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PubMed1990

Alpha 1-adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures.

Binding of [125I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70-85% specific. Various adrenergic agonists and antagonists competed for [125I]HEAT binding according to the potencies of prazosin greater than, yohimbine greater than or equal to, clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showed the Bmax of 209 fmol/mg protein and a Kd of 184 pM for [125I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [125I]HEAT binding sites with a maximal response observed after 8 h at 100 microM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 microM NE. Clonidine expressed differential effects on alpha 1-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 microM clonidine caused a 40% decrease in the Bmax of [125I]HEAT binding without influencing the Kd value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [125I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.

Adrenergic alpha-Agonists↗
PubMed1986

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.

Animals↗
PubMed1989

Calbindin-immunoreactive sensory neurons in dissociated dorsal root ganglion cell cultures of chick embryo: role of culture conditions.

Immunoreactivity to calbindin D-28k, a vitamin D-dependent calcium-binding protein, is expressed by neuronal subpopulations of dorsal root ganglia (DRG) in the chick embryo. To determine whether the expression of this phenotypic characteristic is maintained in vitro and controlled by environmental factors, dissociated DRG cell cultures were performed under various conditions. Subpopulations of DRG cells cultured at embryonic day 10 displayed calbindin-immunoreactive cell bodies and neurites in both neuron-enriched or mixed DRG cell cultures. The number of calbindin-immunoreactive ganglion cells increased up to 7-10 days of culture independently of the changes occurring in the whole neuronal population. The presence of non-neuronal cells, which promotes the maturation of the sensory neurons, tended to reduce the percentage of calbindin-immunoreactive cell bodies. Addition of horse serum enhanced both the number of calbindin-positive neurons and the intensity of the immunostaining, but does not prevent the decline of the subpopulation of calbindin-immunoreactive neurons during the second week of culture; on the contrary, the addition of muscular extract to cultures at 10 days maintained the number of calbindin-expressing neurons. While calbindin-immunoreactive cell bodies grown in culture were small- or medium-sized, no correlation was found between cell size and immunostaining density. At the ultrastructural level, the calbindin immunoreaction was distributed throughout the neuroplasm. These results indicate that the expression of calbindin by sensory neurons grown in vitro may be modulated by horse serum-contained factors or interaction with non-neuronal cells. As distinct from horse serum, muscular extract is able to maintain the expression of calbindin by a subpopulation of DRG cells.

Animals↗
PubMed1996

Human thyroid epithelial cells cultured in monolayers. I. Decreased thyroglobulin and cAMP response to TSH in 12-week-old secondary and tertiary cultures.

An in vitro system of secondary and tertiary cultures of human thyroid epithelial cells (TFECs) in monolayer is described. The function of the cells was evaluated by the second messenger cAMP and the end product thyroglobulin (Tg). The Tg production from the cells was measured in the supernatant by a newly developed enzyme-linked immunosorbent assay. The TFECs in secondary monolayer cultures had preserved the ability to produce Tg and cAMP despite lack of polarization. Furthermore, a preserved ability of TSH-stimulated production of Tg and cAMP in 12-week-old secondary and tertiary cultures was found. However, the Tg and cAMP levels decreased gradually with the age of the cultures. In the secondary culture the TSH-stimulated Tg production decreased from 253 ng/micrograms DNA (205-263) after 3 weeks to 18 ng/micrograms DNA (6-81), P < 0.001, n = 6 after 12 weeks and TSH-stimulated cAMP production from 660 pmol/micrograms DNA (500-840) to 60 (40-200), P < 0.001, n = 6. The decreased responsiveness of long-term cultures results in preference of short-term secondary cultures, which provide a more suitable experimental model for in vitro investigation of human thyroid cell functions.

Cells, Cultured↗
PubMed2006

Cultural competency: Agenda for Cultural Competency Using Literature and Evidence.

