Normal and pathological lymph node tissue cultured in vitro. II. The pathological tissues, and in particular Hodgkin's tissue.
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Mandibular processes from 9- to 13-day-old embryonic mice formed both bone and cartilage when grafted to the chorioallantoic membranes of most embryonic chicks. Isolated ectomesenchyme, taken from 9-day-old embryos did not form bone or cartilage, while older ectomesenchyme formed both. Recombination of the epithelial and ectomesenchymal components confirmed that the presence of the epithelium was a sufficient stimulus for the initiation of both chondro- and osteogenesis. Recombinations between components of mouse and chick mandibular processes showed that 9-day-old mouse ectomesenchyme could respond to chick epithelium but that, although older murine epithelia could initiate osteogenesis from the avian ectomesenchyme, epithelia from 9-day-old embryos could not. These results indicated that an epithelial-ectomesenchymal interaction was responsible for the initiation of both osteo- and chondrogenesis within the mandibular arch of the mouse; that the interaction began at 20 days of gestation; that the ectomesenchyme was capable of responding at 9 days, but that the epithelium did not acquire its ability to act on the ectomesenchyme until 10 days of gestation.
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Currently available data describing the gene expression and regulation, secretion, distribution, and protein chemistry of osteopontin (OPN) all are consistent with the notions of this protein functioning as an inhibitor of mineralization and/or as a mediator of cell-matrix and matrix-matrix/mineral adhesion (cohesion) during the formation, turnover, and repair of normal and pathological mineralized tissues. The properties and overall integrity of mineralized tissues are in part dictated by the nature of their interfaces--sites where organic and inorganic components of the extracellular matrix interact to provide biomechanical strength, regulate mineral ion homeostasis, and influence cellular events involved in mineralized tissue modeling, remodeling, and repair. High-resolution, colloidal-gold immunocytochemistry has been used to characterize the proteinaceous composition of these interfaces and to establish that the phosphorylated sialoprotein, OPN, is a major component found at these sites where it accumulates as a dense, planar "coating" of organic material termed either a cement line or a lamina limitans. Structural/functional features of OPN predict an ability of this protein to regulate calcification in the matrix proper of mineralized tissues and to participate, more specifically, in cell-matrix and matrix-matrix/mineral adhesion in laminae limitantes and cement lines, respectively. From the ultrastructural immunocytochemical data presented herein for OPN illustrating the cellular expression and extracellular matrix distribution of this protein, it is demonstrated that the production of OPN is one of the earliest, and latest, secretory activities of the osteoblast lineage and that this activity manifests itself morphologically as a cement line or a lamina limitans, respectively, at bone matrix interfaces. In laminae limitantes at bone surfaces, OPN appears to be involved in osteoclast adhesion and possibly haptotaxis. An OPN-containing cement line is also present at hard tissue interfaces in rat tooth, against osseointegrated titanium and hydroxyapatite implants and at the margins of surgically created bone defects--and there may influence biological adhesion in a manner similar to that proposed for normal bone. It is suggested, therefore, that in addition to its potential for influencing cell adhesion/dynamics in bones and teeth, OPN in cement lines may act as an interfacial adhesion promoter between apposing substrates, therein maintaining the overall integrity of bone during the bone remodeling sequence and "bonding" dissimilar tissues (or biocompatible materials) together in biological composites such as teeth and osseointegrated implants.
Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.
Telomerase activity in tissues may be related to tumor development, especially malignant conversion, in humans. However, there are few reports about telomeres and telomerase activity in animals. In this study, we examined telomerase activity in rat colon carcinogenesis and in normal rat liver tissue and compared it with that of human colon cancer tissues. This is the first report concerning telomerase activity in rats. F344 rats were used, and colon neoplasms were induced with methylazoxymethanol acetate. There was telomerase activity in not only the induced colon neoplasms but also the colon mucosa and livers of untreated rats, in contrast with the results from normal human somatic tissues in previous reports. Indeed, we also observed negative results in normal human mucosa, despite the positive results in colon-cancer tissues. These findings suggest that there is a difference in the telomerase activities in humans and rats. Because rat telomeres are very long (20-100 kp, average 50 kp) compared with human telomeres (5-15 kp, average 12 kp), the difference in the telomere lengths of rats and humans might be related to their enzyme activities, although this is still unclear. Furthermore, because the inhibition of telomerase has been proposed as a novel cancer therapy for humans, the rat model presented here, in which telomerase is expressed in somatic tissues, may be useful for studies of telomerase inhibition, including inhibition by chemopreventive agents.
We used immunocytochemical localization of calcitonin gene-related peptide (CGRP) to trace the ontogenesis and anatomic distribution of this component of nonadrenergic noncholinergic (NANC) innervation in fetal, neonatal, and mature canine hearts and autonomic ganglia which control cardiac function. Rare varicose CGRP-immunoreactive nerve processes were present in the heart during late gestation. Abundant CGRP-immunoreactive neural tissue in the neonate suggested a burst of NANC innervation around birth. Neonatal, 1-, and 2-month-old animals all had many varicose individual nerve processes in addition to processes within bundles; however, the density of all CGRP-immunoreactive tissue appeared to decrease during this stage of development. Similarly, there were relatively more varicose stained nerve processes in the epicardial ganglia and numerous CGRP-immunoreactive cells and smooth nerve processes in the stellate ganglia of the neonate, as compared with older animals. In the mature animal CGRP-immunoreactive neural tissue in the heart was more sparse and largely confined to heterogeneous nerve bundles in the epicardium. The extramural coronary arteries were virtually the only site of innervation by individual nerve processes; CGRP-immunoreactive neural tissue was not found adjacent to working cardiac muscle fibers. At all developmental stages, the area of the sinoatrial node was the primary focus of CGRP innervation, although the atrioventricular nodal region was also preferentially innervated. In general, the atria contained more CGRP-immunoreactive tissue than the ventricles, which were only sparsely innervated. The perinatal peak in density of CGRP-immunoreactive neural tissue with subsequent decline to reach the adult pattern suggests a developmental role for NANC innervation in the dog heart.
