Isolation of Extracellular Cobalt-free Corrinoid from Methanosarcina barkeri

Cobalt-free corrinoids (CFCs) were isolated from Methanosarcina barkeri Fusaro cells growing on a methanol minimum medium. The methanogen cells excreted a trace of CFCs (9.1 μg/I) into the culture medium when cobalt-deficient methanol medium was used. Several CFCs were separated by column chromatographies on ion exchangers and paper electrophoresis, where a major CFC showed a similar characteristic to that of nucleotide-free corrinoid, Factor B (cobinamide), suggesting to be hydrogenobinamide. By chemical insertion of Co^{2 +}, Cu^{2 +}, and Zn²⁺ into CFCs, the corresponding corrinoid and its metal analogues were observed. Bioassay using Escherichia coli 215 revealed that the major CFC (a yellow product obtained after alkaline treatment) and its copper and zinc analogues were inactive as cobalamin but were active as antimetabolites of cobalamin. However, the CFC greatly stimulated the cell growth of M. barkeri grown under cobalt-deficient conditions.     

Growth of Paenibacillus larvae,the causative agent of American foulbrood in honey bees and Bacillus popilliae,the causative agent of milky disease in beetle larvae in lepidopteran cell cultures

TNM-FH Lepidopteran insect cell culture medium containing 10% fetal bovine serum (FBS), while allowing limited vegetative growth of Paenibacillus larvae (wild-type strain), the causative agent of American foulbrood, contained no viable vegetative cells upon subculture, nor were any heat resistant spores produced in this medium alone. However, TNM-FH medium cotaining embryonic or midgut cells from Trichoplusia ni, hemocytes from Estigmene acrea, ovarian and embryonic cells from Spodoptera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispar, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX-CELL 405 serum-free insect cell culture medium alone appeared to contain the appropriate nutrients required for both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occurred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were any heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores (4,000/ml) were detected in the form of cology-forming units (CFU) on plates following heating to 80°C for 20 minutes. In a parallel study with a wild-type strain of Bacillus popilliae, vegetative cells grew only in TNM-FH medium in the presence of mid-gut BTI-Tn-MG and ovarian (Tn-368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI-Tn-MG and Tn-368 cells were further challenged with four variant cultures of B. popilliae, vegetative growth and limited sporulation were achieved. The BTI-Tn-MG cell line in TNM-FH medium produced as many as 12,000 spores/ml after 21 days in culture.     

Effect of sulphur nutrition on redistribution of sulphur in vegetative barley

Xenobiotics directed against sterol biosynthesis have proved to be useful tools in the determination of which sterol molecules are necessary for successful plant cell growth. However, the exact mode of action by which sterols are able to trigger cell growth remains to be elucidated. Previous studies using the triazole paclobutrazol, demonstrated that in Apium graveolens (cv. New Dwarf White) suspension cultures, sterol and phosphatidylcholine biosynthesis are co-ordinately regulated (C. E. Rolph and L. J. Goad 1991, Physiol. Plant. 83: 605–610). The studies presented herein, were designed to investigate the possible role of phosphatidylcholine in the stimulation of plant cell growth. Sterol biosynthesis, and hence cell growth, was inhibited by the use of the azole xenobiotic miconazole. Treatment of the cultures with miconazole lead to compositional changes in the free sterol content of the cells. For example, 30 μM miconazole treatment led to a reduction in the stigmasterol/sitosterol ratios from 1.53 to 1.24. In contrast, the phospholipid content of the cells remained relatively unchanged with phosphatidylcholine accounting for approximately 25% of the total phospholipids present in both control and miconazole-treated cells. The cytostatic effect of miconazole could be partially counteracted by supplementation of the growth medium with the phytosterol stigmasterol and/or the unsaturated fatty acids oleate and linoleate. The activity of CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15), a rate-limiting enzyme in phosphatidylcholine biosynthesis, was significantly reduced in cells whose growth had been arrested by miconazole treatment. In miconazole-treated cultures whose growth had been partially restored by supplementation with either specific sterols or unesterified fatty acids, the activity of this key enzyme was increased. In the case of stigmasterol, oleate and linoleate supplementation, the microsomal activity was found to be similar to that exhibited by control cultures. From these studies, it may be concluded that certain phytosterols and unsaturated fatty acids play key roles with respect to phosphatidylcholine biosynthesis and that phosphatidylcholine biosynthesis via the CDP-base pathway is an important pre- and/or co-requisite for successful culture growth.     

