In archaebacteria, there is a doxorubicin efflux pump similar to mammalian P-glycoprotein.

We selected for study an anthracycline-resistant mutant from the archaebacteria Haloferax volcanii. This resistance was reversed by a Ca(2+)-channel antagonist, nifedipine (NDP). This resistance and its reversal by NDP suggest P-glycoprotein (Pgp) to be responsible for maintaining an anticancer drug concentration below the cytotoxic level. Using rhodamine 123 (RH123) as a substrate for Pgp, we then examined whether the resistance to anthracyclines in this bacteria might involve a Pgp-like anthracycline efflux pump. RH123 accumulation by the bacteria was determined with flow cytometry. A steady-state RH123 accumulation by the resistant cells revealed approx. one-fifteenth of that by the wild-type cells, which could be remarkably enhanced by NDP. The other modulators of Pgp, diltiazem and verapamil, also enhanced RH123 accumulation in resistant cells. The uncoupler FCCP completely restored RH123 accumulation in resistant cells to the wild-type cell level. RH123 unidirectional efflux from resistant cells after its preloading revealed much greater than that from wild-type cells, which was remarkably inhibited by FCCP. These confirmed that RH123 low accumulation involves its active efflux mechanism. Taken together, the present study indicated that lower evolutionary archaebacteria might also express a Pgp-like protein very similar to mammalian Pgp.     

In archaebacteria,there is a doxorubicin efflux pump similar to mammalian P-glycoprotein

We selected for study an anthracycline-resistant mutant from the archaebacteria Haloferax volcanii. This resistance was reversed by a Ca²⁺-channel antagonist, nifedipine (NDP). This resistance and its reversal by NDP suggest P-glycoprotein (Pgp) to be responsible for maintaining an anticancer drug concentration below the cytotoxic level. Using rhodamine 123 (RH123) as a substrate for Pgp, we then examined whether the resistance to anthracyclines in this bacteria might involve a Pgp-like anthracycline efflux pump. RH123 accumulation by the bacteria was determined with flow cytometry. A steady-state RH123 accumulation by the resistant cells revealed approx. one-fifteenth of that by the wild-type cells, which could be remarkably enhanced by NDP. The other modulators of Pgp, diltiazem and verapamil, also enhanced RH123 accumulation in resistant cells. The uncoupler FCCP completely restored RH123 accumulation in resistant cells to the wild-type cell level. RH123 unidirectional efflux from resistant cells after its preloading revealed much greater than that from wild-type cells, which was remarkably inhibited by FCCP. These confirmed that RH123 low accumulation involves its active efflux mechanism. Taken together, the present study indicated that lower evolutionary archaebacteria might also express a Pgp-like protein very similar to mammalian Pgp.     

Co-cultivation of astroglial enriched cultures from striatum and neuronal containing cultures from substantia nigra

A co-cultivation system was developed with neuron-containing (neuron-specific enolase (NSE) positive) primary cultures from the substantia nigra of 15 to 17-day old embryonic rats which were grown 1 mm apart from astroglial-enriched (glial fibrillary acidic protein (GFAp) positive) primary cultures from the striatum of neonatal rats. The astroglial cells went through a morphological differentiation with extension of processes after co-cultivation with the immunohistochemically-identified neuronal cells. The astroglial-enriched striatum cultures showed a higher active uptake of 3H-L-glutamate after co-cultivation for one week, compared to control cultures from striatum. Vmax (nmol X mg protein-1 X min-1 X was 58.4 +/- 8.3 after co-cultivation and 37.2 +/- 6.3 for control cultures. The glutamine synthetase (GS) activity was slightly increased after co-cultivation. The validity and specificity of the results were ensured. The data suggest that astroglial cells in a primary culture are influenced by co-cultivation with fetal neuron containing cultures resulting in morphological differentiation, and increases in 3H-L-glutamate uptake and GS activity.     

