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Results for “Functional differentiation”

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At least 289 records · Page 16Linked to original sources
PubMed2003

Functional differentiation of peroxisomes revealed by expression profiles of peroxisomal genes in Arabidopsis thaliana.

It is well known that peroxisomal matrix proteins contain one of two targeting signals, PTS1 and PTS2. We comprehensively surveyed genes related to peroxisomal function and biogenesis in the entire Arabidopsis genome sequence. Here, we identified 256 gene candidates of PTS1- and PTS2-containing proteins and another 30 genes of non-PTS-containing proteins. Of these, only 29 proteins have been reported to be functionally characterized as peroxisomal proteins in higher plants. We extensively investigated expression profiles of genes described above in various organs of Arabidopsis: Statistical analyses of these expression profiles revealed that peroxisomal genes could be divided into five groups. One group showed ubiquitous expression in all organs examined, while the other four were classified as showing organ-specific expression in seedlings, cotyledons, roots and in both cotyledons and leaves. These data proposed more detailed description of differentiation of plant peroxisomes.

Arabidopsis↗
PubMed2006

Saethre-Chotzen syndrome caused by TWIST 1 gene mutations: functional differentiation from Muenke coronal synostosis syndrome.

The Saethre-Chotzen syndrome (SCS) is an autosomal dominant craniosynostosis syndrome with uni- or bilateral coronal synostosis and mild limb deformities. It is caused by loss-of-function mutations of the TWIST 1 gene. In an attempt to delineate functional features separating SCS from Muenke's syndrome, we screened patients presenting with coronal suture synostosis for mutations in the TWIST 1 gene, and for the Pro250Arg mutation in FGFR3. Within a total of 124 independent pedigrees, 39 (71 patients) were identified to carry 25 different mutations of TWIST 1 including 14 novel mutations, to which six whole gene deletions were added. The 71 patients were compared with 42 subjects from 24 pedigrees carrying the Pro250Arg mutation in FGFR3 and 65 subjects from 61 pedigrees without a detectable mutation. Classical SCS associated with a TWIST 1 mutation could be separated phenotypically from the Muenke phenotype on the basis of the following features: low-set frontal hairline, gross ptosis of eyelids, subnormal ear length, dilated parietal foramina, interdigital webbing, and hallux valgus or broad great toe with bifid distal phalanx. Functional differences were even more important: intracranial hypertension as a consequence of early progressive multisutural fusion was a significant problem in SCS only, while mental delay and sensorineural hearing loss were associated with the Muenke's syndrome. Contrary to previous reports, SCS patients with complete loss of one TWIST allele showed normal mental development.

Acrocephalosyndactylia↗
PubMed2005

Experimental models to analyze differentiation functions of cultured keratinocytes in vitro and in vivo.

In this chapter, we present technical details for the generation of in vitro skin equivalents consisting of collagen gels with incorporated fibroblasts covered by proliferating and differentiating keratinocytes. Epithelial-mesenchymal interactions are clearly manifest in these skin equivalents. Therefore, they have proven to be suitable experimental tools for a broad range of applications, e.g., for studies on the the paracrine regulation of keratinocyte differentiation and proliferation. On the other hand, in vivo assays cannot be abandoned totally, in particular, when such properties as malignant growth potential, disturbed differentiation control in carcinogenesis, and impact on angiogenesis are concerned. For that reason, we additionally describe xenotransplantation techniques to graft human keratinocytes and skin equivalents, respectively, onto the dorsal muscle fascia of thymus-aplastic mice.

Animals↗
PubMed2004

Differential functional properties of Ca2+ stores in pulmonary arterial conduit and resistance myocytes.

In smooth muscle cells, oscillations of intracellular Ca2+ concentration ([Ca2+]i) are controlled by inositol 1,4,5-trisphosphate (InsP3) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca2+ oscillations are regulated differentially by InsP3 and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca2+ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP3 production or activation of InsP3 receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP3-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca2+ free solutions did not prevent the ATP-induced Ca2+ release from InsP3-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP3- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca2+ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.

Action Potentials↗
PubMed1985

Neurite formation by neuroblastoma-glioma hybrid cells (NG108-15) in defined medium: stochastic initiation with persistence of differentiated functions.

Neurite formation and proliferation by NG108-15 cells were studied in short-term serum-free N2 medium. Neuritogenesis by individual cells was observed at widely differing times, suggesting a stochastic component to initiation of differentiation. Cells with and without neurites could also enter one or more rounds of proliferation at varying times. The initial choice between these divergent behaviors influenced subsequent growth. Cells initially extending neurites had a high probability of continuing neuritic elongation. Cells initially dividing had a high probability of yielding further progeny. Addition of dibutyryl cyclic AMP to cells cultured in N2 medium rapidly increased the probability of differentiating and decreased the probability of proliferating. To test whether or not cells with highly differentiated morphologies had irreversibly lost the capacity for proliferation, induced cultures were washed and challenged by the addition of serum-containing medium. The length of time required for individual cells to divide increased with increasing time of preincubation in the induction medium. However, few cells appeared to be permanently removed from the proliferative pool. These observations suggest that differentiating cells exhibit persistence, a tendency to continue on the differentiation pathway. Persistence is extinguished following one round of proliferation in serum-containing medium.

