Decreasing Cell Concentration Research Articles

The YBR056W-A and Its Ortholog YDR034W-B of S. cerevisiae Belonging to CYSTM Family Participate in Manganese Stress Overcoming

The CYSTM (cysteine-rich transmembrane module) protein family comprises small molecular cysteine-rich tail-anchored membrane proteins found in many eukaryotes. The Saccharomyces cerevisiae strains carrying the CYSTM genes YDRO34W-B and YBR056W-A (MNC1) fused with GFP were used to test the expression of these genes under different stresses. The YBR056W-A (MNC1) and YDR034W-B genes are expressed under stress conditions caused by the toxic concentrations of heavy metal ions, such as manganese, cobalt, nickel, zinc, cuprum, and 2.4-dinitrophenol uncoupler. The expression level of YDR034W-B was higher than that of YBR056W-A under alkali and cadmium stresses. The Ydr034w-b-GFP and Ybr056w-a-GFP proteins differ in the cellular localization: Ydr034w-b-GFP was mainly observed in the plasma membrane and vacuolar membrane, while Ybr056w-a-GFP was observed in the cytoplasm, probably in intracellular membranes. The null-mutants in both genes demonstrated decreased cell concentration and lytic phenotype when cultivated in the presence of excess manganese. This allows for speculations about the involvement of Mnc1 and Ydr034w-b proteins in manganese stress overcoming.

Effects of Refrigeration at 5°C for Long Periods of Time on Bovine Ear Skin as a Strategy to Transport Biological Material and Isolate Fibroblasts to Use in the Nuclear Transfer.

Animal cloning is an important technique used to produce clones from valuable farm animals, to rescue animals in risk of extinction, and for producing transgenic animals. The objective of this work was to evaluate the effects of refrigeration on bovine ear skin as a strategy to transport biological material for long periods of time to isolate viable fibroblasts. Ears from eight cows were collected after death and stored for 30 days at 5°C. On days 0, 2, 4, 7, 14, 21, and 30, skin biopsies were cultured in vitro for fibroblast isolation. The time for first fibroblast outgrowth, time to reach 100% confluence. and cell concentration before freezing were observed for each period. In addition, plasma membrane integrity, cell apoptosis, and necrosis in cells were evaluated through fluorescent colorant combination in a flow cytometer from all periods after thawing. Fibroblasts obtained after 30 days of storage, considered a critical period, were tested for embryo production using nuclear transfer (NT) with micromanipulators. All time points allowed for cell culture. The time of cell growth onset was longer in samples refrigerated for 14, 21, and 30 days. The time to reach confluence also increased with longer refrigeration periods. Cells from day 0 reached confluence in 24 ± 2 days, while day 30 cells took 31 ± 0 days. Cell concentration and viability dropped with increased storage time and freezing/thawing, respectively. It was found that a long period of sample storage results in cell damage, making cultivation more difficult and decreasing cell viability post-thawing and cell concentration. However, when cells from day 30 were used as nuclei donors in NT, a 26.05% blastocyst rate after 7 days in culture was obtained. In conclusion, refrigeration at 5°C was shown to be efficient in maintaining viable tissue for up to 30 days, and fibroblasts isolated can be used for cloned embryo production.

Proteasome inhibition rapidly exacerbates photoinhibition and impedes recovery during high light stress in Chlamydomonas reinhardtii

