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PubMed2003

Rectoanal mucosal swab culture is more sensitive than fecal culture and distinguishes Escherichia coli O157:H7-colonized cattle and those transiently shedding the same organism.

Enrichment and direct (nonenrichment) rectoanal mucosal swab (RAMS) culture techniques were developed and compared to traditional fecal culture for the detection of Escherichia coli O157:H7 in experimentally infected and naturally infected cattle. Holstein steers (n = 16) orally dosed with E. coli O157:H7 were sampled after bacterial colonization starting 15 days postinoculation. Enrichment RAMS cultures (70.31% positive) were more sensitive than enrichment fecal cultures with 10 g of feces (46.88% positive) at detecting E. coli O157:H7 (P < 0.01). Holstein bull calves (n = 15) were experimentally exposed to E. coli O157:H7 by penning them with E. coli O157:H7-positive calves. Prior to bacterial colonization (1 to 14 days postexposure), enriched fecal cultures were more sensitive at detecting E. coli O157:H7 than enriched RAMS cultures (P < 0.01). However, after colonization (40 or more days postexposure), the opposite was true and RAMS culture was more sensitive than fecal culture (P < 0.05). Among naturally infected heifers, enriched RAMS or fecal cultures were equally sensitive (P = 0.5), but direct RAMS cultures were more sensitive than either direct or enriched fecal cultures at detecting E. coli O157:H7 (P < 0.01), with 25 of 144, 4 of 144, and 10 of 108 samples, respectively, being culture positive. For both experimentally and naturally infected cattle, RAMS culture predicted the duration of infection. Cattle transiently shedding E. coli O157:H7 for <1 week were positive by fecal culture only and not by RAMS culture, whereas colonized animals (which were culture positive for an average of 26 days) were positive early on by RAMS culture. RAMS culture more directly measured the relationship between cattle and E. coli O157:H7 infection than fecal culture.

Anal Canal↗
PubMed2001

Influence of culture system and medium enrichment on sulfotransferase and sulfatase expression in male rat hepatocyte cultures.

The expression of sulfotransferase and steroid sulfatase was studied in rat liver using the most promising culture models of hepatocytes, including monolayer culture with a pyruvate (30 mM) enriched medium, co-culture with rat epithelial cells from primitive biliary origin and collagengel sandwich culture. In the latter, addition of dexamethasone (1 microM) to the medium was examined. Phenol sulfotransferase enzymes (SULT1) were studied by measuring activities towards 4-methylphenol and estradiol, hydroxysteroid sulfotransferase (SULT2A) activity was determined towards dehydroepiandrosterone (DHEA). Microsomal steroid sulfatase activity was measured towards estrone sulfate. Western blot analysis was carried out using polyclonal antibodies raised against rat phenol sulfotransferase SULT1A1 (ASTIV), estrogen sulfotransferase SULT1E1 (EST) and hydroxysteroid sulfotransferase (HST). SULT2A activity towards DHEA was maintained at a high level during the whole culture time. In the co-culture it even reached the level of freshly isolated cells. Addition of pyruvate had no positive effect on the activity measured in monolayer cultures. High SULT1A1 activity towards 4-methylphenol was found in the co-culture system. In the monolayer culture, the activity initially decreased with 35% but was then kept at a constant level, while in the sandwich culture low activities were measured. For dexamethasone, an inducing effect on the various SULT activities could not be detected. Independently of the culture model used, the SULT1E1 activity towards estradiol decreased to 20% and 5% of the initial activity after four and seven days of culture, respectively. Microsomal steroid sulfatase activity was best maintained in collagengel sandwich cultures. During the first four days in culture it retained 73% of the initial activity, afterwards it decreased to 40% of the activity found in freshly isolated hepatocytes, irrespective of the culture conditions. High expectations exist for collagengel sandwich cultures, however, in our study the results were rather disappointing. Monolayer is a suitable culture model for short-term purposes. For long-term in vitro biotransformation studies, co-culture is preferred but is rather complex.

Albumins↗
PubMed1991

Effect of culture process on alkaloid production by Catharanthus roseus cells. II. Immobilized cultures.

