Ultrasonic tissue differentiation.
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Adipose Tissue↗
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Control of glycosaminoglycan synthesis and connective tissue differentiation in culture by solubilized bone matrix fractions.
Implantation of decalcified bone matrix in rodents induces the formation of new cartilage and bone by responding cells. Extraction of the bone matrix with high ionic strength solutions, followed by solubilization of part of the extract in isotonic salt solution, yielded material which stimulated glycosaminoglycan (GAG) synthesis by monolayers of fibroblastic cells. Bone is greatly enriched in this stimulatory activity compared to other connective tissues. However, fibroblastic cells from different sources respond to the stimulatory effect of low concentrations of the solubilized material. Kinetic studies of the GAG synthesized and secreted by the cultured cells revealed that a major stimulation of GAG secretion occurred at the cell surface as an early event following stimulation with the bone matrix extract. GAG accumulation in the culture medium occurs at a greater rate in cultures that have been stimulated with the bone matrix extract than in controls.
Animals↗
Protected regions in the chicken alpha 2(1) procollagen promoter in differentiated tissues.
The higher ordered structure of the chicken alpha 2(1) procollagen gene was analyzed in chromatin isolated from expressing (lung) and nonexpressing (reticulocyte and erythrocyte) tissues. Digestion of DNA with methylation sensitive restriction endonucleases revealed that this gene was methylated in all tissues examined and that no differences existed in the promoter methylation patterns between expressing and nonexpressing tissues. DNAse 1 hypersensitive sites were located between 100-300 bp upstream from the transcription initiation site and within the first intron. These sites were also hypersensitive to the single-strand specific S1 nuclease, implying that this region of the gene in the chromatin is either in an unfolded single-stranded conformation or under severe conformational stress. These differences in the alpha 2(1) chromatin structure were confirmed by the finding that the promoter was more accessible to restriction endonuclease digestion in the expressing tissues than in the nonexpressing tissues. Digestion of chromatin with Pst I and Sma I revealed that some of these sites in the promoter were differentially protected by DNA-binding proteins in the two tissue types. These protected sites were located as far upstream as -1,600 and downstream within the first intron at +800.
Animals↗
The murine decorin. Complete cDNA cloning, genomic organization, chromosomal assignment, and expression during organogenesis and tissue differentiation.
Decorin, a proteoglycan known to interact with collagen and growth factors, may play key roles during ontogenesis, tissue remodeling, and cancer. We have deciphered the complete protein sequence of the murine decorin by cDNA cloning, elucidated its gene structure and chromosomal location, and investigated its expression in the developing embryo. The decorin protein and the gene were highly conserved vis à vis the human counterpart; however, the murine gene lacked a leader exon, exon Ib, which was found only in the human. Using interspecific backcrossing, we assigned the gene to chromosome 10 just proximally to the Steel gene locus. In situ hybridization studies of developing mouse embryos showed a distinct pattern of expression with a progressive increase of decorin mRNA during ontogenesis. At early stages (day 11 postconception), decorin was detectable only in the floor plate region. Subsequently (days 13-16 postconception), decorin expression was especially prominent in the meninges and mesothelial linings of pericardium, pleura, and coelomic cavity, as well as in the dermis and subepithelial layers of the intestine and urinary bladder. In contrast, the major parenchymal organs were only weakly positive for decorin mRNA. These findings suggest that decorin may play a role in epithelial/mesenchymal interactions during organ development and shaping.
3T3 Cells↗
Rapid changes in gene expression in differentiating tissues of Drosophila.
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Animals↗
Physiological and cell biological aspects of perfusion culture technique employed to generate differentiated tissues for long term biomaterial testing and tissue engineering.
Optimal results in biomaterial testing and tissue engineering under in vitro conditions can only be expected when the tissue generated resembles the original tissue as closely as possible. However, most of the presently used stagnant cell culture models do not produce the necessary degree of cellular differentiation, since important morphological, physiological, and biochemical characteristics disappear, while atypical features arise. To reach a high degree of cellular differentiation and to optimize the cellular environment, an advanced culture technology allowing the regulation of differentiation on different cellular levels was developed. By the use of tissue carriers, a variety of biomaterials or individually selected scaffolds could be tested for optimal tissue development. The tissue carriers are to be placed in perfusion culture containers, which are constantly supplied with fresh medium to avoid an accumulation of harmful metabolic products. The perfusion of medium creates a constant microenvironment with serum-containing or serum-free media. By this technique, tissues could be used for biomaterial or scaffold testing either in a proliferative or in a postmitotic phase, as is observed during natural development. The present paper summarizes technical developments, physiological parameters, cell biological reactions, and theoretical considerations for an optimal tissue development in the field of perfusion culture.
Animals↗
Upper limb tissue differentiation in the human embryo.
The major histologic process throughout the fetal period is differential growth. Most congenital deformities occur in the third through the seventh weeks, when the tissues are rapidly developing. After the seventh week, certain types of insults may result in deformity, but the intricate structures of the limb have been determined in the time between the third and eighth postovulatory weeks.
Arm↗
Tissue differentiation in malignant synovial tumours.
