Effective gene transfer of lacZ and P0 into Schwann cells of P0-deficient mice

PC12 cells are used as a model system to study neuronal differentiation. Nerve growth factor (NGF) triggers a differentiation pathway in PC12 cells. Neurite outgrowth (a morphological marker of differentiation) in PC12 cells is significantly reduced in the presence of the NOS inhibitor l-NAME, but not d-NAME, implicating NOS in the differentiation process. Previously we have shown that the neuronal NO synthase (nNOS) isoform is induced in PC12 cells in the presence of NGF. Thus, we wished to further evaluate the role of nNOS and NO in PC12 cell differentiation. When a dominant negative mutant nNOS expression vector was transiently transfected into NGF-treated PC12 cells, it significantly reduced PC12 cell neurite outgrowth. Thus, we concluded that the NO required for PC12 cell differentiation, in response to NGF, is produced by nNOS. NO alone was insufficient to induce differentiation as cells treated with the NO donor, sodium nitroprusside did not produce neurites. Treatment of PC12 cells with oxyhemoglobin (an NO scavenger) was also found to significantly reduce the number of neurites produced by PC12 cells treated with NGF. Thus, NO appears to be necessary, but not sufficient, to induce differentiation, and its mode of action appears to be extracellular. A well documented action of NO is to activate soluble guanylate cyclase. Thus, we determined the role of soluble guanylate cyclase activation as a means by which NO induces PC12 cell differentiation. However, in the presence of NGF (to prime PC12 cells for differentiation) and l-NAME (to specifically remove the NO component), 8Br-cGMP (a cGMP analog) failed to induce PC12 cell differentiation. In addition, blockade of sGC activity with specific inhibitors failed to block NGF-induced PC12 cell differentiation. We conclude that the NO required for PC12 cell differentiation is produced by nNOS and that the NO exerts its effects on surrounding PC12 cells in a sGC/cGMP independent manner.     

Effects of short-term treatment with prednisolone and calcitriol on bone and mineral metabolism in normal men

PURPOSE: This study was conducted to clarify the relationship among the frequencies of micronuclei (MN) and apoptosis, and clonogenic cell survival after irradiation. MATERIALS AND METHODS: The frequencies of MN and apoptosis were compared in the surviving fraction in three human tumour cell lines and two rodent cell lines at various irradiation doses. RESULTS: The SHIN-3, DU-145 and CHO-K1 cells showed dose-dependent increases of MN per binucleate cell and an excellent correlation between the MN frequency and surviving fraction after irradiation. The F9 and COLO 320DM cells did not show this correlation. The number of apoptotic cells increased according to the increase in radiation dose in the F9 and COLO 320DM cells, but not in the SHIN-3, DU-145 or CHO-K1 cells. CONCLUSIONS: The detection of the MN frequency alone is insufficient to measure cellular intrinsic radiosensitivity. The simultaneous use of the MN assay and the detection of apoptotic cells would be more reliable as a method for predicting cell survival after radiation.     

Recombinant human phenylalanine hydroxylase: novel regulatory and structural properties

In earlier studies we found that treatment with interferon-gamma (IFN-gamma) produced an 8- to 11-fold increase in choline acetyltransferase (ChAT) in cultured cells taken from Embryonic Day 16 (E16) septal nuclei with adjacent basal forebrain (SN/BF). Since younger cultures responded even more profoundly to IFN treatment, we have tested the possibility that the action of IFN (or its intermediate; see below) is to prompt the cholinergic differentiation of neuronal precursors. SN/BF cultures of various ages were labeled with a retrovirus engineered to express beta-galactosidase (Lac-Z), and ChAT-positive descendants of the retrovirally labeled precursors were counted. IFN-gamma treatment of cultures caused as much as an 8.8-fold increase in the proportion of ChAT-positive cells present in Lac-Z-positive clones, suggesting that IFN promoted cholinergic differentiation in precursor populations. By contrast, bFGF increased clone size but did not change the proportion of ChAT-positive cells. NGF affected neither. Only ameboid microglia present in the cultures responded to IFN with characteristic nuclear translocation of the signal transducing molecule p91, suggesting that a microglial-derived molecule may mediate the action of IFN. Consistent with this hypothesis, conditioned media from cultures of enriched, activated microglia also increased ChAT activity in a dose-dependent fashion. Conditioned media from an unstimulated macrophage/monocyte cell line (RAW 264.7) also proved extremely efficacious in raising ChAT activity. In addition, conditioned media from both activated microglia and RAW 264.7 cells increased the proportion of ChAT-positive cells in retrovirally labeled clones to the same extent as IFN itself, suggesting the possibility that they contain the molecule(s) that mediates the action of IFN. Preliminary characterization of this molecule suggests that it is a very stable and large protein. Together these data suggest that a molecule promoting cholinergic differentiation is produced by activated microglia and other macrophage-like cells. The identity of this molecule and its precise role in normal development await its further purification.     

