Amount Of Viable Cells Research Articles

Uniform Potent Activity of the Antiproliferative Metal Chelate Trans-Bis(resorcylaldoximato)copper(II) against a Large Panel of Human Tumor Cell Lines in vitro

Background: The copper chelate trans-bis(salicylaldoximato)copper(II), CuSAO₂, is a powerful antitumor agent in vivo, being capable of drastically increasing the life span of mice bearing Ehrlich ascites carcinoma, and has in many cases even a curative effect. This compound as well as some congeners, most notably the 4-hydroxylated analogue trans-bis(resorcylaldoximato)copper(II), CuRES₂, also have potent antiproliferative activity against tumor cells in vitro. Methods: CuRES₂ was tested in vitro against a panel of 37 different types of human tumor cells (leukemias, small cell and non-small cell lung tumors, melanomas, as well as colon, breast, central nervous system, ovarian and renal tumors). The cells were incubated on microculture plates for 6 days, and the amount of viable cells was determined with the agent XTT, whose metabolic reduction gives a colored formazan compound. Results: CuRES₂ inhibited the proliferation of all the 37 cell lines studied. The IC₅₀ values ranged between 0.575 and 8.57 µg/ml and the IC₉₀ values between 1.74 and 20.6 µg/ml. Thus, it had distinct and very potent activity against all the cell lines studied, and the differences between the susceptibilities of different tumor types and cell lines were not very large. Discussion: Clearly, CuRES₂ is active against all the tumor cell lines studied. None of the cell lines (tumor types) demonstrates a significantly higher or lower susceptibility, but some trends appear to exist. Thus, most (but not all) of the leukemia and melanoma cell lines, the two ovarian cancer lines and one small cell lung cancer and one CNS cancer cell line were somewhat more susceptible than the cell lines on average. The results obtained for the melanomas are of special interest, since CuRES₂ (and even more so CuSAO₂) have very low aqueous solubilities. In cutaneous melanomas, their administration might, however, be very easy, since both compounds are soluble in dimethyl sulfoxide and could thus easily be administered topically in this solvent whose ability to be absorbed through the skin is well known. Conclusion: The present results on human tumor cell lines are in line with previous observations that CuSAO₂ and CuRES₂ essentially totally inhibit the proliferation of leukemia L1210 and Ehrlich ascites carcinoma cells in vitro in a concentration as low as ∼5 µg/ml.

Cytotoxicity of different intracavernous vasoactive drugs on cultured endothelial cells of human corpus cavernosum penis

Objectives To investigate the cytotoxic effect of prostaglandin E 1 (PGE 1), a standard combination of papaverine/phentolamine, and a triple mixture of these agents on human cavernosal endothelial cells using a cell culture model. The endothelial layer of the corpus cavernosum plays an important role in signal transduction of penile erection and is directly exposed to vasoactive agents after intracavernous injection for erectile dysfunction. Methods Primary endothelial cells were obtained from the corpus cavernosum of 13 potent patients undergoing penile surgery. Cultured cells were exposed for 30 minutes to physiologic dilutions of 20 μg PGE 1, 30 mg papaverine/1 mg phentolamine, or the same dosages of the triple mixture of these agents, each dissolved in 5 to 50 mL sodium chloride. Lactate dehydrogenase release as a cytotoxicity marker was measured 6 hours after drug exposure, and the total cell metabolic activity was quantified after 48 hours with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Additionally, the amount of viable cells was identified with a dual fluorescent staining procedure. Results The initial release of lactate dehydrogenase was elevated up to 3.2-fold in the concentrated papaverine/phentolamine and triple mixture group compared with PGE 1 and the control. After 48 hours, the papaverine-containing formulations led to a significant dose-dependent decrease in the viable cell count and metabolic activity of the cultures that was not noticed with PGE 1. Conclusions These in vitro data strongly suggest an unfavorable effect of vasoactive agents containing papaverine on cavernosal endothelial cells. Before fibrotic changes of the smooth muscle stroma, the functionally important endothelium of the corpus cavernosum might suffer significantly from intracavernous injection therapy. Therefore, papaverine should no longer be used for this indication.