Effect of sediment elutriate on the chemical composition of Gymnodinium sp. (Dinophyceae). II.

Abstract

The chemical composition of Gymnodinium sp. was studied in cells grown in batch cultures with sediment elutriate, enriched or unenriched with F/2 as the culture medium. The cellular protein, chlorophyll a, nitrogen, carbon and phosphorus content were determined in conjunction with cell density. For the enriched media, a high density of biomass was always measured; differences in cellular composition between control and elutriate cells were not observed and the atomic C:N:P ratios were typical of unstarved phytoplankton. Unenriched media produced different effects on the algal growth, which also affected the biochemical composition of the cells. Relative to the control, some elutriates reduced the algal growth, affected the chemical composition of the cells which showed very high levels of carbon, nitrogen and protein, and resulted in an aberrant morphology. The stimulating growth elutriate affected the chemical composition of the cell in another manner: the greatest effect was a decrease of carbon content, whereas the other compounds remained similar to the control; aberrant forms were not observed.     

The Neutral Lipids of Paramecium tetraurelia: Changes with Culture Age and the Detection of Steryl Esters in Ciliary Membranes1

Neutral lipids, particularly triglycerides, accounted for the major decrease in the total lipid content in Paramecium cells that occurs with culture age. Sterols, triglycerides, and steryl esters were the major classes of neutral lipids in cells and isolated cilia. Free as well as high concentrations of esterified sterols were detected in purified ciliary membrane preparations. Stigmasterol and 7-dehydrostigmasterol were the major components of both free and esterified sterols of cells and cilia; however, when cholesterol was present in the growth medium, it was desaturated to 7-dehydrocholesterol and incorporated into cellular and ciliary lipids. Free fatty acids from cells and triglycerides from cells and cilia were low in polyunsaturated fatty acids and reflected the composition of fatty acids in the culture medium. An exception was the reduced concentration of stearate in triglycerides from whole cells. Greater than 50% of triglyceride fatty acids from cilia were saturated. The fatty acid compositions of cellular triglycerides and ciliary steryl esters did not change with culture age, but those of cellular steryl esters and ciliary triglycerides did change. In comparison with phospholipids, these neutral lipid fatty acid compositional changes were smaller. The sensitivity of these stigmasterol-containing cells to polyene antibiotics indicated that they were killed by nystatin > filipin > amphotericin B. The unexpected finding of high concentrations of steryl esters in ciliary membrane preparations is discussed.     

The Role of Intercellular Contacts in the Initiation of Growth and in the Development of a Transiently Nonculturable State by Cultures of Rhodococcus rhodochrous Grown in Poor Media

It was found that the growth of Rhodococcus rhodochrous cells in a modified Saton’s medium strongly depends on the rate of culture agitation in the flask: agitation at 250 rpm in flasks with baffles stops cell multiplication, whereas slight agitation leads to pronounced culture growth. The growth retardation phenomenon was reversible and did not manifest itself in exponential-phase cultures or when the cells were grown in a rich medium; furthermore, it was not connected with the degree of culture aeration. When agitated at a moderate rate, the bacterial cells formed aggregates in the lag phase, which broke up into single cells in the exponential phase. The inhibitory effect of vigorous agitation was removed by the addition, to the medium, of the supernatant (SN) of a log-phase culture grown in the same medium with moderate agitation. Vigorous agitation is thought to interfere with cell contact, whose establishment is necessary for the development of an R. rhodochrous culture in a poor medium, which occurs in the form of (micro) cryptic growth. When grown in a modified Saton’s medium, R. rhodochrous cells were capable of transition, in the prolonged stationary phase, to a resting and transiently nonculturable state. Such cells could be resuscitated by incubation in a liquid medium with the addition of the supernatant or the Rpf secreted protein. The formation of transiently nonculturable cells was only possible under the conditions of a considerable agitation rate (250–300 rpm), which prevented secondary (cryptic) growth of the culture. This circumstance indicates the importance of intercellular contacts not only for the initiation of growth but also for the transition of the bacteria to a dormant state.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 489–797.Original Russian Text Copyright © 2005 by Voloshin, Shleeva, Syroeshkin, Kaprelyants.     