BackgroundProteasomes remove regulatory proteins in eukaryotic cells, and control a variety of plant processes. Proteasomes are localized to the cytosol and nuclear, but their role in plant biology has recently been extended to chloroplasts, where it regulates TOC complex. This is turn controls the import of nuclear-encoded chloroplastic proteins, which remodels the chloroplast proteome and facilitates proper developmental transitions. Proteasomal regulation of the TOC complex also alleviates stressors that generate reactive oxygen species. These recent advances motivated us to determine if proteasome inhibition rapidly alters photosynthetic processes stemming from photoinhibition induced by high light.ResultsThe short-term effects of proteasome inhibition on photosystem II during light stress was measured in Chlamydomonas reinhardtii, which allowed the dual monitoring of both chlorophyll fluorescence and cell viability. After 48 h at low light, proteasome inhibition did not affect viability or photochemistiry, but decreased cell concentration and increased cell volume. Two hours of high light stress impaired the efficiency of photosystem II in proteasome-inhibited cells, as determined by a decrease in Fv/Fm and the electron transport rate. Elevated photoinhibition in proteasome inhibited cells was not caused by a decrease in cell viability or chlorophyll content. Recovery from photoinhibition was attenuated in MG132-treated cells, and suppressed growth of a reestablished culture. Proteasome inhibition decreased de novo protein synthesis, which possibly constrained the ability to remodel the plastid proteome, and thus hampering the ability to adjust to high light stress.ConclusionThe proteasome is implicated in protecting photosystem II from photoinhibition. In addition to high light stress, other stressors- including metals, drought, and salt- are also known to generate reactive oxygen species localized to the chloroplast. Therefore, proteasome maintenance in plants may help protect photosynthesis during abiotic stress, which could increase crop yield during adverse conditions.

Effect of laser fluence, nanoparticle concentration and total energy input per cell on photoporation of cells

Intracellular delivery of molecules can be increased by laser-exposure of carbon black nanoparticles to cause photoporation of the cells. Here we sought to determine effects of multiple laser exposure parameters on intracellular uptake and cell viability with the goal of determining a single unifying parameter that predicts cellular bioeffects. DU145 human prostate cancer cells in suspension with nanoparticles were exposed to near-infrared nanosecond laser pulses over a range of experimental conditions. Increased bioeffects (i.e., uptake and viability loss determined by flow cytometry) were seen when increasing laser fluence, number of pulses and nanoparticle concentration, and decreasing cell concentration. Bioeffects caused by different combinations of these four parameters were generally predicted by their cumulative energy input per cell, which served as a unifying parameter. This indicates that photoporation depends on what appears to be the cumulative effect of multiple cell–nanoparticle interactions from neighboring nanoparticles during a series of laser pulses.

A study of synthesis and properties of poly‐3‐hydroxybutyrate/diethylene glycol copolymers

This study investigates synthesis of poly(3-hydroxybutyrate)/diethylene glycol copolymers (P3HB/DEG) by Cupriavidus eutrophus B-10646 cells as related to DEG concentration in the medium and the time when it is added to the culture of cells synthesizing P3HB. The study determines the limits of physiological effect of DEG on C. eutrophus cells, showing that at DEG concentrations above 30 g/L, it inhibits cell growth, decreasing cell concentration and total P3HB/DEG yield and inducing an increase in the degree of saturation of fatty acids in lipids of cell cytoplasmic membrane. A series of copolymers containing different molar fractions of DEG (between 0.13 and 3.0 mol%) have been synthesized and their physicochemical, physical/mechanical, and biological properties have been investigated as related to the chemical composition and proportions of DEG monomers of the polymers. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1017-1028, 2016.

Cytotoxic potency of H 2O 2 in cell cultures: Impact of cell concentration and exposure time

Using C6 glioma cells in this study we investigated in detail how exposure time and cell concentration affect the cytotoxic potency of H 2O 2 in vitro. Median cytotoxic concentrations (EC 50) decreased from 500 to 30 μM with increasing incubation time from 1 to 24 h. Twenty-four hours proved to be sufficient to determine incipient cytotoxic concentrations of H 2O 2. The incipient EC 50 values were linearly related to the cell concentration. A cell concentration-independent median cytotoxic cell dose (ED 50) of 430 nmol/mg cell protein or 860 nmol/10 7 cells was derived. Median cytotoxic H 2O 2 concentrations were completely eliminated from the culture medium at a rate proportional to both the H 2O 2 and the cell concentrations. In contrast to EC 50 values the corresponding areas under the concentration versus time curve (AUC) were independent of the cell concentration and amounted to 1800 μM × min. With decreasing cell concentration the H 2O 2 elimination decelerates and, thus, exposure to H 2O 2 applied as a bolus approaches a continuous exposure to a steady H 2O 2 concentration. Taken together, our results indicate that the cytotoxic potency of H 2O 2 administered to cultured cells as a bolus is characterized by the AUC, which depends on its initial concentration, the ability of the cells to eliminate H 2O 2, and the cell concentration. We recommend expressing the toxic potency of H 2O 2 in vitro by the incipient toxic cell dose (e.g., nmol H 2O 2/mg cell protein or nmol H 2O 2/10 7 cells), in particular for comparative purposes.

