FLUORESCENCE SPECTROSCOPY ON HUNTINGTON''S FIBROBLASTS

Abstract– 8-Anilino-1-naphthalene sulfonic acid (ANS), a fluorescent probe specific for membrane polar-apolar interfaces, has been used to investigate differences in corrected excitation, emission and scanned excitation polarization spectra of intact age, sex and passage number matched Huntington's disease (HD) and normal fibroblasts grown in cell culture. At passage number 4, the HD fibroblasts, compared to normals, exhibited a 30-fold increase in excitation and emission intensity, a 30 nm blue shift of the emission maximum and a 0.18 polarization unit increase. These findings suggest that the ANS molecule experiences either increased binding sites or an increased quantum yield, a more nonpolar environment, and a more restricted motion in HD fibroblasts. Continued subculture at higher passages (10) showed a change of these findings towards normals. These fluorescent studies support the concept that a membrane abnormality is present in HD.     

继代周期和接种量对葡萄细胞培养的影响

在每种不同的继代周期和接种量条件下,葡萄细胞在连续10次继代培养过程中的生物量、花青素含量、胞内糖、胞内蛋白及胞内总磷均表现出不同程度的波动。不同接种量对培养不稳定性的影响比不同继代周期大;在所考察的条件中,7d继代周期与1.60g接种量组合的继代条件下花青素合成相对稳定;花青素合成与胞内蔗糖或胞内总磷水平呈负相关。     

Modulation of collagen synthesis in normal and osteoarthritic cartilage

In osteoarthritic cartilage, chondrocytes are able to present heterogeneous cellular reactions with expression and synthesis of the (pro)collagen types characteristic of prechondrocytes (type IIA), hypertrophic chondrocytes (type X), as well as differentiated (types IIB, IX, XI, VI) and dedifferentiated (types I, III) chondrocytes. The expression of type IIA procollagen in human osteoarthritic cartilage support the assumption that OA chondrocytes reverse their phenotype towards a chondroprogenitor phenotype. Recently, we have shown that dedifferentiation of mouse chondrocytes induced by subculture was associated with the alternative splicing of type II procollagen pre-mRNA with a switch from the IIB to the IIA form. In this context, we demonstrated that BMP-2 favours expression of type IIB whereas TGF-beta1 potentiates expression of type IIA induced by subculture. These data reveal the specific capability of BMP-2 to reverse the program of chondrocyte dedifferentiation. This interesting feature needs to be tested with human chondrocytes since cell amplification is required for the currently used autologous chondrocyte transplantation.     

Evaluation of the viability of cultured corneal endothelial cells by quantitative electron probe X-ray microanalysis

Construction of artificial organs and tissues by tissue engineering is strongly dependent on the availability of viable cells. For that reason, the viability and the physiological status of cells kept in culture must be evaluated before the cells can be used for clinical purposes. In this work, we determined the viability of isolated rabbit corneal endothelial cells by trypan blue staining and quantitative electron probe X-ray microanalysis. Our results showed that the ionic content of potassium in cultured corneal endothelial cells tended to rise initially, but significantly decreased in cells in the fifth (and final) subculture, especially in comparison to cells in the fourth subculture (P < 0.001). However, the concentration of sulfur was higher in the fifth subculture than in the fourth subculture (P < 0.001), with a nonsignificant increase in sodium in the fifth subculture (P = 0.031). These data imply a remarkable decrease in the K/Na ratio from the fourth to the fifth subculture. Our microanalytical results, along with the morphological differences between cells in the last two subcultures, are compatible with an early phase of the preapoptotic process in the fifth subculture, and suggest that cells of the first four subcultures would be better candidates for tissue engineering.     