Two processes for the production of indole alkaloids 2 l surface-immobilized bioreactor cultures of Catharanthus roseus cells using Zenk's Alkaloid Production Medium (APM) were evaluated. The 1-stage process consisted of inoculating APM containing bioreactors and incubating for 15 d. The 2-stage process involved inoculating growth medium-containing bioreactors, growing the immobilized cultures for a certain period of time and subsequently replacing this medium with APM. The production stage which lasted for 15 d. High production in 2-stage cultures required the replacement of the growth regulator 2,4-dichlorophenoxyacetic acid by indole-3-acetic acid in the growth medium and a growth stage of 6 d (late exponential phase) before production initiation. Growth, main nutrient consumption and alkaloid production were monitored. Both culture regimes resulted in similar biomass production, dw (10-13 g l-1). The 2-stage cultures yielded biomass richer in organic nutrients (200-300%) and with higher respiratory activity (approximately 250%), indicated by their lower biomass-to-carbohydrate yields (31% and 26%), as compared to 1-stage cultures (41%). Two-stage cultures produced more known products (10 as compared to 6) at yields (5 to 4800 micrograms g-1) 3 to 5 times higher than 1-stage cultures. More alkaloids were alkaloids released in the medium of 2-stage cultures, under non-lysing conditions, (20 to 4700 micrograms l-1) than in 1-stage cultures (20 to 460 micrograms l-1). These results were compared to those obtained from shake flask cultures performed at the same time, with the same C. roseus cell line and under similar regimes and reported previously. Suspension and immobilized cultures performed according to the 1-stage regime showed similar total production. However, release of known alkaloids was 2 to 3 times higher in immobilized than in suspension cultures. Total alkaloid production of 2-stage suspension cultures was 3.8-fold higher than 2-stage immobilized cultures. Two stage immobilized cultures released 4 more known alkaloids than the 2-stage suspensions. Lower oxygen availability in the 2 l immobilized cultures may explain lower specific growth rates (0.15-0.22 d-1) and total alkaloid production levels, compared to 200 ml suspension cultures (0.2-0.4 d-1) reported in our previous paper.

Alkaloids↗
PubMed2006

Development of PDMS microbioreactor with well-defined and homogenous culture environment for chondrocyte 3-D culture.

Perfusion cell culture is believed to provide a stable culture environment due to the continuous supply of nutrients and removal of waste. However, the culture scales used in most cases were large, where the culture conditions can not be regarded as homogenous because of chemical gradients. To improve this, the concept of miniaturization is applied to 3-D cell culture. In this study, a simple perfusion microbioreactor was developed based on mass transport simulation to find out the reasonable culture scales with relatively lower chemical gradients. Besides, PDMS surface was treated with surfactant solution to reduce non-specific serum protein adsorption, which keeps the culture conditions steady. Chondrocyte 3-D culture using the proposed microbioreactors was compared with similar perfusion culture with a larger culture scale. Results showed that surfactant-treated PDMS surface could reduce serum protein adsorption by 85% over the native one. Also, microbioreactors were proved to provide a stable culture environment (e.g. pH) over the culture period. Cell culture scale of 200 microm thick culture construct was justified to have relatively lower chemical gradients than the larger scale perfusion culture. As a whole, the proposed culture system is capable of providing a well-defined and homogenous culture environment.

Animals↗
PubMed1971

Human sarcomas in culture. Foci of altered cells and a common antigen; induction of foci and antigen in human fibroblast cultures by filtrates.