Vectorial competence of Glossina tachinoides Westwood and Glossina palpalis gambiensis Vanderplank infected by Trypanosoma brucei brucei EATRO 1125]

In this work the relationship between the proliferation of bovine corneal epithelial cells and PGE2 has been studied. Our data indicate that PGE2 plays an important role in the growth of corneal epithelial cells. Actually, epithelial cells cultured on a keratocyte feeder-layer and exposed to indomethacin, a cyclooxygenase inhibitor, have shown a decrease in growth rate at drug concentrations which otherwise did not induce a reduction in the viability of the keratocytes as well as in epithelial cells in separate cultures. This effect has been reversed by an exogenous PGE2 addition to the culture media. Moreover, significant increases have been found in the growth of epithelial cells cultured in the presence of keratocytes, with basal medium and with conditioning medium after adding exogenous PGE2 at concentrations equal to or lower than 10(-6) M. Significant decreases in the dimensions of the corneal epithelial cells have been found only when PGE2 has been added to basal and to conditioning medium, suggesting that the autacoid maintains cell dimension and morphology. The appearance of keratins with high molecular weight (54 and 57 kDa) coupled with the tendency to stratification of the cells cultivated with media supplemented with PGE2, indicates that the autacoid could favour cell differentiation. The action of PGE2 on the corneal epithelial cells does not seem to be influenced by the presence of the fibroblasts and their products, since PGE2 has induced increases in cell growth and morphological variations, independent of cultural conditions and therefore also only in the presence of basal medium.     

Important variables that influence base-line micronucleus frequency in cytokinesis-blocked lymphocytes-a biomarker for DNA damage in human populations

The cytokinesis-block micronucleus (CBMN) assay has been adopted by numerous laboratories as a means for rapidly assessing base-line chromosome damage (breakage and loss) in human populations. However, the appropriate implementation of this assay requires a thorough understanding of both experimental variables and biological factors that can have impact on micronucleus (MN) frequency. The paper describes, with the help of experimental data the from the author's laboratory as well as other data, the impact of these variables. With regards to experimental variables, the scoring of micronuclei on slides by different technicians has been identified as an important factor; however, the use of different culture media, namely RPMI 1640 and McCoy's medium, did not have a significant effect on base-line frequencies. The paper also describes results showing that the MN index in cytokinesis-blocked cells, measured once every three months over a 12-month period for 53 healthy subjects, remains constant and the data measured on these occasions were significantly and positively correlated (R=0.477 to 0.684, P<0. 0001) with each other thus indicating the reliability and intra-individual variability of the assay over time. Inter-individual variation for males and female subjects has been estimated for each decade of age between 20 and 80 years; the difference between the 25th and 75th percentile of MN frequency varied between 1.4 fold and 2.3 fold and the minimum and maximum values for MN frequency varied by a factor of 4.7 and 12.5 depending on the age group. Age and gender are the most important demographic variables impacting on the MN index with MN frequencies in females being greater than those in males by a factor of 1.2 to 1.6 depending on the age group. For both sexes, MN frequency was significantly and positively correlated with age (R=0.62 in males and R=0.65 in females) and the slope of the regression line in males was 0.314 (P<0.0001) and in females it was 0.517 (P<0.0001). The main dietary factors influencing the MN index in subjects who are not folate deficient are plasma B12 (R=-0.315, P=0.0127) and plasma homocysteine (R=0.415, P=0.0086). In addition, it was proposed that the MN index is likely to be influenced by the propensity of an individual's cells to undergo apoptosis when damaged so that one might expect the MN frequency to be negatively correlated with apoptotic rate although this has yet to be tested. The above indicates the importance of maintaining an international network of scientists working with the CBMN assay to ensure appropriate quality control and for the development of standard experimental and documentation protocols. The human micronucleus (HUMN) project launched in 1997 is briefly described and proposed as the vehicle for these activities.     