Establishment and comparison of fibroblast cell lines from the medial collateral and anterior cruciate ligaments of the rabbit

Summary We have developed a procedure to explant fibroblasts from the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) of the rabbit knee, and have optimized conditions for maintaining them in culture. Maximal growth for both ACL and MCL cells was obtained with Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum and 250 μM ascorbate. ACL and MCL fibroblasts displayed intrinsic differences in their responses to changes in culture parameters. Specifically, they displayed different growth responses when plated at different densities and responded to RPMI 1640 medium in very different ways. There were also biochemical differences between the cell types. Both cell types produced similar amounts of collagen in culture, but the ratio of type I to type III, the major collagen subtypes produced by these cells, were different. ACL fibroblasts produced 86.7% type I and 13.3% type III, and MCL fibroblasts produced 71.1% type I and 28.9% type III. In addition, total protein produced by ACL fibroblasts was higher than that produced by MCL cells. This confirms the suggestions of previous researchers that such differences might exist. This work was funded by a grant-in-aid from Medtronic of Canada, by an R&D Grant from the Alberta Ministry of Technology, Research and Telecommunications, and by the Alberta Heritage Foundation for Medical Research.     

Comparative sequence analysis of bacterial symbionts from the marine sponges Geodia cydonium and Ircinia muscarum

Marine sponges (Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Recently, several studies indicated that sponges are the most prolific source of biologically-active compounds produced by symbiotic microorganisms rather than by the sponges themselves. In the present study we characterized the bacterial symbionts from two Demospongiae, Ircinia muscarum and Geodia cydonium. We amplified 16S rRNA by PCR, using specific bacterial-primers. The phylogenetic analysis revealed the presence of nine bacterial clones from I. muscarum and ten from G. cydonium. In particular, I. muscarum resulted enriched in Bacillus species and G. cydonium in Proteobacterium species. Since these bacteria were able to produce secondary metabolites with potential biotechnological and biopharmaceutical applications, we hypothesized that I. muscarum and G. cydonium could be a considered as a “gold mine” of natural products.     

Inhibition of biofilm populations of Nitrosomonas europaea

The influence of surface attachment and growth on inhibition of the ammonia oxidizing bacterium, Nitrosomonas europaea, by nitrapyrin was investigated in liquid culture in the presence and absence of glass slides. Significant attachment to glass slides occurred in the absence of ammonia, but the extent of attachment was not affected by nitrapyrin, nor by previous culture of cells in medium containing nitrapyrin. The presence of glass slides affected neither the specific growth rate of N. europaea, measured by changes in nitrite concentration, nor inhibition by nitrapyrin. Inhibitory effects of nitrapyrin on increases in nitrite concentration and in free cell concentration were similar, but greater effects were observed on changes in attached cell concentration. Established biofilms on glass slides grew at a lower specific growth rate than freely suspended cells. Both biofilm cells, and those detached from the biofilm, were protected from inhibition. A mechanism for protection of biofilm populations is proposed involving reduced sensitivity of slowly growing cells producing extracellular polymeric material. Offprint requests to.: J. I. Prosser.     

Establishment and characterization of a betaxanthin-producing cell culture from Portulaca grandiflora

Cell cultures derived from three yellow flowering Portulaca grandiflora genotypes contained betacyanins rather than betaxanthins. A betaxanthin-producing cell culture was obtained by subculturing orange cell clusters isolated from the red-violet cell culture of a violet flowering P. grandiflora genotype. Selection of the most strongly yellow coloured cell material reduced the portion of betacyanins considerably and resulted in a P. grandiflora cell culture characterized by a high concentration of betaxanthins and the occurrence of free betalamic acid. Vulgaxanthin I was the main compound. Besides these pigments carotenoids and flavonoids were detectable.Betaxanthin biosynthesis strongly dependend on light. Product accumulation reached its maximum during the stationary phase of the growth cycle. Excretion of pigments, especially of betalains, could not be detected. Vulgaxanthin I as found in the cell culture was identical with one of two main betaxanthins in the yellow petals of P. grandiflora.The yellow P. grandiflora cells grew well on solid modified Murashige and Skoog medium but failed to grow in liquid medium after a few subcultures. In contrast, white P. grandiflora suspension cultures could be established repeatedly.Abbreviations A absorbance - BX I and BX II betaxanthin fractions - DOPA dihydroxyphenylalanin - DW cell dry weight - molar extinction coefficient - SS solvent system     