Fluorescence spectroscopy for detection of malignancy: H-ras overexpressing fibroblasts as a model

Autofluorescence from intracellular chromophores upon illumination of cells by monochromatic light has been studied towards the development of novel noninvasive and sensitive technology for the early detection of cancer. To investigate the relationship between biochemical and morphological changes underlying malignant disease and resulting fluorescence spectra, an in vitro model system of a paired normal and malignant murine fibroblasts cell lines, differing in cancer-associated H-ras expression was employed. A comparison of fluorescence excitation and emission spectra of proliferative cells revealed that fluorescence intensity of malignant cells was significantly less than that of normal cells upon excitation at 290 nm. Fluorescence of both cell lines decreased with decreasing cell concentration, but at each concentration, normal cells had higher fluorescence intensity than malignant cells. Similar differences between the cell lines were observed when brought to quiescence or at stationary phase. Results suggested that the chromophore contributing most significantly to these spectra is tryptophan and its moieties in proteins. This model system demonstrates the specific contribution of H-ras to subcellular chromophores, resulting in a significant difference in their autofluorescence intensity, and implies the potential use of the technique for cancer detection. This model system is potent for analysis of the contribution of other oncogenes and their combinations towards spectral detection of cancer.

Production of Bacillus thuringiensis biopesticides using wastewater sludge as a raw material: effect of inoculum and sludge solids concentration

Studies were conducted to optimise inoculum quantity and total solids concentration for the production of Bacillus thuringiensis (Bt) based biopesticides using wastewater sludge as a raw material in shake flasks and 15 l fermenters. The sludge used was a mixture of primary and secondary sludges. Two different Bt strains were employed; Bacillus thuringiensis kurstaki HD-1 (Btk) and a new strain (Bta) isolated from sewage sludge. Preparation of inoculum in the same sludge as that used for the production medium resulted in higher cell and spore counts, specific growth rate and entomotoxicity value. The final cell count and entomotoxicity achieved at the end of fermentation depended on the amount of inoculum used. A cell count of 2.2×10 9 and 3.6×10 9 CFU/ml was observed at 2% (v/v) and 3% (v/v) inoculum volume, respectively. A lower cell count was recorded at 1, 4 and 5% (v/v) inoculum concentration. Bioassay results also revealed that a 2%(v/v) inoculum furnished higher entomotoxicity (12 900 IU/μl) against spruce budworm. The sludge solids concentration significantly influenced growth and entomotoxicity yield. Solids concentration of 46 g/l using Btk resulted in decreased cell concentration (5.4×10 8 CFU/ml), spore concentration (4.8×10 8 CFU/ml) and low entomotoxicity (9743 IU/μl). The sporulation rate was 89%. The same phenomena was observed when a lower sludge solids concentration (10 g/l) was used, resulting in cell and spore concentrations of 4.3×10 8 and 3.9×10 8 CFU/ml, respectively, and potency of 8231 IU/μl. The optimum total solids concentration was 26 g/l, which resulted in an improved potency of 12 970 IU/μl, cell and spore concentrations of 5.0×10 9 and 4.8×10 9 CFU/ml, respectively, and a sporulation rate of 96%. A similar trend of growth and entomotoxicity at different sludge solids concentration was also observed with new Bt strain isolated from sewage sludge. The fermenter results showed that one of the reasons for inferior entomotoxicity at higher sludge solids was oxygen transfer limitation.

Culture conditions for the detection of allergen-specific T-cell reactivity in cord blood: influence of cell number.