Belongingness in Early Secondary School: Key Factors that Primary and Secondary Schools Need to Consider

It is unknown if, and how, students redefine their sense of school belongingness after negotiating the transition to secondary school. The current study used longitudinal data from 266 students with, and without, disabilities who negotiated the transition from 52 primary schools to 152 secondary schools. The study presents the 13 most significant personal student and contextual factors associated with belongingness in the first year of secondary school. Student perception of school belongingness was found to be stable across the transition. No variability in school belongingness due to gender, disability or household-socio-economic status (SES) was noted. Primary school belongingness accounted for 22% of the variability in secondary school belongingness. Several personal student factors (competence, coping skills) and school factors (low-level classroom task-goal orientation), which influenced belongingness in primary school, continued to influence belongingness in secondary school. In secondary school, effort-goal orientation of the student and perception of their school’s tolerance to disability were each associated with perception of school belongingness. Family factors did not influence belongingness in secondary school. Findings of the current study highlight the need for primary schools to foster belongingness among their students at an early age, and transfer students’ belongingness profiles as part of the hand-over documentation. Most of the factors that influenced school belongingness before and after the transition to secondary are amenable to change.     

Cytokinin production by Asparagus shoot apex cultured in vitro

The cytokinin producing capacity of asparagus (Asparagus officinalis L.) shoot apex was examined by means of shoot apex culture in vitro, where adventitious roots were never formed. The cultured shoot apices continued to diffuse a small but constant amount of cytokinin into the medium throughout five passages of subculture. The cytokinin content in the apices at the end of the subculture was not different from that at the beginning of the subculture. These results indicate a production of cytokinin by the apices. However, the finding is not in conflict with the hypothesis that the root tip is the major source of cytokinin supply, because the root tip of asparagus produced more cytokinin than the shoot apex and the decline of shoot growth observed during the subculture was partially restored by an application of zeatin into the medium.     

THE GROWTH OF LACTIC ACID STREPTOCOCCI IN MIXED STARTER CULTURES

SUMMARY: An attempt has been made so to combine rapid acid-producing streptococci that on daily subculture the individual components in the mixture will remain present in even proportions. Some such combinations have been made, but frequently the proportions of the components alter until one culture becomes dominant. When one culture dominates rapidly this is due to the production of antibiotic factors.     

Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions

Summary The understanding of pathways associated with differentiated function in human epithelial cells has been enhanced by the development of methods for the short-term culture of human epithelial cells. In general these methods involve the use of serum. The subculture and maintenance of epithelial cells in long-term culture has been more problematic. A serum-free medium developed for human bronchial epithelial cells was slightly modified and found to be useful for the subculture and long-term maintenance of not only bronchial epithelial cells, but also tracheal, nasal polyp, and sweat gland epithelial cells from either normal or cystic fibrosis individuals. The cells maintained epithelial-specific characteristics after multiple subcultures. Monolayers of epithelial cells showed junctional complex formation, the presence of keratin, and micro villi. Functional studies with Ussing chambers showed short circuit current (I_sc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells. This work is supported by grants HL41928 and DK39619 (DCG), HL24136 (CBB), and HL42368 (JHW and DCG) from the National Institutes of Health, Bethesda, MD.     

Direct and indirect experiences with heterosexism: How slurs impact all students

Students targeted by homophobic discrimination are at risk for poor academic outcomes, yet few studies have examined how witnessing discrimination affects students. This study examined the impact of direct and indirect experiences of heterosexism on feelings of safety, belongingness, and connectedness among a sample of 1,702 students at a public university in the Western United States. Sexual minority students were more likely to be a target of and witness to discrimination, and they reported significantly lower levels of safety and belongingness. Hierarchical multiple regression analyses revealed that witnessing discriminatory acts contributed significantly to lower levels of safety, belongingness, and connectedness among heterosexual students, even after accounting for personal experiences with discrimination. For sexual minority students, witnessing discrimination was not associated with school outcomes, but hearing peers intervene against slurs was associated with increased belongingness. Perceptions of the campus climate were robustly associated with student outcomes, regardless of sexual orientation.     