Animals↗
PubMed2006

Evidence for a differential functional regulation of the two beta(3)-integrins alpha(V)beta(3) and alpha(IIb)beta(3).

The functional regulation of integrins is a major determinant of cell adhesion, migration and tissue maintenance. The binding of cytoskeletal proteins to various sites of integrin cytoplasmic domains is a key mechanism of this functional regulation. Expression of recombinant integrin alpha(IIb)beta(3) and alpha(M)beta(2) lacking the GFFKR-region in CHO cells results in constitutively activated integrins. In contrast, CHO cells stably expressing either a GFFKR-deleted alpha(V(del))beta(3) or a FF to AA-substituted alpha(V(AA))beta(3) do not reveal a constitutively activated integrin. Adhesion to immobilized fibrinogen is strongly impaired in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells, whereas it is not impaired in alpha(IIb)beta(3) and alpha(M)beta(2), both lacking the GFFKR-region. In a parallel plate flow chamber assay, alpha(V)beta(3)-expressing cells adhere firmly to fibrinogen and spread even at shear rates of 15 to 20 dyn/cm(2), whereas alpha(V(del))beta(3) or alpha(V(AA))beta(3) cells are detached at 15 dyn/cm(2). Actin stress fiber formation and focal adhesion plaques containing alpha(V)beta(3) are observed in alpha(V)beta(3) cells but not in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells. As an additional manifestation of impaired outside-in signaling, phosphorylation of pp125(FAK) was reduced in these cells. In summary, we report that the GFFKR-region of the alpha(V)-cytoplasmic domain and in particular two phenylalanines are essential for integrin alpha(V)beta(3) function, especially for outside-in signaling. Our results suggest that the two beta(3)-integrins alpha(IIb)beta(3) and alpha(V)beta(3) are differentially regulated via their GFFKR-region.

Animals↗
PubMed1983

Involvement of collagen formation in the hormonally induced functional differentiation of mouse mammary gland in organ culture.

In mammary gland organ culture from midpregnant mice, synergistic actions of insulin, cortisol, and prolactin stimulate differentiation of mammary epithelium and induce the synthesis of the milk proteins casein and alpha-lactalbumin. In the present study we examined the production of collagen and its function in the hormone-dependent development of mammary gland in vitro. The measurement of collagen production by the hydroxyproline assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the accumulation of collagen type I and type III in cultured mammary explants increased with the addition of insulin, cortisol, and prolactin to a chemically defined medium. When an analog of proline, L-azetidine-2-carboxylic acid (LACA), was added at a concentration of 80 microgram/ml with insulin, cortisol, and prolactin at the beginning of culture, collagen production in cultured tissue was inhibited by 75% during a 3-day incubation period. This agent also inhibited the synthesis of casein and alpha-lactalbumin by about 77 and 70%, respectively. The inhibitory effect of LACA could be prevented by L-proline; concomitant addition of L-proline (80 microgram/ml) with LACA (80 microgram/ml) resulted in complete restoration of milk protein synthesis to normal levels. Measurement of the amount of milk protein mRNAs in mammary explants by a cell-free translation assay demonstrated that LACA reduced the hormone-stimulated accumulation of casein mRNA and alpha-lactalbumin mRNA by 79 and 76%, respectively. LACA, however, produced little inhibition of DNA synthesis in cultured tissue. These results suggest that collagen production may be involved in the phenotypic expression of milk protein genes during hormonal induction of mammary epithelial differentiation in vitro.

Animals↗
PubMedDate not supplied

Liver-specific physiology of immortal, functionally differentiated hepatocytes and of deficient hepatocyte-like variants.

Five different immortalized transgenic hepatocyte cell lines derived from mice were investigated with respect to their potential to maintain the physiological properties of primary hepatocytes using chemically defined medium. This research completes a previous study by Klocke and coworkers in 2002, using gene expression analysis of the same cell lines by the respective physiological analysis for investigating the hepatocyte-like function. Three transgenic cell lines harboring a fusion gene derivative (construct 202) consisting of the complete SV40 early region, including the coding sequences for the transforming large and small tumor antigens, placed under the control of the murine metallothioneine 1-promotor/enhancer element, showed a hepatocyte-like function and physiology. They grew as a monolayer with a polygonal cell shape, consumed lactate, and secreted albumin at a cell-specific rate of 1.5 pg/h, which is in the range of primary hepatocytes. In addition, the potential of detoxifying ammonium could be maintained. Ammonium was metabolized and urea was produced and released into the medium. A complete urea cycle could be determined. A cell line established from neonatal transgenic mice and expressing a secretory variant of the human epidermal growth factor (IgEGF) under the control of the albumin promoter was characterized by an incomplete urea cycle. Another cell line isolated from the liver of homozygote neonatal p53-knockout mice showed no hepatocyte-specific functions but only properties of continuous cell lines. Specific nucleoside triphosphate (NTP) and uridine (U) ratios were used to characterize the differentiation status of the particular cell lines. A low NTP-U value was found for the three cell lines containing construct 202, which was identical to that observed for primary hepatocytes. In contrast, the cell line harvested from the liver of homozygote neonatal p53-knockout mice presented a NTP-U ratio characteristic for continuous cell lines. This study demonstrates that the four transgenic and the p53-knockout hepatocyte-derived cell lines can be used as models for investigating the conservation of tissue-specific functions in immortalized cells.