Role of endothelin and endothelin A-type receptor in adaptation of the carotid body to chronic hypoxia

Chronic exposure in a low-PO(2) environment (i.e., chronic hypoxia, CH) elicits an elevated hypoxic ventilatory response and increased hypoxic chemosensitivity in arterial chemoreceptors in the carotid body. In the present study, we examine the hypothesis that changes in chemosensitivity are mediated by endothelin (ET), a 21-amino-acid peptide, and ET(A) receptors, both of which are normally expressed by O(2)-sensitive type I cells. Immunocytochemical staining showed incremental increases in ET and ET(A) expression in type I cells after 3, 7, and 14 days of CH (380 Torr). Peptide and receptor upregulation was confirmed in quantitative RT-PCR assays conducted after 14 days of CH. In vitro recordings of carotid sinus nerve activity after in vivo exposure to CH for 1-16 days demonstrated a time-dependent increase in chemoreceptor activity evoked by acute hypoxia. In normal carotid body, the specific ET(A) antagonist BQ-123 (5 microM) inhibited 11% of the nerve discharge elicited by hypoxia, and after 3 days of CH the drug diminished the hypoxia-evoked discharge by 20% (P < 0.01). This inhibitory effect progressed to 45% at day 9 of CH and to nearly 50% after 12, 14, and 16 days of CH. Furthermore, in the presence of BQ-123, the magnitude of the activity evoked by hypoxia did not differ in normal vs. CH preparations, indicating that the increased activity was the result of endogenous ET acting on an increasing number of ET(A). Collectively, our data suggest that ET and ET(A) autoreceptors on O(2)-sensitive type I cells play a critical role in CH-induced increased chemosensitivity in the rat carotid body.     

Radiation-enhanced murine sarcoma virus genome rescue activity.

Although the doses of X-ray (312-2,500 R) used for irradiation of cells caused impairment of DNA synthesis and cell replication, co-cultivation of X-irradiated MuLV- carrier cells with un-irradiated nonproducer cells of MSV-induced tumour resulted in as much as 20-fold increase in MSV retrieval compared with the un-irradiated control. The enhancement was apparent also as a 3-fold increase in the number of cells producing MSV (infectious centers) in the co-cultivation plate. This suggested that the MSV genome rescue efficiency in terms of MSV per cell, as well as the number of cells producing MSV, increased markedly. By uridine-3H-labeling and focus assay experiments, evidence was presented which suggested that an increase in MSV/MuLV ratio in the culture fluid of co-cultivation plates was obtained when the MuLV-carrier cells were pre-irradiated. By contrast, X-irradiation of the nonproducer cells prior to co-cultivation caused only reductions of MSV genome rescue efficiency. However, use of X-irradiated MuLV-carrier cells for co-cultivation with X-irradiated nonproducer cells restored this efficiency to some extent. The dose-survival curve of the nonproducer cells was not much different from those of the MuLV-carrier cells after X-irradiation. It was suggested that the viability of nonproducer cells was required for replication of MuLV transferred from the carrier cells and for subsequent MSV genome rescue.     

Effect of chronic hypoxia on agonist-induced tone and calcium signaling in rat pulmonary artery

The effect of chronic hypoxia (CH) for 14 days on Ca2+ signaling and contraction induced by agonists in the rat main pulmonary artery (MPA) was investigated. In MPA myocytes obtained from control (normoxic) rats, endothelin (ET)-1, angiotensin II (ANG II), and ATP induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) in 85-90% of cells, whereas they disappeared in myocytes from chronically hypoxic rats together with a decrease in the percentage of responding cells. However, both the amount of mobilized Ca2+ and the sources of Ca2+ implicated in the agonist-induced response were not changed. Analysis of the transient caffeine-induced [Ca2+]i response revealed that recovery of the resting [Ca2+]i value was delayed in myocytes from chronically hypoxic rats. The maximal contraction induced by ET-1 or ANG II in MPA rings from chronically hypoxic rats was decreased by 30% compared with control values. Moreover, the D-600- and thapsigargin-resistant component of contraction was decreased by 40% in chronically hypoxic rats. These data indicate that CH alters pulmonary arterial reactivity as a consequence of an effect on both Ca2+ signaling and Ca2+ sensitivity of the contractile apparatus. A Ca2+ reuptake mechanism appears as a CH-sensitive phenomenon that may account for the main effect of CH on Ca2+ signaling.     