BACKGROUND: Cultural competency has been recognized as an important issue relevant to all health professions. A research agenda is needed to establish a systematic approach to developing an understanding of factors relevant to the delivery of culturally competent health care. OBJECTIVE: Within the context of existing literature, evidence-based, concrete recommendations are developed as an Agenda for Cultural Competency Using Literature and Evidence (ACCULTURE). METHODS: First, key points representing opportunities for intervening in promotion of cultural competent health care are discussed. Following is a review of existing literature with a focus on identifying next steps for future research. Recommendations for licensing, education, and continuing education requirements suggest developing educational research establishing course content and delivery strategies that have measurable impact on improving cultural competency. In addition, existing initiatives need to be evaluated regarding effectiveness in recruiting, retaining, and preparing a diverse workforce. Patient care recommendations focus on further developing an understanding of the factors impacting health outcomes for culturally diverse patients. RESULTS: Further work is needed for translating theoretically-based research into concrete curricula maintaining evidence-based outcomes. It is important to continue with promoting policies ensuring that research and clinical trials include diverse samples and a broad range of variables implicated in differential outcomes. CONCLUSIONS: Based on connections between cultural competency and workforce diversity established within existing literature, data are needed regarding the effectiveness of existing initiatives promoting scholarships, grants, and incentives for improving workforce diversity and funding research on diversity issues. Finally, additional research is needed to evaluate existing and new policies for funding services and access for health services.

Clinical Competence↗
PubMed1999

A cell culture system for the study of amyloid pathogenesis. Amyloid formation by peritoneal macrophages cultured with recombinant serum amyloid A.

A murine macrophage culture system that is both easy to employ and amenable to manipulation has been developed to study the cellular processes involved in AA amyloid formation. Amyloid deposition, as identified by Congo red-positive, green birefringent material, is achieved by providing cultures with recombinant serum amyloid A2 (rSAA2), a defined, readily produced, and highly amyloidogenic protein. In contrast to fibril formation, which can occur in vitro with very high concentrations of SAA and low pH, amyloid deposition in culture is dependent on metabolically active macrophages maintained in neutral pH medium containing rSAA2 at a concentration typical of that seen in acute phase serum. Although amyloid-enhancing factor is not required, its addition to culture medium results in larger and more numerous amyloid deposits. Amyloid formation in culture is accompanied by C-terminal processing of SAA and the generation of an 8.5-kd fragment analogous to amyloid A protein produced in vivo. Consistent with the possibility that impaired catabolism of SAA plays a role in AA amyloid pathogenesis, treatment of macrophages with pepstatin, an aspartic protease inhibitor, results in increased amyloid deposition. Finally, the amyloidogenicity exhibited by SAA proteins in macrophage cultures parallels that seen in vivo, eg, SAA2 is highly amyloidogenic, whereas CE/J SAA is nonamyloidogenic. The macrophage culture model presented here offers a new approach to the study of AA amyloid pathogenesis.

Amyloid↗
PubMed2000

Culture-bound syndrome and a culturally sensitive approach: from a viewpoint of medical anthropology.

Some aspects of the culture-bound syndrome are presented for discussion. From the psychiatric and medical anthropological viewpoints, kamidaari is described as an initiatory illness for seeing a shaman, and focus on clinical realities developing between different therapeutic subcultures in the same culture and the complementary practices of two epistemological ones, namely, the shamanistic and modern psychiatric system in the shamanistic climate. It is suggested that the culture-bound syndrome that reflects cultural influences on disease patterns and renders them difficult to place in a universal classificatory system should be seen as a vernacular bricolage or as tactics used by people within the web of their own local culture of origin. Therapists who treat patients in a cross-epistemological milieu should be aware of the subcultural-epistemological issues that may affect the clinical process. It should be recognized that, depending on the nature of a particular psychiatric crisis, the clinical encounter is straddling the boundaries of multiple clinical realities. At every stage in the clinical field, there is an intersection, consonance, or interruption of rejoinders in the open dialog by all those engaged in the clinical time. Aspects of climatic, culturally sensitive psychotherapy will be described, and the concept of the culture-bound syndrome will be reconsidered. Our approach could be seen as 'situation- and fudo-bound'.