Albumins↗
PubMed1996

Functional differentiation of the human red blood cell and kidney urea transporters.

The recent cloning of two urea transporters will allow to better understand their role in the urinary concentrating mechanism. This physiological approach needs to be sustained by a knowledge of their functional characteristics. We compared the pharmacological properties of the human red blood cell and kidney urea transporters (HUT11 and HUT2) in the Xenopus oocyte expression system. Both proteins allow the rapid transfer of urea but not of water. Both are inhibited by phloretin, although with different half-maximal inhibitory concentrations (IC50; 75 microM, for HUT11 and 230 microM for HUT2). Whereas para-chloromercuribenzene sulfonate inhibits HUT11 with an IC50 of 150 microM, it does not inhibit HUT2, whatever the concentration used. We demonstrate that thiourea diffuses through HUT11 with a Michaelis constant (Km) of 40 mM, but not through HUT2. In contrast, it inhibits urea transport through both proteins. This identification of a substrate binding site independent from the transport activity is the first step in the understanding of the molecular events underlying urea transport.

4-Chloromercuribenzenesulfonate↗
PubMed1987

Sequential expression and differential function of multiple adhesion molecules during the formation of cerebellar cortical layers.

We have correlated the times of appearance of the neural cell adhesion molecule (N-CAM), the neuron-glia cell adhesion molecule (Ng-CAM), and the extracellular matrix protein, cytotactin, during the development of the chicken cerebellar cortex, and have shown that these molecules make different functional contributions to granule cell migration. Immunofluorescent staining showed distinct spatiotemporal expression sequences for each adhesion molecule. N-CAM was present at all times in all layers. However, the large cytoplasmic domain polypeptide of N-CAM was always absent from the external granular layer and was enriched in the molecular layer as development proceeded. Ng-CAM began to be expressed in the premigratory granule cells just before migration and later disappeared from cell bodies but remained on parallel fibers. Cytotactin, which is synthesized by glia and not by neurons, appeared first in a speckled pattern within the external granular layer and later appeared in a continuous pattern along the Bergmann glia; it was also enriched in the molecular layer. After we established their order of appearance, we tested the separate functions of these adhesion molecules in granule cell migration by adding specific antibodies against each molecule to cerebellar explant cultures that had been labeled with tritiated thymidine and then measuring the differential distribution of labeled cells in the forming layers. Anti-N-CAM showed marginal effects. In contrast, anti-Ng-CAM arrested most cells in the external granular layer, while anti-cytotactin arrested most cells in the molecular layer. Time course analyses combined with sequential addition of different antibodies in different orders showed that anti-Ng-CAM had a major effect in the early period (first 36 h in culture) and a lesser effect in the second part of the culture period, while anti-cytotactin had essentially no effect at the earlier time but had major effects at a later period (18-72 h in culture). The two major stages of cerebellar granule cell migration thus appear to be differentially affected by distinct adhesion molecules of different cellular origins, binding mechanisms, and overall distributions. The results indicated that local cell surface modulation of adhesion molecules of different specificities at defined stages and sites is essential to the formation of cerebellar cortical layers.

Animals↗
PubMed1996

First biochemical evidence of differential functional effects following Gamma Knife surgery.

Clinical experience with radiosurgery for epilepsy on lesions located in highly functional areas has suggested the possibility of Gamma-Knife-induced functional effects without deterioration of the underlying cerebral cortex. To investigate these hypothetical functional changes, we have developed a special frame dedicated to small-animal radiosurgical experimental models, allowing purely atlasguided protocols. The left striatum of the first series of rats was targeted with high doses (200 Gy maximum) for validation of this new device. The same target was used with lower doses (50 Gy at the 50% isodose) in the second series to evaluate the biochemical changes and their chronology. The main biochemical changes occurred between 59 and 90 days after Gamma Knife irradiation, with different amplitudes depending on the biochemical parameter observed. Differential effects were first observed between glutamate decarboxylase and choline acetyltransferase, and secondarily between excitatory amino acids (AAs) and non-excitatory AAs, particularly gamma-aminobutyric acid. These preliminary results need to be confirmed and completed by further experimental studies. However, Gamma-Knife-induced differential biochemical effects provide the basis for a promising new concept for functional radiosurgery and particularly the Gamma Knife surgery of epilepsy.

Animals↗