Chlorella vulgaris (Chlorellaceae) does not secrete autoinhibitors at high cell densities

Filtrates (conditioned medium) from high-density Chlorella vulgaris cultures in photobioreactors were obtained and tested for autoinhibitory activity under different conditions. Exponentially growing cells were inoculated at low initial cell concentration (2 × 10⁵ cells/ml) in 90% conditioned medium (CM) supplemented with 10% fresh medium (FM) at low (atmospheric) CO₂ levels. The time sequence of DNA histograms of cells in CM cultures showed that there is an accumulation of cells with two and four DNA equivalents in the culture over a period of time, signifying a blockage of cells at the division stage of the cell cycle. Examination of the chemical composition of CM showed the presence of high concentrations (> 10 mM) of bicarbonate. Adding similar bicarbonate concentrations to FM were found to have similar effects as CM cultures, causing blockage of cell division, though the intensity of the blocking effect was lower. The bicarbonate-free CM did not show any cell cycle modulating or inhibitory activity. The growth of cells cultivated at high (5%) CO₂ levels in 90% CM supplemented with 10% FM was comparable to 10% FM cultures, indicating nutrient limitation in 90% CM culture. When the 90% CM culture was supplemented with 100% nutrients, the growth rate and final cell concentration was similar to 100% FM culture. Based on these results we conclude that C. vulgaris does not secrete any autoinhibitor(s) or cell cycle modulating compound(s) under the conditions from which the CM was obtained.     

Stable expression of recombinant human coagulation factor XIII in protein-free suspension culture of Chinese hamster ovary cells

The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture system with a spin filter is employed. This revised version was published online in August 2006 with corrections to the Cover Date.     

GROWTH INDUCTION IN CULTURES OF HAPLOPAPPUS GRACILIS. I. THE BEHAVIOR OF THE CULTURED CELLS

Blakely , L. M., and F. C. Steward . (Cornell U., Ithaca, New York.) Growth induction in cultures of Haplopappus gracilis. I. The behavior of the cultured cells. Amer. Jour. Bot. 48(5): 351–358. Illus. 1961.—Because of the unusual cytology of Haplopappus gracilis (2n = 4), a study has been made of the growth of its stem tissue in culture. Although growth may occur on a basal medium supplemented in various ways, it was stimulated for present purposes by the use of a basal medium containing casein hydrolysate, coconut milk (2–10% by volume) and naphthaleneacetic acid (0.5 p.p.m.). A definite synergism between coconut milk and naphthaleneacetic acid was demonstrated. The general form of the colonies so obtained responds to the composition of the medium, and certain effects of pigmentation indicate that the biochemistry of the cultured tissue is also a function of the conditions. The Haplopappus cultures were maintained in liquid culture either in the form of free cells or of the small cell clusters to which they readily gave rise. The form of typical cells and cell clusters is described, and stress is laid upon the range of growth forms that are encountered. Variations in suspensions of cells, or small cell clusters, may be investigated by the application of simple microbiological techniques. Haplopappus gracilis is thus a useful material for the further study of growth and morphogenesis by tissue culture techniques.     

Chemical and Physical Factors Influencing Growth of Phytophthora parasitica var. nicotianae on Vegetable Oils

Factors influencing the growth of Phytophthora parasition var. nicotiona on vegetable oil liquid media were studied. This fungus grew poorly on oleic acid, triolein, and tristearin. Cholesterol and α-tocopherol reversed the toxicity of oleic acid. Growth was often stimulated by addition of mineral oil to a glucose medium, and sterols dissolved in mineral oil frequently stimulated grwoth. Shaking produced a 5-fold or more increase in growth on either glucose or cottonseed oil media. Cholesterol (20 μ/ml further increased growth on glucose meidum in shake culture by at least 100%. Growth was usually better at pH 6.7 than at 5.4 or 7.7. Growth in shake culture on a glucose medium supplemented with cholesterol and α-tocopherol approached that occuring on vegetable oil media.     