Co-cultivation of antifungal Lactobacillus plantarum MiLAB 393 and Aspergillus nidulans, evaluation of effects on fungal growth and protein expression

The fungal inhibitory effects of strain Lactobacillus plantarum MiLAB 393, producing broad-spectrum antifungal compounds, were evaluated. A co-cultivation method was set up to monitor effects on fungal growth and protein expression of growing Aspergillus nidulans with L. plantarum MiLAB 393. The effects of inhibitory metabolites produced by L. plantarum MiLAB 393, cyclo(l-Phe-l-Pro), lactic acid and 3-phenyllactic acid, were also investigated by addition of pure compounds to the growth medium of A. nidulans. The co-cultivation strongly affected the morphology of the fungal mycelium and decreased the biomass to 36% of control. Co-cultivation with Lactobacillus coryniformis MiLAB 123 gave only marginal morphological changes and minor biomass reduction, suggesting specific effects of L. plantarum MiLAB 393. The amount of several A. nidulans-proteins was increased during co-cultivation and by all of the inhibiting substances. This study shows that the growth of A. nidulans is inhibited during co-cultivation with L. plantarum MiLAB 393 and that the expression of fungal proteins is altered.     

Protein malnutrition mitigates the effects of a high-fat diet on glucose homeostasis in mice

Nutrient malnutrition, during the early stages of development, may facilitate the onset of metabolic diseases later in life. However, the consequences of nutritional insults, such as a high-fat diet (HFD) after protein restriction, are still controversial. We assessed overall glucose homeostasis and molecular markers of mitochondrial function in the gastrocnemius muscle of protein-restricted mice fed an HFD until early adulthood. Male C57BL/6 mice were fed a control (14% protein-control diet) or a protein-restricted (6% protein-restricted diet) diet for 6 weeks. Afterward, mice received an HFD or not for 8 weeks (mice fed a control diet and HFD [CH] and mice fed a protein-restricted diet and HFD [RH]). RH mice showed lower weight gain and fat accumulation and did not show an increase in fasting plasma glucose and insulin levels compared with CH mice. RH mice showed higher energy expenditure, increased citrate synthase, peroxisome-proliferator-activated receptor gamma coactivator 1-alpha protein content, and higher levels of malate and α-ketoglutarate compared with CH mice. Moreover, RH mice showed increased AMPc-dependent kinase and acetyl coenzyme-A (CoA) carboxylase phosphorylation, lower intramuscular triacylglycerol content, and similar malonyl-CoA levels. In conclusion, protein undernourishment after weaning does not potentiate fat accumulation and insulin resistance in adult young mice fed an HFD. This outcome seems to be associated with increased skeletal muscle mitochondrial oxidative capacity and reduced lipids accumulation.     

In vitro selection of cell variants resistant to macrophage and hydrogen peroxide cytotoxic activity in spontaneous transformed cells of the STHE strain]

The new cell variants were selected in vitro from the low-malignant Syrian hamster embryo cells (STHE strain) spontaneously transformed in vitro. Ten cycles of in vitro co-cultivation of the parental STHE cells with the normal Syrian hamsters peritoneal exudate cells (PEC) (resident and LPS-activated PEC) used as a selecting agents (PERr, PECa) were performed. The susceptibilities of thus selected STHE cell variants as well as parental STHE cells to macrophage cytotoxicity and H2O2 dagame were tested with 3H-TdR assays. It has been demonstrated that all five STHE cell variants selected in vitro with PECa, in contrast to the parental STHE cells and three STHE cell variants selected with the use of PECr, were significantly more resistant to H2O2-mediated cytotoxicity. The increased resistance was acquired already after the 1-st cycle of the selection procedure. In contrast, only one STHE cell variant selected with PECr appeared to be semiresistant (demonstrated in 4 out of 6 experiments) to H2O2 damage after 10th cycle of in vitro co-cultivation.     