Particulated bone grafts--effectiveness of bone cell supply.

The objective of this study was to measure the amount of viable bone cells present in different types of bone graft. Bone chips were harvested from the trabecular or cortical bone of the mandible or the iliac crest and either milled or not. The average size of unmilled bone particles was 5 x 5 x 5 mm and that of milled was 2 x 2 x 2 mm. Drill sludge was obtained using either a ball reamer, a diamond ball or an implant drill (the latter from mandibular bone and of average dimension 1 x 1 x 1 mm). A measure of 0.5 g of each category was cultured in Dulbecco's modified Eagle's medium with additives for four weeks. Cell counts were performed. An analysis of the osteocalcin synthesis, the alkaline phosphatase (ALP) activity, the collagen types and the concentration of bone-specific collagen cross-links in medium supernatants was performed. Cells stained positively for osteocalcin and ALP in all groups. Bone-specific collagen cross-links could be quantified and collagen of types I and V was present with no difference in all groups. Unmilled spongy bone chips revealed greater cell counts than milled (P<0.05). Spongy bone chips revealed greater cell counts than cortical bone chips (P<0.05). Drill sludge obtained by hard alloy ball reamer showed the least amount of viable cells (P<0.05). Bone milling reduces the quantity of osteoblasts. Bone obtained by the ball reamer supplies a smaller number of cells than bone obtained by other methods. Unmilled spongy bone chips appear to offer the greatest amount of viable osteoblasts.

VDAC1 Is a Transplasma Membrane NADH-Ferricyanide Reductase

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1-GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis.

Effects of cell damage and glycosaminoglycan degradation on available extravascular space of different dextrans in a rat fibrosarcoma

Drug delivery to solid tumors may be enhanced through increasing the available volume fraction ( K AV ) of drugs. Therefore, two approaches were investigated that may increase K AV of dextrans in a rat fibrosarcoma: (a) damaging cells in tumours via ex vivo incubation of tumour tissues, and (b) degrading tumour glycosaminoglycans (GAGs) with exogenous hyaluronidase. The molecular weights of dextrans used in this study were ~ 10 000 (D10), 70 000 (D70) and 2 000 000 (D2000), respectively. It was found that GAG degradation had minimal effects on K AV of dextrans. Ex vivo incubation at 37°C for up to 3 h caused only minor cell damage and had minimal effects on K AV of D10 and D70. However, the ex vivo incubation reduced K AV of D2000 ( p < 0.05). When the incubation at 37°C was maintained for 20 h, the amount of viable cells in tumours was reduced by 56% and K AV of all dextrans were significantly increased ( p < 0.05). Ex vivo incubation at 41°C for 3 h caused similar cell damage to that at 37°C for 20 h, but only K AV of D10 and D70 were increased significantly ( p < 0.05). There was no significant change in K AV of D2000, although it was higher than that in tumours incubated at 37°C for 3 h ( p < 0.05). These data suggest that cell damage is a more effective approach than GAG degradation for increasing K AV of macromolecules and that the amount of increase depends on the degree of cell damage and the size of molecules.

Effect of process parameters on the production and drying of Leuconostoc mesenteroides cultures.

