor-product relationship described by the VLDL----IDL----LDL cascade. In vitro lipolysis studies support the conclusion that not all HTG-VLDL can be metabolized to LDL. While all NTG subfractions yield products that are LDL-like in size, density, and composition, only HTG-VLDL-III, whose composition is most similar to normal, does so. HTG VLDL-I and VLDL-II products are large and light populations that are highly enriched in cholesteryl ester. We suggest that this abnormal enrichment of HTG-VLDL with cholesteryl ester results from the prolonged action of core-lipid transfer protein on the slowly metabolized VLDL mass. This excess cholesteryl ester load, unaffected by the process of VLDL catabolism, remains entrapped within the abnormal particle. Therefore, lipolysis yields an abnormal, cholesteryl ester-rich product that can never become LDL.     

Growth and buoyant density of Escherichia coli at very low osmolarities.

The growth and buoyant densities of two closely related strains of Escherichia coli in M9-glucose medium that was diluted to produce osmolarities that varied from as low as 5 to 500 mosM were monitored. At 15 mosM, the lowest osmolarity at which buoyant density could be measured reproducibly in Percoll gradients, both ML3 and ML308 had a buoyant density of about 1.079 g/ml. As the osmolarity of the medium was increased, the buoyant density also increased linearly up to about 125 mosM, at which the buoyant density was 1.089 g/ml. From 150 up to 500 mosM, the buoyant density again increased linearly but with a different slope from that seen at the lower osmolarities. The buoyant density at 150 mosM was about 1.091 g/ml, and at 500 mosM it was 1.101 g/ml. Both strains of E. coli could be grown in M9 medium diluted 1:1 with water, with an osmolarity of 120 mosM, but neither strain grew in 1:2-diluted M9 if the cells were pregrown in undiluted M9. (Note: undiluted M9 as prepared here has an osmolarity of about 250 mosM.) However, if the cells were pregrown in 30% M9, about 75 mosM, they would then grow in M9 at 45 mosM and above but not below 40 mosM. To determine which constituent of M9 medium was being diluted to such a low level that it inhibited growth, diluted M9 was prepared with each constituent added back singly. From this study, it was determined that both Ca2+ and Mg2+ could stimulate growth below 40 mosM. With Ca2+ - and Mg2+ -supplemented diluted M9 and cells pregrown in 75 mosM M9, it was possible to grow ML308 in 15 mosM M9. Strain ML3 would only haltingly grow at 15 mosM. Four attempts were made to grow both ML3 and ML308 at 5 mosM. In three of the experiments, ML308 grew, while strain ML3 grew in one experiment. While our experiments were designed to effect variations in medium osmolarity by using NaCl as an osmotic agent, osmolarity and salinity were changed concurrently. Therefore, from this study, we believe that E. coli might be defined as an euryhalinic and/or euryosmotic bacterium because of its ability to grow in a wide range of salinities and osmolarities.     

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.     

球形棕囊藻生长的营养需求研究

以球形棕囊藻(Phaeocystis globosaScherffel)香港株(Hongkong strain,HK)和汕头株(Shantou strain,ST)为材料,采用正交实验方法研究了氮、磷、铁、微量元素及维生素等营养因子对其生长的影响。磷是球形棕囊藻香港株和汕头株生长的首要限制因子,在添加磷的培养基中,球形棕囊藻生长较快,在无磷培养液中,藻细胞生长较慢,其死亡速率超过生长速率,一般不形成正常生长曲线。汕头株的比生长速率极差值远高于香港株,表明汕头株棕囊藻的生长对营养盐更为敏感。磷酸盐对球形棕囊藻生长的影响极为显著(p<0.01),硝酸盐的影响达到显著水平(p<0.05),而铁、微量元素及维生素对球形棕囊藻生长影响不显著,表明自然海水中铁、微量元素及维生素的含量对球形棕囊藻的生长不构成限制。各因素影响的主次次序为P>N>微量元素>铁>维生素。本研究结果提示,海水富营养化是我国南海海域球形棕囊藻赤潮发生的关键诱因之一。     

Immersible probe for continual monitoring of the population density of microorganisms grown in liquid media.

A common technique of measuring population density of microorganisms grown in liquid media is to withdraw a sample of the suspension and measure its apparent optical density with a spectrophotometer. The device we describe is capable of continually and automatically monitoring the population density of microorganisms grown in suspension.     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.     

Utilization of ascites plasma very low density lipoprotein triglycerides by Ehrlich cells

Much of the lipid present in the ascites plasma in which Ehrlich cells grow is contained in very low density lipoproteins (VLDL). Chemical measurements indicated that triglycerides were taken up by the cells during in vitro incubation with ascites VLDL. When tracer amounts of radioactive triolein were incorporated into the ascites VLDL, the percentage uptakes of glyceryl tri[1-(14)C]oleate and triglycerides measured chemically were similar. The cells also took up [2-(3)H]glyceryl trioleate that was added to VLDL, but the percentage of available (3)H recovered in the cell lipids was 30-40% less than that of (1 4)C from glyceryl tri[1-(1 4)C]oleate. This difference was accounted for by water-soluble (3)H that accumulated in the incubation medium, suggesting that extensive hydrolysis accompanied the uptake of VLDL triglycerides. Radioactive fatty acids derived from the VLDL triglycerides were incorporated into cell phospholipids, glycerides, and free fatty acids, and they also were oxidized to CO(2). Triglyceride utilization increased as the VLDL concentration was raised. These results suggest that one function of the ascites plasma VLDL may be to supply fatty acid to the Ehrlich cells and that the availability of fatty acid to this tumor is determined in part by the ascites plasma VLDL concentration. Although Ehrlich cells incorporate almost no free glycerol into triglycerides, considerable amounts of [2-(3)H]glyceryl trioleate radioactivity were recovered in cell triglycerides. This indicates that at least some VLDL triglycerides were taken up intact. The net uptake of VLDL protein and cholesterol was very small relative to the triglyceride uptake, suggesting that intact triglycerides are transferred from the ascites VLDL to the Ehrlich cells and that hydrolysis occurs after the triglyceride is associated with the cells.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.