Raised T-cell proliferation of cord blood mononuclear cells (CBMC) in response to various ingestant and inhalant allergens has been reported in newborns, suggesting a prenatal allergen contact. In general, for in vitro proliferation assays a concentration of 50 x 10(3) or 100 x 10(3) cells/well are used. The aim of this study was to analyze whether cell concentration influences T-cell reactivity in cord blood cells and to study differences of T-cell reactivity triggered by inhalant and ingestant allergens. CBMC from 51 neonates (34 females: 22 with and 29 without a family history of allergy, i.e. FH+ or FH-) were incubated with interleukin-2 (IL-2), beta-lactoglobulin (beta-LG), ovalbumin (OVA), house dust mite allergen Dermatophagoides pteronyssinus (Der p 1), and timothy grass allergen Phleum pratense (Ph1 p 1) for 7 days. The cell concentration ranged from 6.25 x 10(3) to 100 x 10(3) cells/well. Proliferation was assessed by incorporation of [3H]-thymidine and was expressed as counts per minute (c.p.m.). In unstimulated cells, a decreasing cell concentration paralleled a steep drop of background activity. In response to IL-2, a decreasing cell concentration led to a slow decrease of c.p.m. The corresponding mean stimulation indices (SI) were 9, 32, 77, 47, and 21 for 100 x 10(3), 50 x 10(3), 25 x 10(3), 12.5 x 10(3), and 6.25 x 10(3) cells/well, respectively. In addition, the highest number of positive proliferative responses to specific allergens were obscured at lower cell concentrations. For beta-LG, the maximal number of positive responses were obtained between 25 x 10(3) (n = 44) and 12.5 x 10(3) (n = 46) cells/well, for OVA at 25 x 10(3) (n = 3) cells/well, for Der p 1 at 50 x 10(3) (n = 5) cells/well, and for Ph1 p 1 between 25 x 10(3) and 12.5 x 10(3) (n = 5) cells/well. Positive proliferation in at least one of the tested assays was observed in 100% of samples in response to beta-LG, in 22% in response to Ph1 p 1, and in 14% in response to OVA and Der p 1. T-cell reactivity did not differ between samples of newborns with or without a family history of atopy. Therefore, sensitivity of T-cell proliferation measurement is highly influenced by background proliferation of unstimulated cells. Hence, proliferation assays with lower cell numbers unmask T-cell reactivity in response to ingestant and inhalant allergens. We suggest the use of concentrations of 12.5 x 10(3)-50 x 10(3) cells/well in proliferation experiments.

Ultrasonically induced hemolysis at high cell and gas body concentrations in a thin-disc exposure chamber

Ultrasound image contrast may be enhanced by injecting gas bodies into the blood. This in vitro study was undertaken to assess the potential for induction of hemolysis due to ultrasonic activation of the contrast agent gas bodies. Canine whole blood with Albunex ® (Mallinckrodt Medical, St. Louis, MO, USA) was exposed to near-field ultrasound beams in 1-mm-thick chambers held stationary ( i.e., not rotated) in a 37°C water bath. At 2.25 MHz, statistically significant hemolysis occurred in 0.5 hematocrit, 50% Albunex ® suspensions for 0.28-MPa, 1-s continuous exposure and for 0.58-MPa, 100-s exposures with 10-μs pulses and 1.0-ms pulse repetition period. Continuous exposure durations as short as 10 ms produced about 4.5% hemolysis, which only increased slightly to about 5.5% after 100 s. At a constant 1.6 MPa, hemolysis increased with increasing gas body concentration and with decreasing cell concentration. Hemolysis decreased with increasing frequency in a 50 50 mixture of whole blood and Albunex ®, with thresholds rising from 0.12 MPa continuous (1 s) and 0.47 MPa pulsed (10 μs:1.0 ms for 100 s) at 1.06 MHz to 0.47 MPa continuous and 1.9 MPa pulsed at 5.3 MHz.

Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout (Oncorhynchus mykiss). II. Adaptive response of glucose 6-phosphate dehydrogenase activity to high-carbohydrate/low-protein and high-fat/non-carbohydrate diets