Leuconostoc mesenteroides BLAC was grown on MRS broth or on a carrot juice medium, and the effects of sugar concentration, type of pH control, aeration and fermentor size on viable counts were examined. The effect on viability of the type of centrifuge used to concentrate the bacterial culture was also examined. When the MRS broth had the traditional 110 mM glucose, pH control did not increase the final population. However, using a zone pH control mode, increasing the glucose content of MRS both from 110 to 220 mM almost doubled the population. In MRS broth, the amount of acetic acid produced was the same for all treatments, and was proportional to the amount of citrate consumed. There was a significantly lower cell yield in the carrot juice medium when the pH was not regulated. In the carrot juice medium, pH had a more pronounced effect on the final population level than did aeration, even though the quantity of viable cells was greater when the culture was aerated. In MRS broth, glucose was completely consumed during fermentation, but this was not the case in carrot juice medium. Aeration resulted in increased acetic acid content of the fermented medium. Viable counts were not affected by scaling the volume of the fermentation from 2 to 15 l,or by the type of centrifuge used to concentrate the cells. Cells were concentrated by a factor of 10, but in both centrifuge types, viable counts showed only an eightfold average increase. However, freeze-dried powders obtained from the continuous pilot-plant-centrifuged cultures had, on the average, 33% lower populations than those obtained from the laboratory unit.

Annexin V expression in apoptotic peripheral blood lymphocytes: an electron microscopic evaluation.

Loss of plasma membrane asymmetry, resulting in the exposure of phosphatidylserine (PS), is considered to be an 'early' event in apoptosis. It is generally accepted to precede nuclear condensation, independent of the apoptosis inductive agent. In the present study we focus on 2 apoptotic parameters: PS exposure in comparison with morphological alterations. Peripheral blood lymphocytes were irradiated in vitro (5 Gy Co-gamma-rays) or incubated with staurosporine (1 microM, 6 hours). PS exposure was measured flow cytometrically using FITC-labelled annexin V, combined with PI. Morphological alterations were evaluated by electron microscopy (EM). Results are based on 3 independent experiments. For the irradiated lymphocytes the amount of viable cells (annexin V-/PI-) as scored by flow cytometry was comparable or slightly lower than the number of viable cells as scored by EM (75% compared to 79%). However, for the staurosporine treated lymphocytes only about 24% of the cells were designated as viable by EM, whereas by flow cytometry about 65% of the cells were annexin V-/PI-. Examination by EM showed about 40% cells with a morphology distinct from that of a normal viable cell, but without the clear-cut characteristics of apoptotic cells. Time studies revealed that these cells went into apoptosis after prolonged incubation times up to 18 hours. Application of biotinilated annexin V for EM detection with gold-conjugated anti-biotin, showed that only clear-cut apoptotic, apoptotic necrotic and oncotic cells showed the gold-label at their membranes. Cells that could be detected under the EM as 'non-viable' but without the clear-cut characteristics of apoptotic cells, were not labelled. Data indicate that, dependent on the apoptosis inductive mechanism, morphological alterations can occur before PS exposure.

Survival of Micromycetes and Actinobacteria under Conditions of Long-Term Natural Cryopreservation

Almost all of the investigated samples of the Arctic and Antarctic permafrost sediments of different genesis with ages from 5–10 thousand to 2–3 million years were found to contain viable micromycete and bacterial cells. The maximum amounts of viable cells of fungi (up to 104CFU/g air-dried sample) and bacteria (up to 107–109CFU/g air-dried sample) were present in fine peaty sediment samples taken from different depths. The identified micromycetes belonged to more than 20 genera of the divisions Basidiomycota, Ascomycota, and Zygomycota, and some represented mitosporic fungi. Thawing the samples at 35 and 52°C allowed the number of detected fungal genera to be increased by more than 30%. Aerobic heterotrophic prokaryotes were dominated by coryneform, nocardioform, and spore-forming microorganisms of the order Actinomycetales.Analysis of the isolated fungi and actinomycetes showed that most of them originated from the microbial communities of ancient terrestrial biocenoses.