In a study of human sarcomas maintained in culture for periods up to two years, the following observations were made. The most prominent cell type in serially cultured osteosarcomas was fibroblastic in appearance. After 16-20 wk in culture some lines spontaneously developed foci of altered cells resembling the foci produced in monolayer cultures by oncogenic viruses. The presence of these foci in the sarcoma cultures was transient, and usually they did not reappear; but in one instance they recurred with a characteristic periodicity of several weeks. From one of the sarcoma lines, in which foci appeared after 5 months in culture, two subcultures were established from stored frozen cells and these both exhibited foci after approximately the same lapse of time. The same phenomenon has been seen with another line, suggesting that the time of appearance of foci is characteristic for particular sarcomas. Foci of similar type could sometimes be induced in monolayer cultures of human fibroblasts by filtered medium from cultured sarcomas; this bore no relation to the presence or absence of foci in the sarcoma cultures at the time the filtrate was prepared. Electron microscopy of the spontaneous and induced foci, and of the sarcoma cultures, revealed no demonstrable virus. 12 out of 15 sarcoma cultures contained an antigen (S) demonstrable by indirect immunofluorescence with human sera. It was not present in any of the original sarcoma specimens, nor in any culture lines other than sarcomas. At least 3-4 wk in culture appear to be required for its demonstration. The antigen was cytoplasmic, occurred in only a small proportion of the cells, and was unpredictably variable in its expression, even in the same culture line. It could be induced in monolayer cultures of human fibroblasts by filtrates of medium from sarcoma cultures. As with the foci, the induction of S antigen in indicator cultures was not dependent upon the expression of antigen in the sarcoma line from which the filtrates were obtained. There was no association between the presence of foci and of antigen, nor was there any apparent relation between the ability of filtrates to induce foci and their ability to induce antigen. 80% or more of the general population have S antibody, and the titer of antibody in patients with sarcoma is no higher than in normal subjects. Thus, as in the case of Burkitt's lymphoma antigen, it appears that most individuals have been exposed to S antigen. But unlike Burkitt's lymphoma, no relation has so far been established between any particular disease and a corresponding high titer or frequency of occurrence of S antibody. The occurrence of foci of altered cells and of a common antigen, and the transmission of these two characters to indicator cells by filtrates, are all suggestive of a virus specifically associated with human sarcomas, one to which the general population is widely exposed, as indicated by the prevalence of antibody.

Adenocarcinoma↗
PubMed2005

Effect of group culture and embryo-culture conditioned medium on development of bovine embryos.

We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 mul/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P < 0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p < 0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos.

Animals↗
PubMed1994

CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells.

Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence-activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane ("stroma-noncontact" culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma-noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA-DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP-1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.

Animals↗
PubMed2006

Effect of stratified culture compared to confluent culture in monolayer on proliferation and differentiation of human articular chondrocytes.

With conventional tissue culture of cells, it is generally assumed that when the available 2D substrate is fully occupied, growth ceases or is greatly reduced.However, in nature wound repair mostly involves proliferation of cells that are attracted to the defect site in a 3D environment.Hence, proliferation continues in 3D until the defect site is filled with cells contributing to repair tissue. With this in mind,we examined the growth behavior of human articular chondrocytes during stratified culture as opposed to routine culture to confluency. Additionally, we studied the influence of growth factors on proliferation during stratified culture and differentiation thereafter. Chondrocytes were cultured in monolayer on tissue culture plastic to confluency or stratified for an additional 7 days. Culture medium was based on DMEM with 10% serum and either supplemented with high concentrations of nonessential amino acids (NEAA) and ascorbic acid (AsAP), or instead with basic fibroblastic growth factor (bFGF), platelet-derived growth factor (PDBF-BB), and/or transforming growth factor beta1 (TGF-beta). After expansion, cells were harvested, counted, and their differentiation capacity was examined in pellet culture assay. It was shown that chondrocytes, cultured stratified proliferate exponentially for up to an additional 4 days and that cell yield increased 5-fold. Furthermore, during stratified culture the number of cells increased further in the presence of bFGF, PDBF-BB, and TGFbeta1 or high concentrations of NEAA and AsAP. Depending on donor variation and factors supplemented the cell yield ranged from 0.06 up to 1.1 million cells/cm2 at the second passage. During stratified culture in the presence of either bFGF and PDGF or high concentrations of NEAA and AsAP, exponential growth continued for up to 7 days. Finally, cells maintained their differentiation capacity when cultured stratified with or without growth factors (bFGF, TGF-beta, and PDGF), but not when cultured with high levels of AsAP and NEAA. In contrast to other 3D culture techniques like microcarrier or suspension culture, nutrient consumption remained the same as with conventional expansion. Because this allows culturing of clinically relevant amounts of chondrocytes without increasing the amount of serum, chondrocytes can be fully expanded in the presence autologous serum, avoiding the risk of viral and/or prion disease transmission associated with the use of animal-derived serum or serum replacers with animal-derived constituents.