Automatic cell culture quantitation with TRAKCELL: application to cell toxicology and differentiation

An automated system, TRAKCELL, was developed for the quantitation of cells in culture. It enabled cell counting, classification according to morphological cell characteristics and measurement of cell proliferation and differentiation. The system was tested on the toxic effect of ascorbic acid on rat brain catecholaminergic neurons in primary culture. In parallel, the effects of nerve growth factor, dexamethasone and forskolin on cell differentiation were studied using rat pheochromocytoma PC12 cells. The results show that the system permits rapid and reproducible measurements of cell density and of the morphological changes observed following various drug treatments.     

Differentiation and apoptosis of murine neuroblastoma cells N1E115

The mode and the kinetics of differentiation and death of murine N1E115 neuroblastoma cells induced by dimethyl sulfoxide and other nonspecific factors in vitro were investigated. After morphological differentiation neuroblastoma cells die by apoptosis which is indicated by characteristic morphological features and by internucleosomal DNA fragmentation. Durations of both differentiation and apoptosis are dependent on the nature of stimuli used. Protein synthesis inhibitor cycloheximide does not prevent differentiation and apoptosis of neuroblastoma cells induced by dimethyl sulfoxide and even accelerates both processes. The relationship between cell death and differentiation is discussed.     

Postnatal development of type I and type II hair cells in the mouse utricle: acquisition of voltage-gated conductances and differentiated morphology

The type I and type II hair cells of mature amniote vestibular organs have been classified according to their afferent nerve terminals: calyx and bouton, respectively. Mature type I and type II cells also have different complements of voltage-gated channels. Type I cells alone express a delayed rectifier, gK,L, that is activated at resting potential. We report that in mouse utricles this electrophysiological differentiation occurs during the first postnatal week. Whole-cell currents were recorded from hair cells in denervated organotypic cultures and in acutely excised epithelia. From postnatal day 1 (P1) to P3, most hair cells expressed a delayed rectifier that activated positive to resting potential and a fast inward rectifier, gK1. Between P4 and P8, many cells acquired the type I-specific conductance gK,L and/or a slow inward rectifier, gh. By P8, the percentages of cells expressing gK,L and gh were at mature levels. To investigate whether the electrophysiological differentiation correlated with morphological changes, we fixed utricles at different times between P0 and P28. Ultrastructural criteria were developed to classify cells when calyces were not present, as in cultures and neonatal organs. The morphological and electrophysiological differentiation followed different time courses, converging by P28. At P0, when no hair cells expressed gK,L, 33% were classified as type I by ultrastructural criteria. By P28, approximately 60% of hair cells in acute preparations received calyx terminals and expressed gK,L. Data from the denervated cultures showed that neither electrophysiological nor morphological differentiation depended on ongoing innervation.     

Pharmacodynamic effects of St. John''s Wort on rat intracerebral field potentials

In order to investigate the role of Bcl-2 in dopaminergic cells, we established a dopaminergic neuronal cell line (MN9D) stably expressing human Bcl-2 (MN9D/Bcl-2) or neomycin (MN9D/Neo). Overexpression of Bcl-2 in MN9D cells attenuated cell death due to treatment of mitochondrial electron transport inhibitors including N-methyl-4-phenylpyridinium, whereas it did not prevent cell death induced by reagents generating reactive oxygen species including 6-hydroxy-dopamine. Moreover, the rate of glucose uptake in MN9D/Bcl-2 was significantly lower than that in MN9D/Neo after MPP+ treatment. Thus, Bcl-2 may counter aberrations in mitochondrial electron transfer processes by altering energy metabolism within the MN9D cells.     

Negative regulation of the neu promoter by the SV40 large T antigen

We investigated the effect of transforming growth factor-beta 1 (TGF beta) on the proliferation and differentiation of cultured acute promyelocytic leukemia (APL) cells with the chromosomal t(15;17) translocation obtained from four patients to determine the role of TGF beta on growth and differentiation of APL cells. DNA synthesis, determined by 3H-thymidine uptake, was inhibited in the presence and absence of granulocyte colony-stimulating factor (G-CSF) in a dose-dependent manner by TGF beta in APL cells obtained from three of the four cases. TGF beta and G-CSF did not significantly affect the differentiation of APL cells, but all-trans retinoic acid (RA) induced morphological and functional differentiation in all APL cells tested. G-CSF markedly enhanced RA-induced granulocytic differentiation in APL cells obtained from all four cases. In cells in which TGF beta inhibited DNA synthesis, it also inhibited RA-induced granulocytic differentiation of APL cells and, to a greater degree, granulocytic differentiation induced by RA plus G-CSF. These results suggest that TGF beta is a negative regulator of the proliferation and differentiation of APL cells. The significance of TGF beta as an endogenous regulator in differentiation therapy with RA of APL patients is discussed.     

