Drop In Cell Numbers Research Articles

The Great Five\u2014an artificial bacterial consortium with antagonistic activity towards Pectobacterium spp. and Dickeya spp.: formulation, shelf life, and the ability to prevent soft rot of potato in storage

“The Great Five” (GF) is an artificial bacterial consortium developed to protect potato tubers from soft rot caused by Pectobacterium spp. and Dickeya spp. To investigate the commercialization potential of the GF, we developed liquid and powder formulations of the consortium and of each of the comprising strains (Serratia plymuthica strain A294, Enterobacter amnigenus strain A167, Rahnella aquatilis strain H145, Serratia rubidaea strain H440, and S. rubidaea strain H469). To form powders, the cells were lyophilized using a newly developed lyoprotectant: Reagent PS. The shelf life of the formulations stored at 8 and 22 °C was monitored for a period of 12 months. The longest shelf life was obtained for formulations stored at 8 °C; however, the viability of all formulations was negatively affected at 22 °C. For the consortium, a 2.5 log10 cfu (colony forming units) drop in cell number was recorded for the liquid formulation after 6 months, while in case of powders, the drop remained below 1 log10 cfu following 12 months. The ability of the powder formulations to preserve biocontrol activity of the consortium was tested on potato tubers treated with the formulations and a mixture of the soft rot pathogens. The inoculated tubers were stored for 6 months at 8 °C to mimic commercial storage conditions. Soft rot severity and incidence on potato tubers treated with formulations were significantly reduced (62–75% and 48–61%, respectively) in comparison to positive control with pathogens alone. The potential use of the newly developed formulations of “The Great Five” for the biocontrol of soft rot is discussed.Key Points• An innovative reagent to protect bacterial cells during lyophilization was developed.• Powder formulations of “The Great Five” prolonged its shelf life.• The powder-formulated “The Great Five” was active against soft rot bacteria on potato tubers.

Sub‐seafloor biogeochemical processes and microbial life in the Baltic Sea

The post-glacial Baltic Sea has experienced extreme changes that are archived today in the deep sediments. IODP Expedition 347 retrieved cores down to 100 m depth and studied the climate history and the deep biosphere. We here review the biogeochemical and microbiological highlights and integrate these with other studies from the Baltic seabed. Cell numbers, endospore abundance and organic matter mineralization rates are extremely high. A 100-fold drop in cell numbers with depth results from a small difference between growth and mortality in the ageing sediment. Evidence for growth derives from a D:L amino acid racemization model, while evidence for mortality derives from the abundance and potential activity of lytic viruses. The deep communities assemble at the bottom of the bioturbated zone from the founding surface community by selection of organisms suited for life under deep sediment conditions. The mean catabolic per-cell rate of microorganisms drops steeply with depth to a life in slow-motion, typical for the deep biosphere. The subsurface life under extreme energy limitation is facilitated by exploitation of recalcitrant substrates, by biochemical protection of nucleic acids and proteins and by repair mechanisms for random mismatches in DNA or damaged amino acids in proteins.

Effect of bone graft substitute on marrow stromal cell proliferation and differentiation

Marrow stromal cells (MSCs) are ideally suited for tissue engineered bone grafts since they have the potential to regenerate bone, but may also maintain the homeostasis of the repaired tissue through their ability for self-renewal. An ideal bone graft substitute should support MSC self-renewal as well as differentiation to ensure complete bone defect regeneration and maintenance. The purpose of this investigation was to determine the effect of different substrate materials on MSC expansion and differentiation. Calcium polyphosphate (CPP), bone and hydroxyapatite/tricalcium phosphate (HA/TCP) were seeded with rat MSCs and maintained in culture conditions that promote cell expansion. At 0, 3, 7, 14, and 21 days cell numbers were determined by measuring their metabolic activity using a MTT assay and the frequency of cycling cells by 24 hr BrdU incorporation. Osteogenic, chondrogenic, and adipogenic marker expression in these cultures was measured by qRT-PCR. An initial drop in cell numbers was observed on all substrates. CPP and bone, but not HA/TCP supported an increase in proliferating cells at day 14 and 21. In addition, no upregulation of mature bone markers was observed in cells cultured on CPP and bone, which suggests that these substrates support the expansion of undifferentiated MSCs. In contrast, cell numbers on HA/TCP decreased with time and only rare BrdU positive cells were observed. This decrease in proliferation correlated with the down regulation of osteogenic progenitor markers and the substantial increase in mature osteocyte markers, indicating that HA/TCP favors MSC differentiation and maturation along the osteogenic lineage.