Cartilage, Articular↗
PubMed1993

Paradoxical release of insulin by adult pig islets in vitro. Recovery after culture in a defined tissue culture medium.

In this study, in vitro responsiveness to glucose of fresh and cultured islets from adult pigs was tested under both static (incubation) and dynamic (perifusion) conditions. Islets were isolated by an automated method from pancreases of 24-month-old animals and cultured overnight in CMRL 1066 and 10% FCS plus antibiotics; islets, perifused immediately after the overnight culture, showed a paradoxical decrease in insulin release when exposed to an acute glucose stimulus (16.7 mmol/L), and a normal response to acute glucose when isobutylmethylxanthine (IBMX) was added to the perifusing buffer. In addition, an acute reduction of glucose concentration in the perifusate elicited a paradoxical insulin release. At the microscope, islets appeared loose and irregularly shaped after the overnight culture; immunohistochemistry showed loss of peripheral A and other mantle cells. After the overnight culture, islets were divided into 5 groups and were cultured for a further 48 hr in different tissue culture media: CMRL 1066; RPMI 1640 (without glucose); RPMI 1640 (plus 11.1 mmol/L glucose); Ham's F12; and medium 199 (all media were supplemented with 10% FCS and antibiotics). During this period, insulin release was 11.4 +/- 1.1 pg/islet/min in islets cultured in CMRL 1066, 16.2 +/- 2.4 in islets cultured in RPMI 1640 (11.1 mmol/L glucose), 1.8 +/- 0.2 (P < 0.001 vs. all the other groups), and 9.0 +/- 0.6 and 8.4 +/- 0.9 pg/islet/min in islets cultured in RPMI 1640 (without glucose), Ham's F12, and medium 199, respectively. After the 48-hr culture in different media, the islets' responsiveness to an acute glucose stimulus (16.7 mmol/L; static incubation) was evaluated: islets cultured in CMRL 1066 and in RPMI 1640 (with and without glucose) showed no insulin response to the acute glucose stimulus; in contrast, insulin release rose from 0.42 +/- 0.06 to 0.60 +/- 0.12 pg/islet/min (NS) in islets cultured in Ham's F12, and from 0.24 +/- 0.06 to 0.48 +/- 0.06 pg/islet/min (P < 0.001) in islets cultured in medium 199. During perifusions, the paradoxical insulin release in response to an acute fall in glucose concentration disappeared, but a significant increase in response to high (16.7 mmol/L) glucose was observed only in islets previously cultured in medium 199. To assess the possible role of glucagon and of cAMP, additional perifusions were done in islets cultured for 48 hr in CMRL 1066 in the presence of glucagon (10 mumol/L) and IBMX (10 mumol/L); glucagon and IBMX were unable to modify the insulin response to 16.7 mmol/L glucose.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1981

Glucagon production by cultured pancreatic islets: effects of different culture conditions and media.

Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied for their effects on the glucagon production of the cultured specimens. Culture media containing heat-treated bovine calf serum degraded [125I]glucagon to a much less extent than those supplemented with untreated serum. Addition of aprotinin to the heat-treated serum gave a further reduction of the [125I]glucagon degradation in the culture medium. A similar supplementation of Medium 199, used for culture of isolated islets, resulted in the most extensive glucagon accumulation in the culture medium. Islets cultured free-floating or attached to the bottom of the culture dishes contained similar amounts of glucagon. However, the free-floating islets released less glucagon when tested in short-term experiments performed at the end of the 1 wk culture period. A comparison between different culture media showed that islets cultured in RPMI-1640 had the highest glucagon content and released most glucagon to the culture medium. Moreover, these islets responded most actively to an acute arginine challenge at the end of the culture period. The present data suggest that the optimal conditions for culture of isolated islets aimed at studies of glucagon production may be obtained by using a culture medium consisting of RPMI-1640 supplemented with both a proteinase inhibitor and heat-inactivated serum.