A tissue-specific protein fraction has been detected in rat osteogenic tissue. Dissociative extraction of adult rat bone matrix with 4 M guanidinium chloride solution was followed sequentially by gel chromatography and polyacrylamide gel electrophoresis. By the latter procedure a prominent protein component of molecular weight 19,000 was isolated from the low molecular weight fraction, and antibodies directed against this protein were raised in rabbits. The antibodies were mainly against antigenic sites on this protein, as shown by protein blotting techniques. By embedding rat tissues in hydrophilic plastic and by using immunohistochemical procedures the presence of this protein was demonstrated specifically in bone matrix in vivo, in osteogenic tissue developing in diffusion chamber culture, and in a malignant osteoblast cell line (UMR 106). Soft tissues (liver, kidney, spleen, gut, skin, thymus, eye) showed no reactivity with the antiserum and in vitro a further malignant osteoblast cell line (ROS 17/2.8) did not synthesize the 19,000 molecular weight protein. This protein appears to be expressed solely by osteogenic tissue and may be used as a biochemical criterion of osteogenic differentiation.
Red pigment-concentrating hormone (RPCH), an octapeptide found in crustaceans and insects with the sequence pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2, is an N- and C-terminally blocked uncharged peptide. These structural features are shared with many members of the larger adipokinetic hormone (AKH)/RPCH peptide family in insects. We have applied vacuum UV matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron mass spectrometry (FTMS) to the direct analysis of crustacean sinus gland tissues, using 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix, and have found that RPCH is detected in the cationized, [M + Na]+, form under conditions where other peptides in the direct tissue spectra are protonated without accompanying [M + Na]+ or [M + K]+ satellite peaks. The [M + H]+ ion for RPCH is not detected in tissue samples or for an RPCH standard, even when care is taken to eliminate metal ions. This behavior is not unprecedented; however, both direct tissue spectra and SORI-CID spectra provide no clues to suggest that the ionizing agent is a metal cation. In this communication, we characterize the MALDI-FTMS ionization and SORI-CID mass spectra of the [M + Na]+ and [M + K]+ ions from RPCH, and report on the detection of this neuropeptide in sinus gland tissues from the lobster Homarus americanus and the kelp crab Pugettia producta. We describe two strategies, an on-probe extraction procedure and a salt-doping approach, that can be applied to previously analyzed MALDI tissue samples to enhance and unmask sodiated peptides that may otherwise be mistaken for novel neuropeptides.
Proton transverse relaxation rate (R(2)) imaging measurements were made on post mortem iron-loaded human liver tissue samples (both intact and dissected into approximately 1-cm cubes) from a single subject. Iron concentrations for the dissected samples as measured by atomic absorption spectrometry varied from 10.8 to 23.3 mg Fe.g(-1) dry tissue. A significant linear correlation between the mean R(2) and iron concentration of each sample was found (r = 0.95). In addition, regions of liver tissue with micronodular cirrhosis exhibited lower R(2) values, corresponding to the displacement of iron by fibrotic septa. The cirrhotic tissue was clearly identified as a separate peak in the R(2) distribution of the tissue. The relaxivity of the iron did not appear to depend on the microarchitecture of the tissue.
The insulin-like growth factor II (IGF2) is a polypeptide with structure homology to insulin which possesses mitogen activity, and imprinted with paternal allele. In order to elucidate the distribution of imprinting pattern and relationship between allele- and tissue-specific expression of IGF2 in growth and maturation of human tissues, we investigated allele-specific expression of IGF2 in a wide spectrum of normal maturated human tissues by a PCR-based assay and found monoallelic expression in all eight-type tissues tested except human adult liver. Moreover, when a RT-PCR based sensitive allele-specific primer extension for an Apa I polymorphism within exon 9 of IGF2 was used, the analysis revealed the gene was normally imprinted in placenta; in contrast to the finding with placenta, IGF2 transcripts were biallelically expressed in human adult liver. Our results have clearly demonstrated preferential paternal expression and tissue-specific imprinting pattern of IGF2 in all human tissues tested in this study. Collectively, since IGF2 expression in developing fetal and adult liver is specified by distinct promoters, these extensive observations definitively indicate that transcriptional imprinting of IGF2 is more likely a promoter dependent manner.
In vitro tissue binding of clindamycin, trimethoprim and rifampin to serial dilutions of canine tissue homogenates was studied. For all three drugs tested, the percentage of bound drug tended to be linearly related to the logarithm of the percentage of tissue homogenate. Tissue binding studies of antimicrobials looking at a single concentration or tissue homogenate may give erroneous results that do not reflect tissue binding in vivo.