Aneugen-induced micronuclei (MN) in human lymphocytes may be discerned using image analysis techniques when cell-cycle stage is taken into account

We show that for the in vitro cytochalasin-B human lymphocyte micronucleus (MN) test, the quantification of the DNA content of MN and the difference in DNA content between the two macronuclei in the binucleate cells without MN, as measured by image analysis, gives a first estimation of the aneugenic potential of a test compound. Cultures of isolated human lymphocytes were exposed either to gamma-rays as a clastogen or to carbendazim (MBC) as an aneugen. The lymphocytes were stained with Feulgen stain and the MN were analyzed for DNA content with a Magiscan 2A image analyzer. The mean DNA content of MN induced by MBC were statistically higher than gamma-irradiation-induced MN. It was demonstrated that in culture the lymphocytes, as well as the MN, are in different stages of the cell cycle, but this will not affect the discriminating power of the MN DNA content when only G1 cells are considered, or when DNA content of the MN is expressed relative to the total genome. The identification of G1 and G2 cell populations from image analysis data was performed by extrapolation of DNA content data from G1- and G2-sorted lymphocytes with a FacStar plus flow sorter. It was demonstrated that in MBC-treated cells the DNA rearrangement between the macronuclei in binucleates without MN was on the average higher than in gamma-irradiated and untreated cells, which points to aneugenic effects of MBC without the formation of MN. In contrast to DNA content measurements, the area of the MN is not a reliable measure for discriminating clastogens from aneugens.     

Sister chromatid exchange and micronucleus frequency in human lymphocytes of 1,650 subjects in an Italian population: II. Contribution of sex, age, and lifestyle

Sister chromatid exchange (SCE) and micronuclei (MN) analysis was carried out on 1,650 healthy individuals living in Pisa and in two nearby small cities, Cascina and Navacchio (Ca-Na). The effect of smoking on SCEs was linearly correlated with the number of cigarettes per day, and an increase of 7.3% SCEs was detectable for as few cigarettes as 1-10/day. Ex-smokers showed intermediate mean values of SCEs (8.09 +/- 1.88) in comparison with never smokers (7.54 +/- 1.61) and current smokers (8.45 +/- 1.94). Mean values of SCEs of ex-smokers decreased linearly with time of smoking cessation, reaching the mean values of never smokers within 8 years. The extent of SCE decrease was inversely proportional to the number of cigarettes previously smoked. No interaction between smoking habits and coffee or alcohol drinking on SCEs was observed. A borderline (P = 0.053) increase in mean SCE values in coffee drinkers (more than 3 cups/day) was found. The age effect on SCEs was remarkable in Ca-Na, but not in Pisa donors. Job type was not associated with significant modification of mean values of SCEs. Multiple logistic regression analysis revealed a statistically significant association between the proportion of high frequency cells (HCF) outliers and coffee consumption. Age and sex appeared to be by far the most important variables associated with modifications in MN frequency, which increased by 0.04 per thousand and 0.02 per thousand per year in males and females, respectively. Children and young donors (age < or = 40 years) showed lower MN frequency regardless of sex, whereas sex appeared to determine a significantly higher increase of MN only in females older than 40 years. In contrast, in males the MN rate by age tended to level off after the age of 30-50. MN frequencies of Pisa blue- and white-collar workers were statistically significantly higher than in students (+0.71 and +0.55 per thousand, respectively). Smoking did not determine any increase of MN frequency. A total lack of correlation (P = 0.913) between MN and SCEs was observed.     

The role of glutamine, serum and energy factors in growth of enterocyte-like cell lines

BACKGROUND: Glutamine is routinely added to most cell cultures. Glutamine has been found to be the preferential nutrient to the rapidly replicating intestinal mucosa, but whether this is a metabolic effect or due to other properties of this amino acid is not determined. To study the importance of glutamine on the growth of two enterocyte-like cell lines, the effects of depriving the media or supplementing it with glutamine were assessed in media with different serum and energy supplements. METHODS: CaCo-2 and HT-29 cells were grown in serum-free medium, with fetal bovine or synthetic serum, and with or without glucose or galactose. The glutamine content was varied between 0 and 4 mM. All growth assays were performed in triplicate by counting in a hemocytometer. RESULTS: Both cell lines were dependent of serum factors for growth, but displayed distinct requirements on glutamine supplementation. Glutamine was an obligate supplement with dose-dependent correlation to growth (r = 0.87, p < 0.01) for CaCo-2 cells cultured in synthetic, but not in fetal bovine serum. In HT-29 cells, the correlation between glutamine and growth was significant (r = 0.68, p < 0.05) only in fetal bovine serum in the absence of galactose. CONCLUSION: This study shows that glutamine has different growth stimulating effects on two enterocyte-like cell lines studied. This could reflect different modes of action of glutamine on proliferation and differentiation in an enterocyte cell population.     