Animals↗
PubMed1976

Studies on normal human bone marrow cultures in controlled environment thin-film culture systems.

Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first two weeks while an increasing number of monocytes and macrophages appeared in the media of of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO2 concentrations, i.e. 2%, 5%, 10% similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera.

Bone Marrow↗
PubMed2002

Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes.

We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen-collagen or collagen-Matrigel sandwich cultures) or three-dimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)- and UDP-glucuronosyl transferase (UGT)- dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers. During culture, GSH was increased in 2D but not in 3D cultures in accordance with morphological observations; that is that matrix-cell interactions involving GSH, important in 2D, are minimal in 3D cultures. While UGT- and GST-dependent activities were equivalent in cultured hepatocytes and in rat livers, both catalase and GSH-Px activities decreased with time in all culture configurations. Constitutive CYP-dependent activities were drastically decreased in hepatocytes after isolation and attachment and did not recover in any culture configuration tested. Our results highlight that, although 2D sandwich cultures and 3D cultures on Matrigel allow longevity of rat hepatocyte cultures and optimal induction of CYPs, an imbalance in phase I/phase II detoxication processes in cultured rat hepatocytes occurs, whatever the culture configuration.

Animals↗
PubMed2005

Blood culture and conventional media for vitreous culture in infectious endophthalmitis.

OBJECTIVE: To study culture of vitreous fluid specimens in patients with infectious endophthalmitis, using blood culture bottles compared with conventional culture media. MATERIAL AND METHOD: Patients with infectious endophthalmitis, occurred within 6 weeks after ocular trauma or intraocular surgery, were prospectively studied. Vitreous fluid specimens were cultured in both blood culture bottles and conventional culture media. The measured outcome is the yield of positive culture and time to positive culture. RESULTS: The vitreous fluid culture was positive in 14 of 27 eyes (51.85%). Blood culture bottle was positive in 14 of 14 eyes (100%), whereas conventional culture media was positive in 7 of 14 eyes (50%). Most specimens in both techniques showed positive culture within 24 hours. CONCLUSION: Vitreous fluid culture with blood culture bottles is superior to conventional media with statistically significant difference. There was no significant difference in time to positive culture.

Adolescent↗
PubMed2004

Influence of culture passages on growth kinetics and adenovirus vector production for gene therapy in monolayer and suspension cultures of HEK 293 cells.

To characterize the changes in cell growth rate and adenovirus vector (AdV) production capability of 293 cells during culture passages, 293 cells obtained at the 31st culture passage from ATCC (293M #31) were maintained as a monolayer culture and 293 cells obtained at an unknown culture passage from Invitrogen (293S) were maintained as suspension culture. In monolayer culture, the specific growth rate (micro) of 293M cells increased rapidly with culture passage up to passage 65 and thereafter became saturated. The micro of 293M passage 43 (#43) was 0.29 day(-1), while the average micro of 293M from #66 to #86 was 0.74+/-0.01 day(-1) (average +/- standard deviation). It was also noted that the cells became smaller in size during early culture passages. AdV production was also influenced by the number of culture passages. The AdV titer in the culture of 293M #66 was ca. tenfold higher than that of 293M #44, resulting from both a higher cell concentration and a higher AdV titer per cell at #66. In contrast, the micro, cell size, and AdV production of 293S cells in suspension culture did not change significantly as the culture passage number increased up to #40. Taken together, the culture passage influenced cell growth and AdV production of 293M cells in monolayer culture, but not those of 293S cells in suspension culture.

Adenoviridae↗
PubMed1996

Induction of ceramide glucosyltransferase activity in cultured human keratinocytes. Correlation with culture differentiation.