In vitro chemosensitivity testing in acute non lymphocytic leukemia using the bioluminescence ATP assay

The ATP assay is a short term in vitro chemosensitivity assay where the amount of viable cells are determined by their content of ATP. The aim of the study was to compare the in vitro results of six cytostatic drugs to the clinical outcome in 83 acute non-lymphocytic leukemia (ANLL) patients. The secondary ANLL at diagnosis showed an in vitro resistance to daunorubicin that was significantly higher compared to de novo ANLL at diagnosis ( P<0.003). De novo ANLL at diagnosis that achieved complete remission (CR) were significantly more sensitive to daunorubicin compared to those who didn’t achieve CR ( P<0.05). There was an vitro correlation between topoisomerase II active drugs but not between these drugs and ara-C. In vitro ara-C sensitivity (≤ the median of the de novo ANLL at diagnosis) was correlated to poor overall survival ( P=0.02). In vitro sensitivity to daunorubicin and mitoxantrone was associated with prolonged disease free survival ( P=0.03 and P=0.04). We conclude that despite significant correlation to clinical parameters for daunorubicin and mitoxantrone the predictive value of the ATP assay in this material was insufficient for directing therapy.

Regulatory effect and mechanism of gastrin and its antagonists on colorectal carcinoma.

AIM:To explore the effect and mechanism of gastrin and its antagonists prog lumide and somatostatin on colorectal carcinoma and their clinical significance.METHODS:A model of transplanted human colonic carcinoma was established from SW480 cell line in gymnomouse body.The volume and weight of transplanted carcinoma was observed under the effect of pentagatrin (PG), proglumide (PGL) and octapeptide somotostatin (SMS201-995, SMS). The cAMP content of carcinoma cell was determined by radioimmunoassay and the DNA, protein content and cell cycle were determined by flow-cytometry. The amount of viable cells was determined by MTT colorimetric analysis,IP(3) content was determined by radioimmunoassay, Ca(2+) concentration in cell by fluorometry and PKC activity by isotopic enzymolysis. The expression of gastrin, c-myc, c-fos and rasP21 in 48 cases of colorectal carcinoma tissue was detected by the immuno-cytochemistry SP method. Argyrophilia nucleolar organizer regions was determined with argyrophilia stain.RESULTS:The volume,weight, cAMP, DNA and protein content in carcinoma cell, cell amount and proliferation index of S and G(2)M phase in PG group were all significantly higher than those of control group. When PG was at the concentration of 25mg/L, the amount of viable cells, IP(3) content and Ca(2+) concentration in cell and membrane PKC activity in PG group were significantly higher than those in control group; when PGL was at a concentration of 32mg/L, they dropped to the lowest level in PG (25mg/L)+PGL group, but without significant difference from the control group. The positive expression rate of gastrin, c-myc, c-fos and rasP21 in carcinoma tissue was 39.6%, 54.2%, 47.9% and 54.2% respectively and significantly higher than that in mucosa 3cm and 6cm adjacent to carcinoma tissue and normal colorectal mucosa. The positive expression rate of gastrin of highly differentiated adenocarcinoma group was significantly higher than that of poorly differentiated and mucinous adenocar-cinoma groups. The AgNORs count of carcinoma tissue was significantly higher than that in mucosa 3cm and 6cm adjacent to carcinoma tissue and normal colorectal mucosa; and the positive expression of c-myc and c-fos and the AgNORs count in gastrin-positive group was significantly higher than those in gastrin negative group.CONCLUSION:Pentagastrin has a promoting effect on the growth of transplanted human colonic carcinoma from SW480 cell line. PGL has no obvious effect on the growth of human colonic carcinoma SW480 cell line, but could inhibit the growth promoting effect of PG on transplanted carcinoma. Somatostatin can not only inhibit the growth of transplanted human colonic carcinoma from SW480 cell line directly but also depress the growth-promoting effect of gastrin on the transplanted carcinoma. Some colorectal carcinoma cells can produce and secrete gastrin through autocrine, highly differentiated adenocarcinoma express the highest level gastrin.Endogenous gastrin can stimulate the cell division and proliferation of carcinoma cell and promote the growth of colorectal carcinoma regulating the expression of oncogene c-myc, c-fos. Our study has provided experimental basis for the adjuvant treatment using gastrin antagonist such as PGL, somatostatin of patients with colorectal carcinoma.