Prevalence study of serious substance abusers in the Czech Republic

The exfoliated cell micronucleus (MN) assay using fluorescent in situ hybridization (FISH) with a centromeric probe is a rapid method for determining the mechanism of MN formation in epithelial tissues exposed to carcinogenic agents. Here, we describe the use of this assay to detect the presence or absence of centromeric DNA in MN induced in vivo by radiation therapy and chronic arsenic (As) ingestion. We examined the buccal cells of an individual receiving 6,500 rads of photon radiation to the head and neck. Exfoliated cells were collected before, during, and after treatment. After radiation exposure a 16.6-fold increase in buccal cell MN frequency was seen. All induced MN were centromere negative (MN-) resulting from chromosome breakage. This finding is consistent with the clastogenic action of radiation and confirmed the reliability of the method. Three weeks post-therapy, MN frequencies returned to baseline. We also applied the assay to exfoliated bladder cells of 18 people chronically exposed to high levels of inorganic arsenic (In-As) in drinking water (average level, 1,312 micrograms As/L) and 18 matched controls (average level, 16 micrograms As/L). The combined increase in MN frequency was 1.8-fold (P = 0.001, Fisher's exact test). Frequencies of micronuclei containing acentric fragments (MN-) and those containing whole chromosomes (MN+) both increased (1.65-fold, P = 0.07, and 1.37-fold, P = 0.15, respectively), suggesting that arsenic may have both clastogenic and weak aneuploidogenic properties in vivo. After stratification on sex, the effect was stronger in male than in female bladder cells. In males the MN- frequency increased 2.06-fold (P = 0.07) while the frequency of MN+ increased 1.86-fold (P = 0.08). In addition, the frequencies of MN- and MN+ were positively associated with urinary arsenic and its metabolites. However, the association was stronger for micronuclei containing acentric fragments. By using FISH with centromeric probes, the mechanism of chemically induced genotoxicity can now be determined in epithelial tissues.     

Kinetics of conformational changes associated with potassium binding to and release from Na+/K(+)-ATPase

Previous studies have demonstrated that embryonal carcinoma (EC) cells express both fibroblast growth factor-4 (FGF-4) and FGF receptors. It has also been established that differentiation of EC cells represses the expression of the FGF-4 gene. Currently, the role of FGF-4 in the growth and differentiation of EC cells is unclear. In this study, we examined whether the differentiation of EC cells requires the repression of FGF-4 expression. To address this and related questions, F9 EC cells were transfected with an expression vector that uses the human beta-actin promoter to drive the constitutive expression of recombinant FGF-4. Unlike their untransfected counterparts, F9 EC cells transfected with this plasmid continue to produce recombinant FGF-4 after they differentiate. However, constitutive expression of this growth factor does not block morphological differentiation of the cells, nor does it alter the expression of six genes regulated by the differentiation of EC cells. Constitutive expression of recombinant FGF-4 also did not noticeably alter the growth of the transfected F9 EC cells before or after differentiation. Furthermore, unlike immortalized fibroblasts, which are known to grow in soft agar after transfection with FGF-4 expression plasmids, continued expression of recombinant FGF-4 activity did not enhance the ability of the EC-derived differentiated cells to form colonies in soft agar. These findings argue that continuous expression of recombinant FGF-4 activity does not block the differentiation of EC cells and that repression of the FGF-4 gene after EC cells differentiate does not appear, on its own, to be responsible for the loss of tumorigenicity that accompanies the differentiation of EC cells.     