The Spread of Infection by the Microsporidan,Nosema disstriae,in Insect Cell Lines1

Nosema disstriae, a parasite of the forest tent caterpillar, Malacosoma disstria, was cultured with cell lines UMN-MDH-1 (Malacosoma disstria), IPLB-1075 (Heliothis zea), and BTC-32 (Triatoma infestans). Infected cultured cells were used to infect the healthy cell lines. Electron micrographs of thin sections of 6-day-old cultures revealed infected cells that exocytosed vesicles containing vegetative and immature sporulating forms of the parasite. Some of these forms were believed to be responsible for intercellular transmission of the parasite. The spread of infection was augmented by culturing the cells at high densities; if the density was too low, there was little or no cross infection. Cross infection was inhibited, but not blocked completely, by high osmolality of the culture medium. The yield of spores from a confluent cell monolayer at the end of growth was generally 1–4 × 10⁷ per ml of culture medium.     

Effects of medium and substratum conditions on the rates of DNA synthesis in primary cultures of bile ductular epithelial cells

Summary Select medium and substratum conditions were investigated for their effects on semiconservative DNA synthesis in essentially pure primary cultures of bile ductular epithelial cells that were initially isolated from cholestatic rat livers at 6 to 10 wk after bile duct ligation. DNA synthesis in these cultured cells was serum-dependent, being at its highest level when the concentration of fetal bovine serum present in the medium was maintained at 10%. This serum-dependent DNA synthesis was completely inhibited when 10 mM hydroxyurea was also included in the medium, and bile ductular cells cultured in the continued presence of 1.0% fetal bovine serum showed only marginal DNA synthesis during 8 to 10 d of primary culture when compared with no-serum controls. Maximum rates of serum-dependent DNA synthesis were obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with either fibronectin from bovine plasma or type I rat-tail collagen. Cells cultured on plastic coated with basement membrane Matrigel exhibited the lowest levels of DNA synthesis, whereas those on plastic alone had intermediate amounts. Furthermore, the addition of epidermal growth factor (50 ng·ml⁻¹·d⁻¹) to medium supplemented with 1.0% fetal bovine serum greatly enhanced the rate of DNA synthesis in bile ductular cells after 6 d in primary culture on type I collagen-coated plastic over that measured in solvent control cultures. These findings indicate that our bile ductular epithelial cell culture model is potentially useful in the study of biliary cell growth regulation and carcinogenesis. This investigation was supported by USPHS grant RO1 CA 39225 to A. E. Sirica by the National Cancer Institute, Department of Health and Human Services, Bethesda, MD. During the period of this study, G. A. Mathis was a recipient of a Fellowship from the Fund for Academic Career Development of the State of Zurich, Switzerland.     

Stability of the cytokinin requirement in Paul's scarlet rose cells in culture

Summary Cells ofRosa sp. cv Paul's scarlet have been reported to require cytokinin for growth in suspension culture. This report was verified in the present study. However, a rose cell line that was maintained for 2 yr in suspension culture by routine subculturing developed the capacity to grow without exogenous cytokinin. The stability of the cytokinin requirement and the basis for this altered response to cytokinin was investigated. The parental cell line, which has been maintained independently on agar-solidified medium, was subcloned and the cytokinin dependence of the subclones was determined. The subclones were found to exhibit a continuous spectrum of responses, ranging from a high degree of cytokinin dependence for growth to rapid growth upon the initial transfer to cytokinin-deficient medium. The average growth constant (K=1n W/Wo; Wo=initial fresh weight,W=fresh weight after growth for the stated time interval) of 30 subclones grown on medium containing 0.5 μM zeatin was 3.1, with a range of 1.1 to 4.0. The average growth constant of the same subclones when grown on medium lacking a cytokinin was 1.5, with a range of 0 to 3.9. By comparison, the parental cell line exhibited growth constants of 3.5 in the presence of 0.5 μM zeatin and 1.6 in the absence of exogenous cytokinin. Although the growth of some of the subclones after transfer to cytokinin-deficient medium suggested that they were cytokinin autotrophs, this was not the case because none of them grew after a second transfer to medium lacking cytokinin. Culture in medium containing cytokinin conferred upon the cells the capacity for a limited amount of growth after subsequent transfer to medium lacking cytokinin. The extent of this cytokinin-induced growth potential varied from subclone to subclone. Efforts to determine the frequency with which cytokinin autotrophs appeared in a subclone that required cytokinin suggested that it is a rare event and that the cytokinin requirement is a fairly stable phenotypic characteristic of these cells. This research was supported by Grant PCM 7722398 from the National Science Foundation.     