Adult↗
PubMed1995

The characteristics of cultured mucosal cell sheet as a material for grafting; comparison with cultured epidermal cell sheet.

The characteristics of cultured mucosal cells from the oral mucosa were investigated and compared with those of cultured epidermal cells. Total cell counts showed that mucosal cells possessed greater proliferating ability than epidermal cells. The results of 3(4,5-dimethyle-thiazoyl-2-yl)2,5 diphenyltetrazolium bromide assay confirmed this observation and also suggested that the mucosal cells maintained biological activity longer than epidermal cells. The most important morphological characteristics of mucosal cells in culture were their low grade of differentiation. Interestingly, the epidermal cells showed enucleation and keratinization progressively during culture, whereas the mucosal cells showed no obvious enucleation when examined by light microscopy. Transmission electron microscopy showed a smaller number of desmosomes in cultured mucosal cells than epidermal cells. The results of this study reveal cultured mucosal cell sheets to be a possible material for grafting in addition to cultured epidermal cell sheets.

Cell Count↗
PubMed2005

Cell culture block array for immunocytochemical study of protein expression in cultured cells.

Immunocytochemical staining of cultured cells using specific antibodies is a powerful technique to study the expression and subcellular localization of proteins. However, this technique is associated with sample-to-sample variations because samples are handled individually and manually. Cell permeation is needed when intracytoplasmic or nuclear proteins are studied. Storage of cultured cells is difficult, and experiments must be repeated if additional studies are desired later, which introduces more variations. We developed a cell culture block array technique that converts cultured cells into a permanently fixed form identical to tissue sections prepared for pathologic examination. Cells from different cultures can be embedded in a single block. Many identical sections, each containing cells from multiple cultures, may be stained with different antibodies using an automated stainer. As a result, sample-to-sample variation is eliminated. Because cells in these blocks are sectioned by knives, all cellular proteins come into direct contact with antibodies, and cell permeation is not needed. Such blocks can be conveniently stored for years without loss of antigens, providing a constant source for future studies. We demonstrated the utility of this technique by studying the proliferation and neuroendocrine differentiation of prostate cancer-derived LNCaP cells cultured in vitro.

Cell Culture Techniques↗
PubMed2004

Intelligence and culture: how culture shapes what intelligence means, and the implications for a science of well-being.

This paper discusses the relationship between culture and intelligence. The main message of the paper is that intelligence cannot fully or even meaningfully be understood outside its cultural context. Behaviour that is considered intelligent in one culture may be considered unintelligent in another culture, and vice versa. Moreover, people in different cultures have different implicit (folk) theories of intelligence, so may not even mean the same thing by the word. The relationships between different aspects of intelligence can vary across cultures, with correlations that are positive in one setting proving to be negative in another. The paper opens with a general discussion of issues regarding the relationship between the two concepts. It then describes the theory of successful intelligence, which motivates our work on the interface between culture and intelligence. Finally, the article draws some conclusions.

Cognition↗
PubMed1995

Cultured human fibroblasts and not cultured human keratinocytes express a PTH/PTHrP receptor mRNA.