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Humans↗
Controlling cell proliferation in differentiating tissues: genetic analysis of negative regulators of G1-->S-phase progression.
Withdrawal from the cell cycle as cells begin to differentiate is accomplished by the downregulation of cyclin-dependent kinase activities in G1 phase. Recent analysis of loss-of-function mutations in flies, worms, and mice has provided insight into the roles of various negative regulators of G1 phase in developing organisms.
Animals↗
Generation of color composites for enhanced tissue differentiation in magnetic resonance imaging of the brain.
Currently, the diagnostic interpretation of magnetic resonance (MR) images requires that radiologists integrate specific tissue contrast information from several different images obtained at the same anatomic slice position. Each of these images has its own unique tissue contrast patterns which are based on the image acquisition parameters (pulse sequence) selected. The complex contrast patterns observable in these images reflect the inherent biophysical characteristics of the tissues and fluids present in the imaged section. In an effort to increase the diagnostic accuracy and efficiency of MR image interpretation, we have generated color composite images from quantitatively analyzed achromatic MR images of the brain, obtained while utilizing different pulse sequences. By using a DEC MicroVAX II computer with Interactive Digital Language (IDL), this color display method has been applied to images obtained from General Electric Signa and Siemens Magnatom imagers. For this study, our image sets included T1-weighted, T2-weighted, and proton density spin echo sequences as well as both high and low flip angle gradient echo sequences. Advantages of our color composite methods, in contrast to many other image processing techniques that have been described, are that minimal information is lost, computer misclassification of tissues is avoided, and the conspicuity of specific tissues is enhanced. Furthermore, with this method it is possible to produce composite images whose color renditions approach a natural anatomic tissue appearance. Availability of these color composites to radiologists may improve the efficiency and accuracy of the diagnostic interpretation of MR images.
Brain↗
[Fibronectin, its role in the processes of tissue differentiation, regeneration and neoplastic transformation].
The data from literature on certain biological and functional peculiarities of fibronectin which is a high molecular connective tissue glycoprotein of the intercellular matrix are reviewed. Various features of fibronectin, such as its promoting action in cell adhesion, participation in cytodifferentiation, embryogenesis and regeneration are discussed. Several points of view about fibronectin participation in neoplastic transformation are mentioned. Diagnostic and prognostic value of the quantitative and qualitative estimation of plasma fibronectin in the human tumour investigation are emphasized.
Animals↗
Modulation of epidermal differentiation, tissue inflammation, and T-lymphocyte infilitration in psoriatic plaques by topical calcitriol.
Psoriasis is characterized by immune activation, increased proliferation and abnormal differentiation of keratinocytes. The reported anti-psoriatic mechanisms of action in vivo of vitamin D analogues include reduction of keratinocyte proliferation and induction of keratinocyte terminal differentiation. We investigated whether the anti-psoriatic effect of the natural active vatamin D analogue, calcitriol, applied topically, is due to direct effects on keratinocytes alone or also due to immunoregulatory effects of calcitriol. Psoriasis patients were treated with topical calcitriol (0.005%) and a vehicle control for 8 weeks. Disease activity was assessed by a severity index and quantitative histopathological markers. In vitro studies of lymphocyte proliferation and gamma interferon secretion and of keratinocyte proliferation complemented the clinicohistopathologic studies. A heterogeneous response to calcitriol treatment could be segregated based upon elimination of K-16 keratin expression. Calcitriol treatment decreased keratinocyte proliferation, normalized keratinocyte differentiation and decreased immune activation in plaques. The histologic response to vitamin D treatment of psoriasis includes suppression of both immune and keratinocyte activation in situ. These studies provide a basis for rational combination of anti-psoriatic treatments and for the design of new vitamin D analogues to treat psoriasis.
Administration, Topical↗
Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum.
Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.
Antibodies, Monoclonal↗
CD55 in rat male reproductive tissue: differential expression in testis and expression of a unique truncated isoform on spermatozoa.
CD55 is a key regulator of complement activation, expressed on most tissues and cells in man and other mammals. In the rat, alternative splicing in the gene encoding CD55 yields GPI-anchored (GPI-CD55) and transmembrane (TM-CD55) forms. Published Northern blot analysis indicated that while GPI-CD55 was broadly expressed, TM-CD55 was primarily expressed in the testis, although the precise site of expression was not identified. To clarify the distribution of CD55 isoforms in rat reproductive tissues, we first performed immunohistochemistry and Western blot analysis with an anti-rat CD55 mAb that recognized all reported CD55 isoforms, and a polyclonal immunoglobulin specific for TM-CD55. CD55 was absent in testis prior to puberty. Post-puberty, CD55 was expressed at high levels on all spermiogenic cells from step 6 spermatid onward, and on mature spermatozoa focussed on the acrosome, but was absent from support cells and early progenitors. Enzymatic digestion revealed that GPI-CD55 was predominant in testis and spermatozoa. Staining for TM-CD55 with specific immunoglobulin confirmed its absence from mature sperm and expression on spermatids only between steps 11 and 14 of development. GPI-CD55 on spermatozoa was of lower molecular weight than that in testis and other tissues; sequencing from spermatozoal mRNA identified a unique isoform of GPI-CD55 missing short consensus repeat 4. The predominant acrosome expression and presence of a unique, truncated isoform of CD55 on spermatozoa provides further support for the hypothesis that the acrosome is a highly specialized region in which closely regulated complement activation may contribute to reproductive function.