: The influence of long-term administration of high-carbohydrate/low-protein and high-fat/non-carbohydrate diets were studied in relation to kinetic behaviour of glucose 6-phosphate dehydrogenase in liver and kidney of rainbow trout. In all cases studied, the saturation curves of these enzyme showed typical hyperbolic kinetics without evidence of sigmoidicity. After 30 days of feeding with a high-fat diet (170 g kg−1), there was a significant decrease in Vmax and specific activity (45%) as well as catalytic efficiency (39%) without changes in Km or activity ratio of hepatic glucose 6-phosphate dehydrogenase. These changes agree more with a clearly decreased cell concentration than with an inhibition of the pre-existing enzyme. The administration of a high-carbohydrate diet (60 g kg−1), contrary to what was previously thought, decreased Vmax by 21% and specific activity and catalytic efficiency by 30%, without significant changes in the other kinetic parameters of the hepatic enzyme. The kinetic behaviour under these nutritional conditons was due to the rejection of this diet by the fish and thus could be considered a low-feeding situation. On the other hand, no variations in the kinetics of renal glucose 6-phosphate dehydrogenase were found, clearly demonstrating that in this organ, the pentosephosphate pathway showed no adaptive response related to fattyacid and other lipid synthesis. The activity of the renal enzyme was consistently half that of the hepatic enzyme.

Mitochondrial DNA loss by yeast reentry-mutant cells conditionally unable to proliferate from stationary phase.

Double-mutant cells of the budding yeast Saccharomyces cerevisiae harboring the gcs1-1 and sed1-1 mutations are conditionally defective (cold-sensitive) only for reentry into the mitotic cycle from stationary phase. If already proliferating at the permissive temperature (29 degrees C), these reentry-mutant cells continue to proliferate when transferred to the restrictive temperature of 14 degrees C, but under these conditions reentry-mutant cells lose mitochondrial DNA (mtDNA). In addition, upon exhaustion of the nutrient supply at 14 degrees C, these reentry-mutant cells entered stationary phase at a decreased cell concentration and did not accumulate the reserve carbohydrates trehalose and glycogen. Both of these deficiencies were due to the loss of mtDNA, as shown by the responses of wild-type cells also lacking mtDNA. Mitochondrial status did not affect other aspects of the reentry-mutant phenotype. Although mitochondrial activity and the accumulation of carbohydrate reserves are typical features of cells in stationary phase, the reentry-mutant phenotype reveals that neither entry into nor exit from stationary phase need involve mitochondrial function.

Grazing Rates and Food Selection in Diaptomus oregonensis (Copepoda) from Marion Lake, British Columbia

The filtration and ingestion rates of Diaptomus oregonensis females and stage V copepodids were examined in three experimental situations. When unialgal cultures of 20,000 cells per milliliter were offered to D. oregonensis the filtration rates remained constant for cells ranging from 120 to 10,000 µ3. When a mixture of diatoms was offered the animals did not filter cells ranging from 1 to 60 µ3 despite the fact that the cells in this size range were concentrated at about 20,000 per milliliter. When natural phytoplankton from Marion Lake was used as a food source, the filtration rates were low for cells smaller than 100 µ3, increased to a maximum of 12.9 ml per animal per day for cells ranging from 102 to 333 µ3, and remained constant with increased cell volume and decreased cell concentration. Several species of Cyclotella and Merismopedia were not removed from the Marion Lake plankton by D. oregonensis.

Respiration and Glycolysis of Normal Human Lymphocytes

Abstract The metabolism of intact, normal, human lymphocytes in vitro was studied from a total of 80 subjects. Corrected for the metabolism of contaminating red blood cells, the glucose uptake, lactic acid production, and oxygen consumption were 62, 95, and 117 µmoles per 1010 lymphocytes per hour, respectively, provided the cells were incubated at concentrations greater than 40 x 106 lymphocytes per ml. At lower lymphocyte concentrations the oxygen consumption per lymphocyte rose steeply with decreasing cell concentration (crowding effect). A similar but weaker crowding effect was noted for the lactic acid production, but not for the utilization of glucose. The oxygen uptake was lower with 20 per cent than with 100 per cent oxygen as gas phase. Small Pasteur and Crabtree effects were demonstrated. The oxygen consumption and lactic acid production proceeded linear with time, while the glucose utilization was higher during the first 30 minutes of incubation than later on. It is concluded that lymphocytes have a low aerobic glycolysis accounting for 75 per cent of the glucose utilization. The respiration is severely inhibited at high cell concentrations and it is suggested that this is caused by an insufficient availability of oxygen to the cells.