The relationship between aromatase activity and body fat distribution

The metabolism of androstenedione (A) to estrone (E1) and 5 alpha-reduced androgens was studied in stromal cells derived from human adipose tissue from different body sites. The tissue was obtained from non-obese patients undergoing cosmetic liposuction or at the time of surgery for reduction mammoplasty. The conversion of A to E1 per 1x 10(6) cells was between 6- and 30-fold greater in the upper thigh, buttock, and flank than in the abdomen. These differences were present in primary culture and persisted to at least the third subculture. Estrogen formation in breast adipose tissue was similar to that found in cells from abdominal fat. The formation of 5 alpha-reduced metabolites (5 alpha-androstenedione, androsterone, and dihydrotestosterone) varied from patient to patient but was similar in cells from different body sites. These studies show that the regional distribution of fat may influence the metabolism of androgens in adipose tissue, with upper body fat tending to form a lower ratio of estrogens to 5 alpha-reduced androgens than lower body fat.     

The needs of primary care mental health service users: a Q-sort study

Background Within scientific and policy literature, the broader needs and preferences of the service user are usually described as being relatively homogenous for any particular condition. Additionally, despite common adult mental health problems being a huge burden to primary care services, there has been little research into the client's perspective of their needs regarding such difficulties. The aim of this research was thus to uncover the issues and preferences for this sample.Method The attitudes of 28 service users were explored through use of a Q-methodology approach. This procedure involves statistical analysis of the order that participants rank pertinent statements associated with the area of study.Results Five independent factors (clusters of preferences) were identified. These differed over issues associated with: relationships with service providers, access/barriers associated with services, information provision, stigma, choices associated with therapy and the care pathway, medication and complementary approaches, as well as social support.Discussion The study suggests that differing individual needs exist within diagnostic categories which, unless considered, may impede service engagement. Issues associated with the implications for service delivery for these differing clusters of perceived needs and viewpoints are outlined.     

Biological characterization of a cell line derived from the pig oviduct

Summary A cell line has been derived from the inner lining of the pig oviduct. During 33 months in continous culture, the cells have been subcutured 145 times. Early passage cells had an epithelial-like morphology with clear and abundant cytoplasm. A spontaneous morphological alteration occurred at the 50th subculture by emergence of fibroblast-like cells. Evolution towards establishment of a cell line was suggested by the spontaneous morphological alteration, increase of the maximum population density without a significant change in the diploid state (92nd subculture), increase, in size and number of nucleoli, and increase in the nuclear-cytoplasmic ratio and of cytoplasmic basophilia. Evidence of pig origins is subtantiated by the karyotype (38, XX), the persistence of Barr bodies indicating female origin (140th subculture) and the persistence of species specificity by fluorescence (141th subculture). Preliminary studies indicate that nine viruses will replicate in both epithelial-like and fibroblast-like cells.     

Purity,cell viability,expression of GFAP and bystin in astrocytes cultured by different procedures

Primary astrocyte cultures are the most commonly used in vitro model for neurobiological studies. We speculated that different protocols might induce differences not only in the percentage of astrocytes but also in their biological characteristics. In this study, we investigated the effects of four major protocols on the purity of astrocytes, cell viability, expression of glial fibrillary acidic protein (GFAP) and bystin of cultured astrocytes using MTT assay, immunocytochemical staining, and Western blot analysis. We demonstrated that the purity of astrocytes (98.9%) generated by the subculture (SC) procedure is significantly higher than those generated by primary culture (PC), shaken once culture (SK‐1) or shaken twice culture (SK‐2). We also showed that expressions of GFAP and bystin in astrocytes that are purified by the SK‐2 or SK‐1 procedures are significantly higher than those in astrocytes prepared by PC or SC. In addition, astrocytes cultured by SK‐2 or SK‐1 have a higher level of cell viabilities at most time points after ischemia compared with astrocytes cultured by PC or SC. These suggested that physical stimulation induced by “shaken” or culture operation might be able to activate astrocytes and implied that different procedures induce differences not only in the purity but also in the biological characteristics of astrocytes, such as the percentage of activated astrocytes, GFAP, and bystin expressions and responses to ischemia. A more detailed analysis about the effect of “culture protocol factor” on the biological characteristics of astrocytes is absolutely needed. J. Cell. Biochem. 109: 30–37, 2010. © 2009 Wiley‐Liss, Inc.     