Xylitol production in repeated fed batch cultivation

The ability of Candida parapsilosis to produce xylitol was tested using successive substrate supplies, and the importance of the amount of viable cells in enhancing the conversion rate was demonstrated. The suitability of this yeast for the production of xylitol was investigated in repeated fed-batch cultivation, using pure xylose or mixtures of xylose and glucose. The use of this process increased productivity by about 40% compared with simple batch cultivation without loss of yield of product on substrate. The presence of glucose in the culture medium seemed to stimulate the specific growth rate, but had no influence over other fermentative parameters.

Magnesium in Acute MI

It is an axiom of coronary care medicine that infarct size is a pivotal determinant of the prognosis of patients with acute myocardial infarction (AMI). When one is able to limit the size of an AMI, one is rewarded with a reduction in the number of potentially life-threatening complications such as pulmonary edema, cardiogenic shock, ventricular septal defects, papillary muscle rupture, and ventricular tachyarrhythmias.1 Salvage of even a rim of epicardial tissue as the wave front of necrosis spreads from the endocardium might prevent infarct expansion, ventricular remodeling, and congestive heart failure.2 3 Reperfusion that is sufficiently early (within 15 to 20 minutes) after AMI may prevent ischemic zones of myocardium from progressing to infarction. However, with the possible exceptions of an AMI that develops during cardiac catheterization, of patients who receive prehospital thrombolysis, or of the rare patient who occludes a coronary artery in the Emergency Department or coronary care unit and receives extraordinarily prompt and successful thrombolysis, reperfusion in cases of human AMI results in a mixture of necrotic and salvaged myocytes. The amount of myocardium that is irreversibly damaged (area of necrosis divided by area at risk) is directly related to the duration of occlusion. Interventions that delay cell death (eg, β-blockers) may help to protect ischemic myocytes and leave a greater quantity of viable cells that can subsequently be rescued by reperfusion. More than a decade ago, Braunwald and Kloner2 emphasized that reperfusion therapy, while beneficial in terms of myocardial salvage, may come at a cost because of a process referred to as reperfusion injury. Four aspects of reperfusion injury have been recognized4 : (1) lethal reperfusion injury (reperfusion-induced death of cells that were still viable at the moment of restoration of blood flow), (2) vascular reperfusion injury (no-reflow phenomenon and loss …

Distribution and function of central somatostatin (SRIF) receptors

The aim of the work was to investigate the effects of somatostatin analogs acting selectively on sst1 (BIM-23926), sst2 (BIM-23120) and sst5 (BIM-23206) receptor subtypes on the viability of “clinically non-functioning” pituitary adenomas in vitro. The effects of native SST (SST-14), a SST/DA chimera (BIM-23A387) and a D2-dopamine receptor agonist bromocriptine (BC) were also examined. The study was performed on 10 surgically removed pituitary macroadenomas, diagnosed before surgery as “non-functioning”. A part of each tumor was mechanically dispersed and digested with collagenase to isolate the tumoral cells. Another part of each tumor was fixed, embedded in paraffin and immunostained to reveal the pituitary hormones and SST receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, sst5). The tumoral cell suspensions were incubated for 24 h with the substances mentioned above. The quantity of viable cells was estimated using the EZ4U system. The results were compared with the immunohistochemical evaluation of the hormonal profile of adenoma and the sst receptor subtype immunoreactivities present.The findings indicate that selective sst1, sst2 and sst5 receptors agonists, SST/DA chimera and D2-dopamine receptor agonist bromocriptine affect the viability of some, but not all, “clinically non-functioning” pituitary adenomas in vitro. The most effective was bromocriptine. The investigated somatostatin analogs including SST/DA chimera exerted roughly similar inhibitory effects. Further studies are needed to fully evaluate the potential usefulness of these compounds in the pharmacological treatment of “non-functioning” pituitary tumors.

Cold storage of peripheral nerves: an in vitro assay of cell viability and function.