Acrosome↗
Expression of cyclooxygenase 1 and cyclooxygenase 2 in human synovial tissue: differential elevation of cyclooxygenase 2 in inflammatory joint diseases.
OBJECTIVE: To compare the expression of the cyclooxygenase (COX) isoforms, COX-1 and COX-2, in synovial tissue samples between patients with inflammatory arthritis (i.e., rheumatoid arthritis [RA], ankylosing spondylitis [AS], or psoriatic arthritis [PsA]) and patients with osteoarthritis (OA). METHODS: Paraffin-embedded sections of synovial tissue from patients with OA (n = 18), RA (n = 35), AS (n = 9), and PsA (n = 16) were immunostained for COX-1 and COX-2. Staining intensity was quantified videodensitometrically from specific synovial cell areas. In addition, samples of OA and RA synovial tissue were analyzed for levels of COX-1 and COX-2 messenger RNA (mRNA) using reverse transcriptase-polymerase chain reaction. RESULTS: Strong COX-2 immunostaining was observed in synovial blood vessel endothelium, synovial lining cells, chondrocytes, and subsynovial fibroblast-like cells in patients with inflammatory arthritides. In the blood vessels, the mean (+/-SD) optical density (MOD) of staining was elevated, especially in AS samples (2.73 +/- 0.63), but also in PsA (1.99 +/- 0.66) and RA samples (1.54 +/- 0.73), in comparison with OA synovial tissue (0.84 +/- 0.30; P < 0.01 versus other groups). COX-1 staining was almost exclusively localized in synovial lining cells, with no significant differences in the MOD between the diseases. COX-2 mRNA expression was higher in RA than in OA samples (P < 0.05). CONCLUSION: The expression of COX-2, but not the expression of COX-1, was found to be elevated in a disease-related pattern in the synovial tissue from patients with RA, AS, or PsA in comparison with OA samples, and was especially high in AS synovial tissue. These results may improve our understanding of the pathogenesis of different arthritic diseases, and may have implications for the use of selective COX-2 inhibitors in the treatment of inflammatory joint symptoms.
Adult↗
Protein kinase C delta- and epsilon-subspecies in rat central nervous tissue; differential distribution and phorbol ester-induced redistribution in synaptosomes.
Distribution of the delta- and epsilon-subspecies of protein kinase C (PKC) in rat central nervous tissues was semiquantitated by an immunoblot analysis. The epsilon-subspecies, which is expressed predominantly in the brain, was abundant in hippocampus and cerebral cortex among rat brain areas, whereas the delta-subspecies, which is expressed ubiquitously in many tissues, showed no significant difference among tissue areas tested. Unlike other subspecies, the delta-subspecies was primarily associated with particulate fractions. Subcellular fractionation analysis revealed that both the epsilon- and delta-subspecies were enriched in the synaptosomal P2 fraction. Treatment with 12-O-tetradecanoyl-phorbol 13-acetate (TPA) induced translocation of the epsilon-subspecies from cytosolic fraction to membrane fraction. In contrast, upon treatment with TPA, the delta-subspecies normally bound to membrane fraction was released into cytosol.
Animals↗
Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies.
Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.
Animals↗
Differential tissue expression of three 35-kDa annexin calcium-dependent phospholipid-binding proteins.
We have purified three 35-kDa calcium- and phospholipid-binding proteins from rat liver. These three calcimedins bind to phosphatidylserine in a calcium-dependent manner and have been termed 35 alpha, 35 beta, and 35 gamma based on their relative charge as determined by isoelectric focusing. Purification of the three 35-kDa calcimedins is achieved by phenyl-Sepharose, ion exchange, and gel filtration chromatography. Antibody was produced against the annexin consensus peptide, Lys-Ala-Met-Lys-Gly-Leu-Gly-Thr-Asp-Glu, which was derived from the sequence of several Ca2+/phospholipid-binding proteins including calpactin, lipocortin, endonexin II, 67-kDa calelectrin, lymphocyte 68-kDa protein, and protein II. Recognition of each 35-kDa calcimedin by anticonsensus sequence antibody places them in this protein family. Antibodies against each 35-kDa calcimedin were raised and purified by antigen-affinity chromatography. Each antibody is monospecific for the respective 35-kDa calcimedin. Immunological cross-reactivity defines 35 alpha, 35 beta, and 35 gamma as lipocortins III, IV, and V, respectively. Surveys by immunoblot analysis using these monospecific antibodies demonstrate a markedly different tissue expression pattern for each 35-kDa calcimedin. Furthermore, the levels of 35 alpha, 35 beta, and 35 gamma are differentially regulated in maturing rat ovary and uterus. Each calcimedin has been localized by indirect immunofluorescence within specific cell types. These results support the concept that mediation of the intracellular calcium signal can occur via multiple pathways through several related yet independent mediator proteins.
Amino Acid Sequence↗