Neplanocin A, a potent inhibitor of S-adenosylhomocysteine hydrolase, potentiates granulocytic differentiation of acute promyelocytic leukemia cells induced by all-trans retinoic acid

Several neplanocin A analogs were synthesized and their growth-inhibiting and differentiation-inducing activities on myelogenous leukemia cells were examined. An adenosine kinase-ineffective analog of neplanocin A was effective in inducing differentiation, suggesting that phosphorylation of the nucleoside is not essential for inducing the differentiation of leukemia cells. Neplanocin A induced functional and morphological differentiation of HL-60 cells, but did not effectively induce differentiation of NB4, a cell line derived from a leukemia patient with t(15;17). However, these cells have been known to undergo granulocytic differentiation upon treatment with all-trans retinoic acid (ATRA), and are used as a model for differentiation therapy in acute promyelocytic leukemia. Preexposure of NB4 cells to low concentrations of neplanocin A greatly enhanced the ATRA-induced differentiation of the cells, whereas representative antileukemic drugs such as cytosine arabinoside and daunomycin did not enhance this differentiation. A clinical strategy that combines intermittent treatment with neplanocin A analogs and a low dose of ATRA may increase the clinical response and decrease the adverse effects of ATRA.     

Skeletal progenitor cells and ageing human populations

PURPOSE: To investigate (1) the radiosensitivity of B versus T lymphocytes with respect to micronucleus (MN) induction and (2) the possible application of the B cell MN assay for biological dosimetry of individuals after acute exposure to low doses of ionizing radiation. MATERIALS AND METHODS: MN analysis was performed in T and B lymphocytes of six healthy volunteers exposed in vitro to gamma-ray doses ranging from 0.05 Gy to 1 Gy. For the MN assay on B cells, peripheral blood mononuclear cells were cultured and stimulated with pokeweed mitogen (PWM). Afterwards the B lymphocytes (characterized by the CD20+ phenotype) were separated with the FACSort flow cytometer and the number of MN in the sorted binucleate cells was scored. For T lymphocytes the standard MN protocol was applied. RESULTS: The number of spontaneous and radiation induced MN were significantly higher in B lymphocytes compared to T lymphocytes in the low dose range up to 1 Gy. An analysis of the present data showed that when the spontaneous MN frequencies are not known, doses from 0.08 Gy could be detected with the B cell MN assay while the conventional MN assay only allowed detection of doses > 0.25 Gy. However, in contradiction to the linear-quadratic dose-response for T cells, for B cells the initial steep increase of the MN yield with the very low dose was followed by a flattening of the curve towards higher doses. CONCLUSION: This study shows that B lymphocytes express a high number of MN for doses up to 1 Gy gamma-rays reflecting the highly radiosensitive behaviour of B cells. The results also point to the possible application of the B-cell MN assay for individual dose assessment. When blood samples can be taken within 24 h after acute accidental overexposure, the B-cell MN assay can be performed but only as a supplementary test to the conventional MN assay.     

Applicability of combination with tirapazamine in boron neutron capture therapy

SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells. After injection of tirapazamine (TPZ), a bioreductive agent, combined with sodium borocaptate-10B (BSH) or dl-p-boronophenylalanine-10B (BPA) administration, the tumors were irradiated with thermal neutrons, and then isolated and incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus (MN) frequency in cells without BrdU labeling (quiescent (Q) cells) was determined by means of immunofluorescence staining for BrdU, and that for total cells was obtained from tumors not pretreated with BrdU. Even when no 10B-compound was administered, TPZ increased the MN frequency of tumor cells including Q cells, resulting in reduction of the difference in MN frequency between total and Q cells, mainly by increasing the MN frequency of Q cells. TPZ increased the MN frequency of Q cells when combined with BPA administration, but TPZ showed no apparent effect on each cell population when combined with BSH. Namely, TPZ reduced the difference in MN frequency between total and Q cells caused by 10B-compound administration, especially when BPA was administered. From the viewpoint of the overall cell killing effect in boron neutron capture therapy (BNCT), combination with TPZ appeared to be useful in BPA-BNCT, but not in BSH-BNCT.     

Modification of cell damage, caused by variable rate heating, by means of changes in osmotic pressure of the medium or using chloramphenicol

We investigated the influence of media with different osmotic pressure and of chloramphenicol on cytotoxic effects of heating with different rate of Escherichia coli B/r and Escherichia coli Bs-1 bacteria, and Zygosaccharomyces bailii yeast cells. It was shown that the hypotonic media appreciably increased cytotoxic action of heating with different rate, and, on the contrary, the hypertonic media inhibited induction of these effects. The inhibitor of protein synthesis chloramphenicol was established not to affect the bacterial thermoresistance in the process of different heating rate. On the basis of analysis of the obtained and literature data, it is proposed that a reason of cell injury dependence of the heating rate may be the availability of dissimilar levels of osmotic homeostasis destabilization in these cells when heated with different rates.