Abstract A72: Assessment of pyuvate dehydrogenase kinase inhibition by dichloroacetate in human colon carcinoma cells by dynamic hyperpolarized 13C MRS and steady state 1H MRS

Abstract Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), which causes the activation of pyruvate dehydrogenase and increases glucose oxidation by promoting the influx of acetyl-CoA into the mitochondria and the Krebs cycle [1]. The aim of this work was to use hyperpolarised 1-13C pyruvate 13C-magnetic resonance spectroscopy (MRS) and conventional 1H MRS to measure lactate formation/production in real-time and in steady state, respectively, in order to study the mechanism of action of DCA and to develop a non-invasive biomarker for response following PDK inhibition. Inhibition of PDK by DCA should cause a drop in lactate production. The hyperpolarised 13C experiment measures the exchange of labeled pyruvate for labeled lactate which has been shown to be proportional to lactate dehydrogenase activity and the availability of substrates as well as cellular NADH, the enzyme co-factor [2]. Conversely, steady state measurements of eupolarised (i.e. unlabelled) lactate production report on the overall flux through glycolysis. Dynamic nuclear polarization (DNP) was used to hyperpolarise labelled 1-13C pyruvate, and to measure lactate formation in real-time in intact cells by 13C-MRS. Intact human HT29 (colon) carcinoma cells were studied after 24 hours of DCA treatment (100mM). Culture media and extracts from the DCA-treated and control cells were analysed by 1H-MRS. Cell cycle analysis was also performed. DCA treatment of HT29 cells caused a drop in cell number (∼40% of controls, p<0.0001) and G1 arrest (p<0.0001). A dramatic drop in lactate formation (0.69+/−0.06 nmol/s/106 cells in control versus 0.06+/−0.01 nmol/s/106 cells in treated cells; p<0.0001) was measured in real-time by DNP 13C-MRS. This could be due to the combined effect of: i) a drop in cellular NADH due to apoptosis, as DCA is known to induce apoptosis [1] which causes NADH loss [2]; and/or ii) an increase in glucose oxidation causing a decrease in NADH available for further metabolism of pyuvate to lactate. 1H spectra of the culture media of DCA treated cells also showed a reduction in steady state eupolarized lactate production (38% of controls; p<0.0001), increased alanine uptake (p<0.0001) and no difference in glucose uptake when compared with controls. Increases in alanine (p<0.0001), glucose (p=0.02), free choline (p=0.02) and glycerophosphocholine (p<0.0001) and decrease in phosphocholine (p<0.0001) were found in DCA-treated cell extracts. The phospholipid changes could be associated with modulation in membrane turnover, as DCA is known to reduce proliferation in cancer cells [1]. DCA treatment resulted in reduced lactate formation/production in HT29 cells, as shown by both real time and steady-state measurements. These changes are consistent with the mechanism of action and cellular response to PDK inhibition. DCA treatment also altered phospholipid metabolism, which could also be used as biomarkers of response. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A72.

Noninvasive Magnetic Resonance Spectroscopic Pharmacodynamic Markers of the Choline Kinase Inhibitor MN58b in Human Carcinoma Models

MN58b is a novel anticancer drug that inhibits choline kinase, resulting in inhibition of phosphocholine synthesis. The aim of this work was to develop a noninvasive and robust pharmacodynamic biomarker for target inhibition and, potentially, tumor response following MN58b treatment. Human HT29 (colon) and MDA-MB-231 (breast) carcinoma cells were examined by proton (1H) and phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment with MN58b both in culture and in xenografts. An in vitro time course study of MN58b treatment was also carried out in MDA-MB-231 cells. In addition, enzymatic assays of choline kinase activity in cells were done. A decrease in phosphocholine and total choline levels (P < 0.05) was observed in vitro in both cell lines after MN58b treatment, whereas the inactive analogue ACG20b had no effect. In MDA-MB-231 cells, phosphocholine fell significantly as early as 4 hours following MN58b treatment, whereas a drop in cell number was observed at 48 hours. Significant correlation was also found between phosphocholine levels (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008) following MN58b treatment. Phosphomonoesters also decreased significantly (P < 0.05) in both HT29 and MDA-MB-231 xenografts with no significant changes in controls. 31P-MRS and 1H-MRS of tumor extracts showed a significant decrease in phosphocholine (P < or = 0.05). Inhibition of choline kinase by MN58b resulted in altered phospholipid metabolism both in cultured tumor cells and in vivo. Phosphocholine levels were found to correlate with choline kinase activities. The decrease in phosphocholine, total choline, and phosphomonoesters may have potential as noninvasive pharmacodynamic biomarkers for determining tumor response following treatment with choline kinase inhibitors.

Repeated Mifepristone And Polyethylene Glycol Exposure Prevent Intestinal Lymphocyte Loss After Treadmill Exercise In Mice