Transient inhibition by cycloheximide of protein synthesis in cultured plant cell suspensions: a dose response paradox

$\text{Rosa}$ cell suspension cultures supplied with high concentrations (>10^?5M) of cycloheximide (CH) rapidly develop a resistance to the drug under conditions in which protein synthesis is initially severely impaired. CH at 10^?6M, while equally effective in inhibiting protein synthesis, is markedly less efficient in inducing resistance. Development of resistance is not due to selection, or to induction of the capacity to metabolize the drug. Cells washed free of CH retain their resistance over several generations before reverting to the CH-sensitive condition.     

Influence of synovial cells on cartilage in vitro: induction of breakdown and inhibition of synthesis.

Organoid or high density cultures of: (1) synovial cells from patients with rheumatoid arthritis, and (2) prechondrogenic mesenchymal cells from limb buds of 12-day-old mouse embryos, were co-cultured for 7-10 days using the Trowell culture system. Depending on the time of commencing co-cultivation, chondrogenesis was inhibited (co-cultivation from the start) or the cartilaginous matrix was partly degraded (co-cultivation after formation of embryonic cartilage, i.e. on day 4). These effects were obtained with cells from synovial fluid as well as from synovial tissue. Matrix degradation and the behaviour of the different cell types could be demonstrated well by electron microscopy under the in vitro conditions applied.     

Cell cycle progression and SCE rate of Bloom syndrome cells with/without co-cultivation in the presence/absence of normal cells

The present study was undertaken to examine cell cycle progression and SCE rate in three types of B-lymphoid cell line, viz., normal (KS-86), high-SCE Bloom syndrome (BS (BS2-2) and dimorphic BS (BS-SYW). In order to compare the dimorphic condition (BS-SYW) with artificial dimorphism (co-cultivation of BS2-2 with KS-86) these experiments were designed to test whether the BS B-lymphoid cell line cultures would influence the cell cycle progression and SCE rates of a normal B-lymphoid cell line, and vice versa. The present study resolved the controversy reported in the literature, by finding a definite time period under co-cultivation conditions when the SCE in normal cells was increased after 8 days of co-culture, whereas SCE in the BS cells decreased immediately with co-cultivation. In the dimorphic BS cell line (BS-SYW) the SCE frequency of a high-SCE cell population was also observed to be lower than that of a non-dimorphic BS cell line (BS2-2), thus corroborating the experimental observations under co-cultivation conditions. The decrease in BS SCE and increase in normal SCE (after a particular time period) is attributed to numerous causes discussed in relation to the cell cycle progression.     

Rate of sister chromatid exchanges in Bloom syndrome fibroblasts reduced by co-cultivation with normal fibroblasts.

Six strains of Bloom syndrome (BlS) fibroblasts responded to co-cultivation with normal fibroblasts at a 1:2 ratio by a reduced rate of sister chromatid exchanges (SCE's) from a mean of 67.5 (range = 59--78) to 28.4 (range = 21--35). The response was dose-dependent in one strain tested at 1:2, 1:1, and 2:1 ratios. In addition, quadriradial exchange figures and other signs of increased chromosomal instability were not found in BlS cells following co-cultivation with control cells. Control cells did not respond to BlS cells and maintained a normal rate of SCEs. Culture medium conditioned for 48 hrs by normal fibroblasts could also reduce the rate of SCEs in BlS fibroblasts, but less than in co-cultivation. We suggest that the reduced rate of SCEs and the lack of chromosomal instability in BlS cells following co-cultivation represent a corrective effect that is related to the basic defect and not dependent on cell-to-cell contact.     