There is increasing evidence that parathyroid hormone (PTH) and PTH-related peptides (PTHrP) are involved in normal skin cell growth; therefore, we investigated whether the PTH/PTHrP receptor was expressed in cultured human keratinocytes and dermal fibroblasts. Northern analyses of poly (A)+ RNA isolated from cultured fibroblasts revealed two PTH/PTHrP receptor transcripts with one major band at 2.5 kb and one minor band at 2.3 kb. These transcripts were consistent with those found in human osteosarcoma cells, which are known to express PTH/PTHrP-R mRNAs. In contrast, after repeated Northern analyses no PTH/PTHrP receptor transcripts were found in poly (A)+ RNA isolated from cultured keratinocytes. Reverse-transcriptase/nested polymerase chain reaction analyses of total RNA isolated from cultured keratinocytes and fibroblasts confirmed the Northern analyses data that the PTH/PTHrP receptor was expressed in cultured fibroblasts but not in cultured keratinocytes. When cultured fibroblasts and keratinocytes were exposed to 10(-7) M PTH (1-34) there was a twofold increase in cAMP levels in the fibroblasts and no demonstrable increase was noted in keratinocytes. These results suggest that skin fibroblasts possess the classical PTH/PTHrP receptor and are target cells for PTH and PTHrP whereas keratinocytes do not have the receptor and are unresponsive to its N-terminal agonist in the stimulation of cAMP formation.

Base Sequence↗
PubMed2006

Growth of nutrient-replete Microcystis PCC 7806 cultures is inhibited by an extracellular signal produced by chlorotic cultures.

The frequency of cyanobacterial blooms has been increasing all over the world. These blooms are often toxic and have become a serious health problem. The aim of this work was to search for population density control mechanisms that could inhibit the proliferation of the toxic bloom-forming genus Microcystis. Microcystis PCC 7806 cultured for long periods in liquid ASM-1 medium loses its characteristic green colour. When a medium of chlorotic cultures is added to a nutrient-replete culture, cell density increase is drastically reduced when compared with controls. Inhibition of cell proliferation occurs in Microcystis cultures from any growth stage and was not strain-specific, but other genera tested showed no response. Investigations on the mechanism of growth inhibition showed that cultures treated with the conditioned medium acquired a pale colour, with pigment concentration similar to that found in chlorotic cultures. Ultrastructural examination showed that the conditioned medium induced thylakoid membrane disorganization, typical of chlorotic cells, in nutrient-replete cultures. An active extract was obtained and investigations showed that activity was retained after heating and after addition of an apolar solvent. This indicates that activity of the conditioned medium from chlorotic cells results from non-protein, apolar compound(s).

Culture Media, Conditioned↗
PubMed1995

Nicotinate catabolism is dispensable and nicotinate anabolism is crucial in Azorhizobium caulinodans growing in batch culture and chemostat culture on N2 as The N source.

When Azorhizobium caulinodans was grown in chemostat cultures with N2 as the N source at a constant dilution rate of 0.1 h-1 in media with a constant concentration (50 mM) of succinate and variable concentrations (1.5 to 585 microM) of nicotinate, neither the growth yield on succinate, the specific rate of O2 consumption, nor the specific rate of CO2 production showed linear regression with the concentration of nicotinate. Moreover, for transient continuous cultures in which the nicotinate concentration was gradually lowered, growth parameters remained unchanged until an apparently critical level of 0.7 microM nicotinate was reached. Below this nicotinate level, an immediate washout of the chemostat population began. A. caulinodans nicotinate hydroxylase-negative mutant 61007, unable to catabolize nicotinate, and the wild type behaved similarly. Thus, for continuous cultures supplied with N2 as the N source, submicromolar concentrations of nicotinate both sustained pyridine nucleotide biosynthesis at sufficient levels and precluded the use of nicotinate as a catabolic substrate. Furthermore, when more nicotinate was provided, dual succinate-nicotinate limitation in continuous cultures did not occur. Finally, when nicotinate is present in suboptimal concentrations, the specific growth rate is directly proportional to the amount of nicotinate present per unit of biomass. By contrast, in batch cultures with different nicotinate concentrations and with either succinate or lactate as the carbon and energy source, anomalous growth curves were obtained. With a low concentration (1.5 microM) of nicotinate, growth on N2 occurred, albeit at low rates. With a high concentration (195 microM) of nicotinate, growth on N2 was temporarily stimulated, but nicotinate was quickly exhausted and growth was thereafter nicotinate limited. Continuous supplementation of batch cultures with nicotinate allowed only transient exponential growth followed by linear growth. Thus, also for batch cultures, nicotinate catabolism is dispensable, although a high concentration of nicotinate temporarily stimulates growth on N2. Ut us concluded that A. caulinodans is a true diazotroph.