Ceramides are the major component of the extracellular lipids that comprise the epidermal permeability barrier. They are derived from glucosylceramides (GlcCer) upon their extrusion from lamellar granules into the extracellular space in the upper layers of the epidermis. To better understand the regulation of the unique pathway for ceramide production in epidermis, we have studied the activity of the enzyme responsible for GlcCer synthesis, ceramide glucosyltransferase (CerGlc transferase), during keratinocyte culture differentiation. Human keratinocyte cultures were expanded in low calcium keratinocyte growth medium (KGM) and then switched to either normal calcium KGM (nKGM) or "complete" Dulbecco's modified Eagle's medium/Ham's F-12 (3:1) supplemented with 10% fetal bovine serum (cDMEM). At 7 and 10 days after the medium switch, electron microscopy revealed that cDMEM cultures were more fully differentiated morphologically and contained numerous lamellar granules. The GlcCer/DNA content of cDMEM cultures increased to 6 times that of day 0 cultures and was nearly 4 times greater than that of nKGM cultures, whereas the total lipid/DNA content of cDMEM cultures increased to only 1.8 times that of day 0 cultures and was approximately 1.2 times that of nKGM cultures. CerGlc transferase activity/DNA increased 6 times in cDMEM cultures but <1.5 times in nKGM cultures. By contrast, beta-glucocerebrosidase activity, which is responsible for the conversion of GlcCer to ceramide, increased to a similar extent in both differentiating culture systems. Treatment of cultures with the reversible CerGlc transferase inhibitor, DL-threo-1-phenyl-2-(palmitoylamino)-3-morpholino-1-propanol, prevented the increase of GlcCer in cDMEM cultures, and blocked conversion of exogenously added ceramide to GlcCer. A low level of CerGlc transferase activity, relative to that in differentiated keratinocytes, was detected in cultures of other human cell types. These results indicate that CerGlc transferase activity is induced during epidermal differentiation and that regulation of this enzyme may be an important determinant of the specialized production and compartmentalization of epidermal sphingolipids.

4-Chloro-7-nitrobenzofurazan↗
PubMed2006

Beyond the 'new cross-cultural psychiatry': cultural biology, discursive psychology and the ironies of globalization.

The 'new cross-cultural psychiatry' heralded by Kleinman in 1977 promised a revitalized tradition that gave due respect to cultural difference and did not export psychiatric theories that were themselves culture bound. In the ensuing years, the view of culture within anthropology has continued to change, along with our understanding of the relationship of biological processes to cultural diversity, and the global political economic contexts in which mental health care is delivered. This article considers the implications of these new notions of culture, biology and the context of practice for theory in cultural psychiatry. The future of cultural psychiatry lies in advancing a broad perspective that: (a) is inherently multidisciplinary (involving psychiatric epidemiology, medical anthropology and sociology, cognitive science and social psychology), breaking down the nature/culture dichotomy with an integrative view of culture as a core feature of human biology, while remaining alert to cultural constructions of biological theory; (b) attends to psychological processes but understands these as not exclusively located within the individual but as including discursive processes that are fundamentally social; and (c) critically examines the interaction of both local and global systems of knowledge and power. Globalization has brought with it many ironies for cultural psychiatry: Transnational migrations have resulted in cultural hybridization at the same time as ethnicity has become more salient; the call for evidence-based medicine has been used to limit the impact of cultural research; and cultural psychiatry itself has been co-opted by pharmaceutical companies to inform marketing campaigns to promote conventional treatments for new populations. Cultural psychiatry must address these ironies to develop the self-critical awareness and flexibility needed to deliver humane care in shifting contexts.

Biology↗
PubMed2006

Characterisation of prototype Nurmi cultures using culture-based microbiological techniques and PCR-DGGE.