0910 Strenuous physical exercise is associated with lymphocytopenia and compromised immune function. Glucocorticoids (GC) and oxidative stress (ROS) produced by exercise are two factors that may be responsible for this lymphocyte cell loss. Purpose: To determine if the GC receptor antagonist mifepristone (MIF) and polyethylene glycol (PEG), commonly used for MIF solubilization and which inhibits ROS production and repairs membrane damage, prevent exercise-induced intestinal lymphocyte cell loss. Methods: One hundred and fifty-three female C57BL/6 mice were given (i) a single s.c. injection of control solution (vehicle), (ii) a single injection of MIF, (iii) repeated injections of MIF solubilized in PEG, or (iv) repeated injections of PEG. Following injection mice were exposed to 90 min of treadmill exercise (28m·min−1, 2° slope) and sacrificed immediately or 24h after exercise. Control mice were exposed to treadmill noise and vibration without running. Following sacrifice intestinal lymphocytes (IL) were isolated and analysed for T and natural kill (NK) subpopulations using flow cytometry. Plasma corticosterone (CORT) was determined by RIA. Results: Plasma CORT was significantly elevated in repeated MIF, nonexercised mice (mean ± SEM; 126.8 ± 3.9ng·ml−1) compared with vehicle (67.9 ± 2.9ng·ml−1), single MIF (77.1 ± 6.8ng·ml−1) and PEG (61.7 ± 6.6ng·ml−1) injected mice (p < 0.001). After exercise there was a significant decrease in total IL recovered from the bowel as well as in CD3+, CD3γδ+, CD8+, CD4+ subsets in vehicle and single MIF conditions. This exercise-associated cell loss was not observed following repeated MIF and PEG injections. However, repeated MIF and PEG failed to inhibit the exercise-associated loss in NK+ cells isolated from the intestine 24h post-exercise. Conclusion: Postexercise IL, but not NK cells, depletion may be mediated by oxidative stress rather than directly by GC: repeated MIF (solubilized in PEG) and PEG alone attenuated the post-exercise drop in cell numbers whereas vehicle and single MIF were associated with pronounced IL depletion. Further studies are needed to confirm whether oxidative stress is the underlying mechanism for depletion in intestinal lymphocytes 24h after exercise. Research supported by a grant from NSERC Canada (LHG).

Effects of brevetoxins on murine myeloma SP2/O cells: aberrant cellular division.

Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.

Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord.

Mesenchymal stem cells (MSCs) have the capability for renewal and differentiation into various lineages of mesenchymal tissues. These features of MSCs attract a lot of attention from investigators in the context of cell-based therapies of several human diseases. Despite the fact that bone marrow represents the main available source of MSCs, the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferative/differentiation capacity with age. Thus, the search for possible alternative MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells and does not contain mesenchymal progenitors. However, MSCs circulate in the blood of preterm fetuses and may be successfully isolated and expanded. Where these cells home at the end of gestation is not clear. In this investigation, we have made an attempt to isolate MSCs from the subendothelial layer of umbilical cord vein using two standard methodological approaches: the routine isolation of human umbilical vein endothelial cell protocol and culture of isolated cells under conditions appropriate for bone-marrow-derived MSCs. Our results suggest that cord vasculature contains a high number of MSC-like elements forming colonies of fibroblastoid cells that may be successfully expanded in culture. These MSC-like cells contain no endothelium- or leukocyte-specific antigens but express alpha-smooth muscle actin and several mesenchymal cell markers. Therefore, umbilical cord/placenta stroma could be regarded as an alternative source of MSCs for experimental and clinical needs.

TGF-β1 inhibits polyamine biosynthesis in K 562 leukemic cells

The present study proved that TGF-beta 1 significantly inhibited the growth of K 562 cells. The drop in cell numbers after 24 h incubation with increasing concentrations of TGF-beta 1 (0.01, 0.1, 1.0, 10.0 ng/ml) was accompanied by significant suppression of the activity of two key enzymes of polyamine biosynthesis: ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). In contrast to ODC and SAMDC activity, TGF-beta 1 did not significantly affect the absolute concentration of spermidine and spermine in K 562 cells. We suppose that the lack of an evident drop in concentration of spermidine and spermine in spite of a significant decrease in ODC and SAMDC activity in K 562 cells exposed to TGF-beta 1 resulted from the uptake of polyamines from the extracellular space.

Growth, copper-tolerant cells, and extracellular protein production in copper-stressed chemostat cultures of Vibrio alginolyticus

The influence of elevated copper concentrations on cell numbers and extracellular protein production was investigated in chemostat cultures of Vibrio alginolyticus. High (20 microM) copper in the medium reservoir resulted in a dramatic drop in cell numbers which was overcome with time. The copper-stressed cultures established a new equilibrium cell concentration slightly (ca. 20%) lower than control cultures. Copper-stressed chemostat populations contained an increased number of copper-resistant cells, but these averaged only 26% of the copper-adapted population. Previously copper-stressed populations exhibited resistance to a second challenge with copper. Proteins with properties identical to those of copper-induced, copper-binding proteins (CuBPs) observed in batch cultures of V. alginolyticus were observed in the supernatants of copper-stressed chemostat cultures and not in controls. CuBPs from batch and chemostat cultures were identical in terms of their induction by copper, molecular weight, and retention volumes on both immobilized copper ion-affinity chromatography and reverse-phase high-performance liquid chromatography columns. The concentration of CuBP in the chemostat was dependent on copper concentration in the medium reservoir. Either one or two forms of CuBP were observed in various analyses from both batch and chemostat cultures. Gel-to-gel variability was implicated as a factor determining whether one or two forms were resolved in a given analysis.(ABSTRACT TRUNCATED AT 250 WORDS)