Individual acquisition of “stick pounding” behavior by naïve chimpanzees

Many studies investigating culture in nonhuman animals tend to focus on the inferred need of social learning mechanisms that transmit the form of a behavior to explain the population differences observed in wild animal behavioral repertoires. This research focus often results in studies overlooking the possibility of individuals being able to develop behavioral forms without requiring social learning. The disregard of individual learning abilities is most clearly observed in the nonhuman great ape literature, where there is a persistent claim that chimpanzee behaviors, in particular, require various forms of social learning mechanisms. These special social learning abilities have been argued to explain the acquisition of the relatively large behavioral repertoires observed across chimpanzee populations. However, current evidence suggests that although low‐fidelity social learning plays a role in harmonizing and stabilizing the frequency of behaviors within chimpanzee populations, some (if not all) of the forms that chimpanzee behaviors take may develop independently of social learning. If so, they would be latent solutions—behavioral forms that can (re‐)emerge even in the absence of observational opportunities, via individual (re)innovations. Through a combination of individual and low‐fidelity social learning, the population‐wide patterns of behaviors observed in great ape species are then established and stably maintained. This is the Zone of Latent Solutions (ZLS) hypothesis. The current study experimentally tested the ZLS hypothesis for pestle pounding, a wild chimpanzee behavior. We tested the reinnovation of this behavior in semi‐wild chimpanzees at Chimfunshi Wildlife Orphanage in Zambia, Africa, (N = 90, tested in four social groups). Crucially, all subjects were naïve to stick pounding before testing. Three out of the four tested groups reinnovated stick pounding—clearly demonstrating that this behavioral form does not require social learning. These findings provide support for the ZLS hypothesis alongside further evidence for the individual learning abilities of chimpanzees.     

Induction of B16 melanoma melanogenesis by a serum-free synthetic medium.

Cultured murine B16 melanoma cells normally grow as spindle-shaped cells firmly attached to tissue culture flasks. Pellets obtained from harvested B16 melanoma cells are white to grey in color. When the same cells were grown in synthetic, serum-free AIM V medium, cellular morphology and pigmentation were radically altered. Within 3 days of subculture in AIM V, cells rounded up and grew in clusters in suspension. Melanin content increased to greater than 30 times and tyrosinase activity was found to be 10-50 times higher in cells grown in AIM V medium compared to those cultured in normal medium. A concomitant increase in the level of immunoreactive tyrosinase was also induced. The individual growth factors and hormones present in AIM V medium were examined to determine which component(s) stimulates melanogenesis. Only those cells grown in the presence of 2.5% human albumin were stimulated to synthesize melanin. These findings suggest that albumin, or a component associated with albumin, has a major effect upon the regulation of melanogenesis in these cells.     

The Personal and Contextual Contributors to School Belongingness among Primary School Students

School belongingness has gained currency among educators and school health professionals as an important determinant of adolescent health. The current cross-sectional study presents the 15 most significant personal and contextual factors that collectively explain 66.4% (two-thirds) of the variability in 12-year old students’ perceptions of belongingness in primary school. The study is part of a larger longitudinal study investigating the factors associated with student adjustment in the transition from primary to secondary school. The study found that girls and students with disabilities had higher school belongingness scores than boys, and their typically developing counterparts respectively; and explained 2.5% of the variability in school belongingness. The majority (47.1% out of 66.4%) of the variability in school belongingness was explained by student personal factors, such as social acceptance, physical appearance competence, coping skills, and social affiliation motivation; followed by parental expectations (3% out of 66.4%), and school-based factors (13.9% out of 66.4%) such as, classroom involvement, task-goal structure, autonomy provision, cultural pluralism, and absence of bullying. Each of the identified contributors of primary school belongingness can be shaped through interventions, system changes, or policy reforms.     

Morningness/eveningness and satisfaction with life in a Polish sample

Previous studies have provided evidence that eveningness could be related to disadvantageous individual characteristics. The hypothesis that more evening-oriented individuals exhibit lower satisfaction with life has been tested by Randler using a sample of 164 German university students, and this showed that eveningness is associated with lower satisfaction with life (r =?.177). The aim of the present study was to answer the question of whether or not this relationship is culturally independent, and would exist if a sample from another geographical location and culture had been studied. A sample of 349 residents (149 men) aged between 13 and 59 yrs from two Polish cities completed the Morningness-Eveningness Questionnaire and the Satisfaction with Life Scale. Analysis revealed that morningness was related to greater life satisfaction (r =?.105), regardless of age and sex. The magnitude of this relationship did not differ significantly between the Polish and the German sample, which suggests that the magnitude and direction of association between morningness/eveningness and satisfaction with life could be independent of culture or geographical location.     