Effects of the medium ammonium-nitrate ratio on competence for asparagus cell division induced by phytosulfokine-α

The effects of medium ammonium-nitrate ratio on cell proliferation were investigated using a low cell-density culture of Asparagus officinalis L., which was triggered by a peptidal plant growth factor, phytosulfokine-α (PSK-α). The asparagus cells proliferated the most in a medium with an ammonium-nitrate ratio of 0:30 mM and could be maintained without significant loss of responsiveness to PSK-α for at least 96 h from the beginning of culture. In this medium, single cells gave rise to microcalli at initial densities as low as 3.2×10² cells/ml as long as PSK-α was present in the medium. Increasing the ammonium-nitrate ratio resulted in severe inhibition of cell proliferation at a low cell density, even if PSK-α was added to the medium. Received: 6 May 1997 / Revision received: 5 August 1997 / Accepted: 2 September 1997     

Establishment and characterization of immortalized cell lines from rat parotid glands

Summary This study reports for the first time the establishment of immortalized cell lines from normal adult rat parotid glands. The freshly prepared cellular clumps obtained from parotid glands of isoproterenol-treated rats were incubated in 0.2% trypsin solution without EDTA. These clumps were transfected with plasmid vectors pSV ₃ ^neo and pSV ₅ ^neo by electroporation and calcium phosphate-Co-DNA-precipitation techniques. The untransfected and transfected cellular clumps were plated in precoated dishes containing modified MCDB-153 medium. Epithelial cells grew from the clumps that were attached. All epithelial cells from untransfected culture died within 6 to 8 wk. Two cell lines which were isolated from transfected cultures subsequently grew on regular tissue culture dishes. One of them, which was isolated from pSV ₅ ^neo transfected cultures, exhibited non-epithelial cell morphology, but at confluency, many cells mature to acinar-like cells containing numerous granules. The other cell line (2RS), which was isolated from pSV ₃ ^neo transfected culture, contained cells of non-epithelial and epithelial morphology. During the initial phase of the growth, MCDB-153 medium was essential; however, at a later time, RPMI medium was better than MCDB-153 or F12 medium for maintaining morphology and growth of these cells. The immortalized cells grew in RPMI with a doubling time of about 25 h, synthesize T-antigen,α-amylase mRNAs of 1176 and 702 bp, andα-amylase and were non-tumorigenic. These amylase-producing cells can be a useful model to study the mechanisms of regulation of growth and differentiation in these cells.     

An evaluation of lipid metabolism in the insect trypanosomatid Herpetomonas muscarum uncovers a pathway for the uptake of extracellular insect lipoproteins

Lipid uptake and metabolism by trypanosomatid parasites from vertebrate host blood have been well established in the literature. However, there is a lack of knowledge regarding the same aspects concerning the parasites that cross the hemolymph of their invertebrate hosts. We have investigated the lipid composition and metabolism of the insect trypanosomatid Herpetomonas muscarum by ³H- palmitic acid and phosphate (³²Pi) and the parasite interaction with Lipophorin (Lp) the main lipid carrying protein of insect hemolymph. Gas chromatography-mass spectrometry (GC–MS) analyses were used to identify the fatty acids and sterols composition of H.muscarum. Furthermore, we investigated the Lp binding site in the plasma membrane of parasite by Immunolocalization. We showed that H. muscarum incorporated 3H-palmitic acid and inorganic phosphate (32Pi) which were readily used as precursor molecules of lipid biosynthetic pathways. Furthermore, H. muscarum was able to take up both protein and lipid moieties of Lp which could be used as nutrient sources. Moreover, we have also demonstrated for the first time the presence of a Lp binding site in the membrane of a parasite. Such results point out the role of describing the metabolic pathways of trypanosomatids in order to provide a better understanding of parasite-host interaction peculiarities. Such studies may enhance the potential form the identification of novel chemotherapeutic targets in harmful parasites.