Interior Archives_1(2013)_000pag_2-6 - RAMI Editorial Board

More documents

Recommendations

Info

VLADIMIR F. NICULESCU at 4°C (up to 30 days). Opsonized bacterial cells are added for the Aa(Sm) culture medium which is now ready for growing amoebae. En- and excystment experiments were started with freshly harvested bacteria. Inoculum The culture was carried out in 10 ml centrifuge glass U-tubes containing 5 ml of CM, 5 mg of A. aerogenes AH8 bacteria and 0.5 or 1.0 x 10 6 amoebae. The bacteria were inhibited by a combination of streptomycin and erythromycin. After addition of the amoeba inoculants, the tubes were centrifuged at 1,000 rpm to gently form a solid breeding sediment on the tube bottom and incubated at 28°C. The antibiotic-repressed bacterial mass provides the redox environments for optimal amoebic growth. Amoe bae consumed bacteria in the sediment. The medium remained transparent. Bacterial superinfections producing opacity and precipitation were therefore easy to detect. Aa(Sm)-CM supports ex-cystment, growth and cyclic encystment of E. invadens. All work was performed under sterile conditions. Quantitative analysis The size of the amoeba population was determined using a hematologic counting chamber. All cell counts were performed under microscope. Cysts were identified visually. In order to evaluate the population density of amoebae the contents of a culture tube needed to be homogenized by stirring. The contents of culture tubes (culture sediment and liquid) were mechanically homogenized by stirring and sucked up and down using a sterile 2 ml glass pipette. The contents of the culture tubes were thus also aerated. This is a serious problem as evaluation of each culture altered the redox environment and influenced later development in the Aa(Sm) culture. The effects of changing redox status due to the aeration caused by the first measurement were examined, by taking a second measurement at a later point. After sampling, the culture sediment was recovered by centrifugation. Later to minimize the impact of counting on population kinetics, many duplicate samples were started in parallel, each of which may be examined only once, then discarded. RESULTS Culture sediments with oxygen consu - ming bacteria provided optimal hypoxic conditions for growth and development of E. invadens Work previous to this study showed that E. invadens preferred low oxygen levels and opsonized A.aerogenes. Aminoglycosides such as streptomycin and macrolides such as erythromycin added in moderate doses to the Aa(Sm) stopped bacterial growth by inhibiting protein synthesis. The advantage of the Aa(Sm) sediment culture is the hypoxic dynamics occurring during the interplay between the antibiotically regulated bacteria and the phagocytic amoebae. Amoebae consume the bacteria by predation reversing the hypoxia. The initial consumption of oxygen by the bacteria and the subsquent consumption of the bacteria by the growing amoeba modulate the hypoxic conditions causing chan - ges in growth phases and development in E. invadens (Fig. 1). The large number of bacteria at the beginning of growth and the rapid oxygen depletion in the early initiation phase (0-2 h) induce mitotically active cells. During the growth phase (4-72 h) when amoebae feed on the oxygen consuming bacteria from the culture sediment, hypoxia is gradually reversed. As opposed to axenic culture media the environmental changes occuring in the Aa(Sm) culture, mimic the extracellular signaling from natural habitats and reveal new insights concerning growth, cell cycle and multicellularity in developmental populations of E. invadens. Growth and differentiation The initiation phase (0 - 4 h)*. The initiation phase begins at t0 and ends at t4 when amoebic population has doubled. In the early initiation phase (0-2 h) dissolved oxygen in the sediment is consumed rapidly by the bacteria. Mitotic activators cause all passaged cells to reenter and continue the cell cycle. In the late subphase (2-4 h) amoeba make the first asymmetric cell division; The growth phase I (5 - ca 30 h) * . With continuing growth the amoeba population increases and the bacteria are consumed decreasing their population. 28 - 30 h after culture * The duration of individual stages of growth indicates average time periods 28