Characterization of the cell kinetics and growth properties of cystic fibrosis diploid fibroblasts in vitro.

The population growth kinetics of human diploid skin fibroblasts derived from cystic fibrosis homozygotes and heterozygotes and from normal subjects were investigated. Our data suggest the following: 1. Population doubling times increase with time in culture, with no significant differences observed among the three genotypes tested, when data were compared at the same subculture times or phases of growth. 2. The fraction of dividing cells in a population decreased with the duration in which the cells were in culture. No significant differences were obtained for cells derived from the three genotypes. 3. Cell cycle times were very similar (18-20 hr) when comparing the normal and cystic fibrosis lines or when comparing cystic fibrosis lines in phases 1 and 2 of growth. 4. No significant variations in population doubling times or growth fractions could be attributed to age or sex of the biopsy donor. 5. Variability in growth fractions and doubling times was minimal through the eighth subculture period but was very great in older cultures (tenth subculture). 6. Changes in growth fractions and doubling times appear to be due to the possibility of "aging" of human diploid fibroblasts in culture rather than to the presence or absence of genes for cystic fibrosis. 7. It is strongly indicated that differences in cell kinetic parameters cannot be used as the basis for differentiation between cells derived from normal or cystic fibrosis genotypes.     

Protein synthesis and processing in cytoplasmic microdomains beneath postsynaptic sites on CNS neurons

Recent studies have shown that protein synthetic machinery consisting of polyribosomes and associated membranous cisterns is selectively localized beneath synaptic sites on neurons. In the present paper, the role of this machinery in neuronal function will be considered. We will: 1. Summarize the studies that characterize the polyribosomes and define their associations with membranous cisterns. Taken together, these observations suggest the existence of a system for the synthesis and posttranslational processing of proteins at individual synaptic sites; 2. Review the evidence that the protein synthetic machinery is particularly prominent during the initial formation of synaptic contacts (during early development), and during lesion-induced synaptogenesis in mature animals. These observations have led to the hypothesis that the polyribosomes produce proteins that play a role in the formation of the synaptic junction; 3. Review evidence that supports the hypothesis that there is a local synthesis of protein within dendrites, as well as local glycosylation; 4. Describe the evidence suggesting that at least some of the protein constituents of the synaptic junction itself are synthesized locally; and 5. Describe our studies that reveal a mechanism for selective dendritic transport of RNA; this transport mechanism permits the delivery of RNA to postsynaptic sites throughout the dendritic arbor. We will advance the hypothesis that neurons position protein synthetic machinery together with the mRNA's that are appropriate for particular synapses beneath synaptic contact regions. At the synaptic site, this machinery could then direct the synthesis of particular proteins that are critical for synapse formation or maintenance. The positioning of protein synthetic machinery at postsynaptic sites permits a rapid local regulation of the production of key proteins by events at individual synapses.     

Establishment and characterization of a human papillary thyroid carcinoma cell line with oxyphilic differentiation (ONCO-DG 1).

In the present study the establishment and characterization of a new oxyphilic papillary thyroid carcinoma cell line--ONCO-DG1- is given. With immunohistological, histochemical and flow cytometric methods, ONCO-DG 1 cells revealed features of epithelial differentiation. Furthermore the cells formed von Kossa-positive deposits resembling psammoma bodies in monolayer and spheroid culture until late passages. The tumor cell line is now in the 40th subculture. Because of the ability to form multicellular tumor spheroids (MCTS), this cell line is a good model for examining the interaction between thyroid tumor cells and confluent human endothelial cells on extracellular matrix in vitro. It is also suitable for xenotransplantation studies, because it is tumorigenic in NMRI nude mice in vivo.