Undefined Nurmi-type cultures (NTCs) have been used successfully to prevent salmonella colonisation in poultry for decades. Such cultures are derived from the caecal contents of specific-pathogen-free birds and are administered via drinking water or spray application onto eggs in the hatchery. These cultures consist of many non-culturable and obligately anaerobic bacteria. Due to their undefined nature it is difficult to obtain approval from regulatory agencies to use these preparations as direct fed microbials for poultry. In this study, 10 batches of prototype NTCs were produced using an identical protocol over a period of 2 years. Traditional microbiological techniques and a molecular culture-independent methodology, polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE), were applied to characterise these cultures and also to examine if the constituents of the NTCs were identical. Culture-dependent analysis of these cultures included plating on a variety of selective and semi-selective agars, examination of colony morphology, Gram-staining and a series of biochemical tests (API, BioMerieux, France). Two sets of PCR-DGGE studies were performed. These involved the amplification of universal and subsequently lactic acid bacteria (LAB)-specific hypervariable regions of a 16S rRNA gene by PCR. Resultant amplicons were subjected to DGGE. Sequence analysis was performed on subsequent bands present in resultant DGGE profiles using the Basic Local Alignment Search Tool (BLAST). Microbiological culturing techniques tended to isolate common probiotic bacterial species from the genera Lactobacillus, Lactococcus, Bifidobacterium, Enterococcus, Clostridium, Escherichia, Pediococcus and Enterobacterium as well as members of the genera, Actinomyces, Bacteroides, Propionibacterium, Capnocytophaga, Proteus, and Klebsiella. Bacteroides, Enterococcus, Escherichia, Brevibacterium, Klebsiella, Lactobacillus, Clostridium, Bacillus, Eubacterium, Serratia, Citrobacter, Enterobacter, Pectobacterium and Pantoea spp. in addition to unculturable bacteria were identified as constituents of the NTCs using universal PCR-DGGE analysis. A number of the sequences detected by LAB-specific PCR-DGGE were homologous to those of a number of Lactobacillus spp., including L. fermentum, L. pontis, L. crispatus, L. salivarius, L. casei, L. suntoryeus, L. vaginalis, L. gasseri, L. aviaries, L. johnsonii, L. acidophilus, and L. mucosae in addition to a range of unculturable lactobacilli. While NTCs are successful due to their complexity, the presence of members of Lactobacillus spp. amongst other probiotic genera, in these samples possibly lends to the success of the NTC cultures as probiotics or competitive exclusion products in poultry over the decades. PCR-DGGE proved to be an effective tool in detecting non-culturable organisms present in these complex undefined cultures. In conclusion, while the culture-dependent identification methods or PCR-DGGE alone cannot comprehensively elucidate the bacterial species present in such complex cultures, their complementarity provides useful information on the identity of the constituents of NTCs and will aid in future development of defined probiotics. Moreover, for the purpose of analysing prototype NTCs during their development, PCR-DGGE overcomes the limitations associated with conventional culturing methods including their low sensitivities, inability to detect unculturable bacteria and unknown species, very slow turnabout time and poor reproducibility. This study demonstrated that PCR-DGGE is indeed more valuable in detecting predominant microbial populations between various NTCs than as an identification methodology, being more applicable as a quality control method used to analyse for batch-to-batch variation during NTC production.

Animals↗
PubMed1992

Change of the terminal oxidase from cytochrome a1 in shaking cultures to cytochrome o in static cultures of Acetobacter aceti.

Acetobacter aceti has an ability to grow under two different culture conditions, on shaking submerged cultures and on static pellicle-forming cultures. The respiratory chains of A. aceti grown on shaking and static cultures were compared, especially with respect to the terminal oxidase. Little difference was detected in several oxidase activities and in cytochrome b and c contents between the respiratory chains of both types of cells. Furthermore, the results obtained here suggested that the respiratory chains consist of primary dehydrogenases, ubiquinone, and terminal ubiquinol oxidase, regardless of the culture conditions. There was a remarkable difference, however, in the terminal oxidase, which is cytochrome a1 in cells in shaking culture but cytochrome o in cells grown statically. Change of the culture condition from shaking to static caused a change in the terminal oxidase from cytochrome a1 to cytochrome o, which is concomitant with an increase of pellicle on the surface of the static culture. In contrast, reappearance of cytochrome a1 in A. aceti was attained only after serial successive shaking cultures of an original static culture; cytochrome a1 predominated after the culture was repeated five times. In the culture of A. aceti, two different types of cells were observed; one forms a rough-surfaced colony, and the other forms a smooth-surfaced colony. Cells of the former type predominated in the static culture, while the cells of the latter type predominated in the shaking culture. Thus, data suggest that a change of the culture conditions, from static to shaking or vice versa, results in a change of the cell type, which may be related to the change in the terminal oxidase from cytochrome a1 to cytochrome o in A. aceti.

Acetobacter↗