Chronic hypoxia-induced spontaneous and rhythmic contractions in the rat main pulmonary artery

The effect of chronic hypoxia (CH; 1-4 wk) on the electromechanical properties of the rat main pulmonary artery (MPA) was investigated. MPA rings obtained from rats exposed for 14 days to hypobaric (50.5 kPa) CH exhibited spontaneous and rhythmic contractions (SRCs) that were never observed in control (normoxic) rats. SRCs were unaffected by tetrodotoxin, phentolamine, BQ-123 and BQ-788, N-nitro-L-arginine methyl ester, or endothelium removal. CH depolarized smooth muscle cells from -58.8 +/- 9 to -38.6 +/- 5.4 mV and increased the resting cytosolic Ca2+ concentration from 67.3 +/- 11.9 to 112.5 +/- 16.4 nM. CH also induced spontaneous spikelike depolarizations. All of these effects were inhibited by external Ca2+ removal or nifedipine (1 microM). Moreover, depletion of intracellular Ca2+ stores with ryanodine (1-5 microM) or cyclopiazonic acid (3 microM) progressively attenuated SRCs. This study demonstrates that CH switches the MPA from a quiescent to a spontaneously active mechanical state. Finally, the fact that SRCs precede the development of right ventricle hypertrophy and disappear when this hypertrophy reaches a maximal value (after 3-4 wk of CH) suggests that SRCs may play a role in the adaptive process of the pulmonary circulation to CH.     

Modulations of food-derived substances on intestinal permeability in Caco-2 cell monolayers

The effects of more than 300 kinds of food extracts on intestinal permeability were investigated in Caco-2 cells with the use of model compounds: Lucifer Yellow (LY) for the paracellular pathway, Fluorescein (FC) for the monocarboxylic acid transporter-mediated pathway, and Rhodamine 123 (RH) for the p-glycoprotein-mediated efflux pathway. With several extracts of increasing or decreasing LY permeation, increasing FC or RH permeation was also observed, indicating modulation by dietary substances in several pathways for intestinal absorption.     

Ecology of cycloheximide-resistant fungi in field soils receiving raw city wastewater or normal irrigation water

The effect of raw city wastewater irrigation on biodiversity and population densities of a cycloheximide-resistant (CH) fungal community was studied in 13 field soils receiving either raw city wastewater or normal irrigation, and in raw city wastewater in the Nablus area, using the hair baiting technique (HBT) and a surface soil dilution plating (SSDP) technique. Three of these fields [one had been receiving raw city wastewater for more than ten years and was designated a heavily polluted field, and the other 2 were cultivated for the first time and were either irrigated with raw city wastewater (newly polluted field) or normal irrigation water (nonpolluted)], were sampled 4–7 times over a 9-month period. The other ten fields, which had been under raw city wastewater irrigation for more than 10 years, were sampled only once. Fifty-seven CH-resistant species belonging to 18 genera were recovered, of which 49 species were recovered from soil habitats and 28 species from raw city wastewater. The HBT had shown to be more efficient in the isolation of pathogenic and potentially pathogenic fungi including dermatophytes. A higher percentage of this group of fungi was recovered from the three main field soils studied using HBT (70% of all isolates), than the SSDP (35.5%); no dermatophytes were recovered by the SSDP method. Two dermatophytes (Microsporum gypseum, and Trichophyton ajelloi), and five more fungi (Arthroderma cuniculi, A. curreyi, Chrysosporium keratinophilum, C. tropicum, and C. pannorum), were recovered from these habitats. Wastewater irrigation seemed to have affected the fungal population densities, with the highest population densities being found in the heavily polluted field soil, while lower population densities were found in the nonpolluted field soil. Increases in organic matter were also observed as a result of sewage effluent irrigation. However, basic similarities in the biodiversity of CH-resistant fungal communities existed in nonpolluted and polluted field soils, and raw city wastewater. Comparable numbers of fungal species were recovered from the three main field soils. The species most commonly found in those habitats included: Alternaria alternata, Aspergillus candidus, Geotrichum candidum, and Paecilomyces lilacinus. Field soils receiving either raw city wastewater or normal irrigation water, were found to be rich in pathogenic and potentially pathogenic CH-resistant fungi, including dermatophytes, with raw city wastewater yielding the highest percentage (81%), followed by the newly wastewater irrigated field (77.7%), the nonpolluted field (67%), and the heavily polluted field (63.4%). Hygienic measures should therefore be taken to control the spread of these fungi in the environment of human communities, and to avoid mycotic infections among farmers. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of the toxic compounds present in brewer's spent grain hemicellulosic hydrolysate on xylose-to-xylitol bioconversion by Candida guilliermondii