The development of a nerve bank as a source of donor material to repair large defects in peripheral nerve injuries requires an understanding of the influence of cold storage on cell viability and function in these potential nerve grafts. Segments of peripheral nerves from both human and rat were stored in University of Wisconsin Cold Storage Solution (UW) at 4 degrees C for < 12 h, 3 days, and 1, 2, or 3 weeks. Cellular viability was initially assessed by the degree of cellular outgrowth from explants of the stored nerves placed in culture, and then further quantitated by dissociating the cultured nerve explants and calculating the type and number of cells per milligram of peripheral nerve. Rat Schwann cells (SCs) obtained from the stored (control and 1 and 2 weeks) nerves were tested for their functional ability to myelinate dorsal root ganglion (DRG) neurons in culture. Our findings indicate that human and rat peripheral nerves contain few viable SCs and fibroblasts after 3 weeks of cold storage with the quantity of viable cells within the human cold stored peripheral nerves decreasing significantly after 1 week of cold storage. Despite their reduced number, some SCs from rat nerves stored up to 2 weeks are capable of myelinating DRG axons in culture. These results suggest that short intervals (< 1 week) of cold storage will result in potential peripheral nerve grafts containing large populations of functional cells, while long-term (> or = 3 weeks) cold stored peripheral nerves will contain few viable cells.

Fermentation properties of intestinal strains of Lactobacillus, of a sour dough and of a yoghurt starter culture in an oat-based nutritive solution

Fermented oatmeal soup containing viable lactic acid bacteria is a product which can be used as a base for a nutritive solution in enteral feeding. Oatmeal soups were individually inoculated with 18 strains of Lactobacillus, isolated from the intestinal mucosa of humans and rat, and with two kinds of commercial starter cultures L. plantarum strain A1 (sour dough) and a mixture of L. bulgaricus and S. thermophilus (yoghurt), and fermented at 37 °C for 15 h. The rate at which the pH decreased depended on the strains used, and the time taken for pH 4·0 to be reached in the soups varied from 6 to 15 h. The viable counts of lactobacilli varied in the final soups, from 1·0×108 to 4·7×109 cfu ml−1. The amount of lactate varied between 0·3 to 0·9 g/100 ml soup and L. plantarum strain A1 produced the highest proportion of D-lactate of the total amount of lactate. 53% of the tested strains produced 0·05 g acetate/100 ml soup, or more. L. plantarum (299, A1) and L. reuteri (108) were shown to have different requirements for amino acids during the fermentation process in the oatmeal soup. L. acidophilus (308) showed the slowest rate of pH decrease, which also gave a low amount of viable cells in the final soup (1×108 cfu ml−1) after 15 h fermentation. There were distinct differences in the aroma among the soups fermented by homofermentative and heterofermentative bacteria. For instance, the aroma of the soups fermented byL. plantarum (299 and A1) was more appetizing than of the soup fermented by L. reuteri (108).

Preparation of renal papillary collecting duct cells for studyin vitro

The mammalian kidney is comprised of a heterogeneous array of tubules and blood vessels. Over the years the function of the individual nephron segments in the production of urine has been reasonably well defined by micropuncture and in vitro microperfusion techniques. Beyond this descriptive step, however, to clarify the mechanisms of solute and water transport, it is necessary to study cellular compartments, specifically. Several techniques are used currently for cell studies, but in each the usefulness is limited. For example, thin slices of kidney tissue contain a variety of cell types as do suspensions of kidney cortex [1]. By dissection in vitro it is possible to isolate specific segments of nephron, but the mass of tissue is insufficient for the limits of resolution of many analytical techniques [2]. The purpose of this report is to describe a method for collecting milligram quantities of viable cells from the papillary collecting duct of the rabbit kidney.

The quantity of viable cells at various stages of gestation

The quantity of viable cells at various stages of gestation was determined with trypan blue. Samples taken between the 12th to 13th weeks of pregnancy may at times contain no viable cells. The success of a culture of amniotic fluid cells depends among other things on the amount of viable cells added. An increase in the amount of dead cells in a culture seems also to impair the result of the culture.