Multicellularity in single-celled eukaryotes t0 t4 t30 t72 t96 initiation growth I growth II pre-stationary stationary 3,5 3,0 2,5 2,0 1,5 1,0 0,5 c v c v c v c v c (cysts) v (vege-tative cells) t 0 24 48 72 96 c( x 104) 0.60 3.00 3.50 3.45 3.50 v( x 106) 0.50 1.08 1.54 2.04 2.01 Ratio(%) 1.20 2.77 2.27 1.69 1.74 Ratio=c/v Fig. 1 - TD6 LT and QD24 LT in the cyst producing LT- culture of E. invadens The LT- passage was F started with 5 ml of culture medium CM and 5 mg of bacteria in the culture sediment. It began with 0.5 x 10 6 large QD24 LT cells and 0.6 x 10 4 TD6 LT cells. A population of about 2 x 10 6 large vegetative cells (v) reached the pre-stationary phase, 72 h after the start. The number of cysts (c) at the end of the growth phase has increased to 3.5 x 10 4 . The total population of large vegetative cells was then up to 98%, which did not form cysts and death in the apoptotic phase. The ratios values showed the relative numbers of cysts in comparison to the prevailing vegetative cells. The different kinetics of the two cell lines has a ratio reduction from 2.77% (t0) to 1.69% (t72) inoculation, decreasing hypoxia arrested proli - fera tion of the cyst-producing subpopulation, as described below. This critical point marks the passage from the more hypoxic growth phase I to the low hypoxic growth phase II; The growth phase II (30 - 72 h)*. In this phase only the cyst-free subpopulation continues proliferation and consumes the bacteria until the hypoxic growth stimuli no longer exist. At the end of this phase 80 % of the cyst-free subpopulation is mitotically inactive as a quiescent cell fraction continuing phagocytosis. The few remaining bacteria provided cannot sustain hypoxic conditions. The pre-stationary phase (72 - 96 h)*. In this phase the number of cell divisions is clear. But in some cases other than Fig. 1 a few symmetric divisions were observed. The stationary phase (97 - 124 h)*. Cultures enter this absolute stationary phase when net growth is zero and the amoebic subpopulations experience oxidative stress and other growth limiting factors. The vegetative death phase begins at the moment when vegetative cell metabolism can no longer be maintained in the culture. 144 h after start, the vegetative cells showed increased lysis, but not encystment. Their senescence was irreversibly programmed. Thus, in the first 3-4 h of recultivation about one-third of the 144 h inoculum went apoptotic. Subpopulations During a period of four years more than 100 passages of multicellular Aa(Sm) culture were examined. In the long term cultures (LT culture), two subpopulations were observed, namely (i) a major significant cystfree subpopulation (90-98% of total population) and (ii) a minor cyst-containing subpopulation which supports cell -cycle associated encystment. An additional metacystic subpopulation was generated by excysted amoebulae, however it was quantitatively insignificant in the culture from Fig. 1 (
Page 1 and 2: ROMANIAN ARCHIVES OF MICROBIOLOGY A
Page 3 and 4: ROMANIAN ARCHIVes OF MICROBIOLOGY A
Page 5 and 6: UNDeRsTANDING THe MOLeCULAR TARGeTs
Page 7 and 8: Understanding the molecular targets
Page 15 and 16: AGeNÞI TeRAPeUTICI CU ACÞIUNe DIR
Page 17 and 18: Agenþi terapeutici cu acþiune dir
Page 25 and 26: GROWTH OF ENTAMOEBA INVADENS IN SED
Page 27: Multicellularity in single-celled e
Page 31 and 32: Multicellularity in single-celled e
Page 37 and 38: seDIMeNTe CU BACTeRII AeROBe RePRIM
Page 39 and 40: Sisteme multicelulare la eucariotel
Page 49 and 50: TWO CAse RePORTs ON VIsCeRAL LeIsHM
Page 51 and 52: Two case reports on visceral leishm
Page 57 and 58: Douã rapoarte de cazuri de leishma
Page 63 and 64: ADeNYLATe CYCLAses INVOLVeMeNT IN P
Page 65 and 66: Adenylate cyclases involvement in p
Page 75 and 76: IMPLICAReA ADeNILAT CICLAZeLOR ÎN
Page 77 and 78: Implicarea adenilat ciclazelor în
Page 79 and 80:
Implicarea adenilat ciclazelor în
Page 81 and 82:
Page 83 and 84:
Page 85 and 86:
show all

Interior Archives_1(2013)_000pag_2-6 - RAMI Editorial Board

Create successful ePaper yourself

Delete template?

Save as template?