Fermentation media containing different concentrations of toxic compounds were prepared from brewer's spent grain (BSG) hemicellulosic hydrolysate, and used for xylose-to-xylitol bioconversion by Candida guilliermondii. Such fermentation media were composed of the hydrolysate in the following ways: raw (RH); concentrated four-fold (CH); concentrated and treated with activated charcoal (TCH); raw supplemented with sugars until a concentration four-fold higher (SRH); concentrated and subsequently diluted but supplemented with sugars until a concentration four-fold higher (SDCH). All media presented an initial xylose concentration of 85 g/l, except RH, which contained 23 g/l xylose. Fermentation results revealed that the sugars supplementation to raw hydrolysate favored the xylitol production. Nevertheless, xylitol production from CH was negatively affected due to the high concentration of toxic compounds present in the medium. The hydrolysate treatment with activated charcoal partially removed the toxic compounds, and the xylitol production was higher than in CH, but not so efficient as in SRH. It was thus concluded that to obtain an efficient xylose-to-xylitol bioconversion from BSG hydrolysate, the sugars concentration must be increased, but the toxic compounds concentration must be reduced to the same level present in the raw hydrolysate.     

Factors affecting transformation efficiency of embryogenic callus of Upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) with <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 10⁸ cells ml^–1 (OD₆₀₀=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l^–1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.     

Influence of Pathogenic and Non-pathogenic Bacteria on Soybean Suspension Cells

Co-cultivations of plant suspension cultures of soybean ( Glycine max ) with compatible phytopathogenic ( Pseudomonas syringae pv. glycinea ), incompatible phytopathogenic ( Pseudomonas syringae pv. tomato ), and different non-pathogenic ( Erwinia herbicola, Escherichia coli ) bacteria were carried out. Growth and viability (triphenyltetrazolium chloride activity) of plant cells and bacteria as well as enzyme activities of peroxidase (PO), polyphenoloxidase (PPO), and phenylalanine ammonialyase (PAL) within the plant cells were investigated over an incubation period of 7 days. The compatible pathogen inhibited growth and viability of the plant cells after 1 day and led to the death of the majority of the plant cells by the seventh day. In contrast, the incompatible pathogen directly reduced growth and viability of the soybean cells and caused a strong induction of enzyme activities of PO and PAL to more than 4 times of the untreated control by the seventh day. The epiphytic bacterium Erwinia herbicola caused a slight inhibition of growth and viability of the plant cells after the second day of co-cultivation. The PO activity increased in the same manner as in the incompatible interaction. The saprophytic Escherichia coli strain had a negligible influence on soybean suspension cells. All the bacteria tested except for Escherichia coli multiplied rapidly during co-cultivation and reached populations of 10⁸-10⁹ colonyfoming units/ml in the stationary phase. The results from this study demonstrate that the soybean suspension cells react differently to compatible, incompatible and saprophytic bacteria.     

Effect of Relative Humidity on Survival of Candida albicans and Other Yeasts

Individual blastospores of Candida albicans were deposited on the surface of 50-mm membranes (Millipore Corp.) and placed within sealed glass chambers at various relative humidities (RH). After 48 hr, virtually all cells maintained at 100 and 10% RH had survived, but 84% of the cells maintained at 60% RH failed to develop into colonies when transferred to Sabouraud medium. No morphological abnormalities could be observed in cells surviving low RH values, but their initial rate of multiplication after transfer to Sabouraud medium was greatly reduced, compared to that demonstrated by cells maintained at 100% RH. At 60% RH, the exposure time required to kill 50% of the blastospores was 2 to 3.5 days. The inimical effect of 60% RH was confirmed in a total of 21 isolates of C. albicans. No deleterious effect was noted when 12 other species of yeasts were subjected to 10, 60, and 100% RH. The single isolate of Candida brumptii and 1 out of the 20 isolates of Cryptococcus neoformans tested also failed to grow after blastospores had been exposed to 60% RH for 4 days.