Bacteriological Techniques↗
PubMed1997

Use of Gen-Probe AccuProbe Group B streptococcus test to detect group B streptococci in broth cultures of vaginal-anorectal specimens from pregnant women: comparison with traditional culture method.

Detection of vaginal-anorectal colonization with group B streptococci (GBS) is critical to the prevention of neonatal GBS disease. The recommended method for the detection of GBS is culture of the distal vagina and anorectum in a selective broth medium followed by subculture to solid media and identification of GBS on the solid media. The purpose of this study was to compare this standard culture method with the detection of GBS directly from an enrichment broth by utilizing the Gen-Probe AccuProbe Group B Streptococcus Culture Identification Test (GPGB). A total of 502 specimens were tested in this study. Both culture and the GPGB detected 90 of 95 positive specimens (sensitivity, 94.7%). There were two false-positive GPGB results (specificity, 99.5%). An analysis of 100 consecutive specimens was performed to compare the costs associated with the use of a primary tryptic soy agar plate with 5% sheep blood (BAP) and a 3-ml tube of Todd-Hewitt broth supplemented with 10 micrograms of nalidixic acid per ml and 15 micrograms of colistin per ml (LIM broth) with subculture to another BAP and the costs associated with the GPGB. Our estimated costs were $3.68 for a negative culture including 7.0 min of labor, $5.41 for a positive culture including 8.9 min of labor, and $5.16 for the GPGB including 3.6 min of labor (based upon a test run of 10 specimens and two controls and a cost of $70.00 for a 20-test GPGB kit). Accessioning and reporting of results are not included in these costs. In conclusion, we found that the GPGB was equivalent in sensitivity to our standard culture method. While overall costs were somewhat higher for the GPGB, the GPGB has lower labor costs and offers the potential for incremental savings with higher test volumes and computer interface capability.

Bacterial Typing Techniques↗
PubMed1981

Normal mammary cells in long term culture. I. development of hormone-dependent functional monolayer cultures and assay of alpha-lactalbumin production.

Mammary cells from normal tissue of virginal, pregnant, and lactating rats have been adapted to long term monolayer culture in plastic culture dishes with retention of hormone-responsive functional activity. The addition of PRL, insulin, and corticosterone resulted in an increase in the proportion of epithelial cells. the development of intracellular lipid droplets, and the ordered aggregations of these cells. In the presence of these hormones, the milk protein, alpha-lactalbumin (a-LA), was secreted into the growth medium at rates of 20-100 ng/mg cellular protein . 24 h. A double antibody RIA for a-LA capable of measuring 0.1 ng a-LA/100 microliter growth medium was developed in our laboratory for these studies. Both intracellular and extracellular a-LA fell below detectability within 2-3 weeks after hormone withdrawal. Intracellular a-LA accounted for less than 3% of the total a-LA accumulated in each culture in 24 h. the production rate of cells continuously given hormones increased 4- to 7-fold over a period of several months in culture, and their output was greater than 100-fold above that of cells not given hormones. These cells were obtained by overnight digestion and dispersion of tissue using selected batches of collagenase in the presence of 5% fetal calf serum. Plating densities of at least 3 X 10(4) cells/cm2 in Minimum Essential Medium supplemented with 14% fetal calf serum were required for optimal functional activity. Despite several months without added hormones, these cultures can retain their hormone responsiveness, since subsequent hormone addition resulted in detectable a-LA production beginning within 7-14 days. Our studies demonstrate for the first time that normal mammary cells can be maintained in a functional hormone-responsive state for extended periods in primary cell culture. These long term cell cultures provide a system with which the effects of these and other hormones on milk production and cell differentiation can be assessed under conditions which minimize the influence of the prior in vivo hormonal millieu. (Endocrinology 108: 573, 1981)

Animals↗
PubMed1991

Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity.

In the developing peripheral nerve, Schwann cells proliferate rapidly and then become quiescent, an essential step in control of Schwann cell differentiation. Cell proliferation is controlled by growth factors that can exert positive or inhibitory influences on DNA synthesis. It has been well established that neonatal Schwann cells divide very slowly in culture when separated from neurons but here we show that when culture was continued for several months some cells began to proliferate rapidly and non-clonal lines of immortalised Schwann cells were established which could be passaged for over two years. These cells had a similar molecular phenotype to short-term cultured Schwann cells, except that they expressed intracellular and cell surface fibronectin. The difference in proliferation rates between short- and long-term cultured Schwann cells appeared to be due in part to the secretion by short-term cultured Schwann cells of growth inhibitory activity since DNA synthesis of long-term, immortalised Schwann cells was inhibited by conditioned medium from short-term cultures. This conditioned medium also inhibited DNA synthesis in short-term Schwann cells stimulated to divide by glial growth factor or elevation of intracellular cAMP. The growth inhibitory activity was not detected in the medium of long-term immortalised Schwann cells, epineurial fibroblasts, a Schwannoma (33B), astrocytes or a fibroblast-like cell-line (3T3) and it did not inhibit serum-induced DNA synthesis in epineurial fibroblasts, 33B cells or 3T3 cells. The activity was apparently distinct from transforming growth factor-beta, activin, IL6, epidermal growth factor, atrial natriuretic peptide and gamma-interferon and was heat and acid stable, resistant to collagenase and destroyed by trypsin treatment. We raise the possibility that loss of an inhibitory autocrine loop may contribute to the rapid proliferation of long-term cultured Schwann cells and that an autocrine growth inhibitor may have a role in the cessation of Schwann cell division that precedes differentiation in peripheral nerve development.

Animals↗
PubMed1989

Protein synthesis and degradation in non-cultured and in-vitro cultured rabbit blastocysts.

In 'pulse-chase' experiments synthesis and half-lives of leucine-labelled proteins were determined in rabbit blastocysts. Embryos were either non-cultured controls or were cultured for 24 h or 48 h in Ham's F-10 medium supplemented with homologous serum or uterine flushings. In control blastocysts protein synthesis increased by a factor of 10 between Day 4 and Day 5. Half-lives of newly synthesized proteins were 32 h in Day-4 and 99 h in Day-5 control blastocysts. In-vitro culture of Day-4 blastocysts led to dramatically shortened half-lives, amounting to 6-10 h. Blastocysts developing in uterine flushing-supplemented media differed significantly from those cultured in serum-supplemented media. Protein synthesis was enhanced and protein degradation was normal for culture times up to 24 h. These results demonstrate (1) that half-lives of proteins in rabbit blastocysts increase with advancing embryonic age, and (2) that a characteristic feature of the altered metabolism of cultured blastocysts is a dramatically accelerated protein degradation, which (3) can be prevented for some time by supplementation of the culture medium with uterine secretions.

Animals↗
PubMed1988

Comparison of tube cultures of Madin Darby canine kidney cells with shell-vial cultures after low-speed centrifugation for influenza virus isolation.

The efficacy of isolation of influenza viruses was compared in two systems--conventional tube cultures of Madin Darby canine kidney cells, and shell-vial cultures after low-speed centrifugation. One hundred and fifty eight throat and nasal swabs were inoculated and cultures screened by hemagglutination on days 1, 2, 3 and 5 post-inoculation. Twenty-four shell-vial cultures (15.2%) and 6 tube cultures (3.8%) were positive for influenza virus type B, indicating that shell-vial cultures with low-speed centrifugation are superior to tube cultures for influenza virus type B isolation (p less than 0.005 by McNemar's test for analysis of matched pairs data).

Animals↗