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Cyclic adenosine 3',5'-monophosphate induces differentiation of mouse embryonic stem cells into cardiomyocytes.

Embryonic stem (ES) cells, derived from blastocyst-stage of early mammalian embryos, have the potential to differentiate into derivatives of all three embryonic germ layers. Here we reported the first evidence that murine pluripotent ES cells could be induced to differentiate into cardiomyocytes by cyclic adenosine 3',5'-monophosphate (cAMP) in vitro. Spontaneously beating of cardiac cell clusters began to be observed within the outgrowths of embryoid bodies (EBs) as early as 2 days after the onset of differentiation. By days 5-8 after induction, a maximum level of cardiomyocyte differentiation could be achieved. Incubation of EBs with cAMP at concentrations ranging from 0.01 mg/L to 1 mg/L resulted in a significant elevation in differentiation rate, reaching a maximum value of 44.0 +/- 1.3% at 0.03 mg/L of exposure. At 0.03 mg/L concentration point, an approximately 8.1-fold increase in cardiomyocyte differentiation was observed in comparison with 5.4 +/- 0.9% of untreated controls. The differentiation rate induced by cAMP was shown to be similar to that of RA/DMSO treated controls, indicating that cAMP has the same inducing effect as RA/DMSO. However, no significant co-inducing effects between cAMP and RA/DMSO were seen. Cardiomyocytes were evident as they expressed cardiac cell specific genes and protein markers including GATA4, Nkx2.5, beta-MHC, atrial natriuretic factor (ANF) and alpha-actin when analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining. The results from the present study suggested a novel role of cAMP in cardiomyocyte differentiation and provided a new research model for the study of cardiac cell biology.

Animals↗

Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.

A zebrafish cDNA encoding a novel keratin protein was characterized and named keratin8, or krt8. krt8 expression was initiated at 4.5 hr postfertilization, immediately after the time of zygotic genome activation. The expression is limited to a single layer of envelope cells on the surface of embryos and, in later stages, it also appears in the innermost epithelial layer of the anterior- and posteriormost portions of the digestive tract. In adult, its expression was limited to the surface layer of stratified epithelial tissues, including skin epidermis and epithelia of mouth, pharynx, esophagus, and rectum but not in the gastral and intestinal epithelia. By using a 2.2-kb promoter from krt8, several stable green fluorescent protein (gfp) transgenic zebrafish lines were established. All of these transgenic lines displayed GFP expression in tissues mentioned above except for the rectum; therefore, the pattern of transgenic GFP expression is essentially identical to that of the endogenous krt8 mRNAs. krt8-GFP fusion protein was also expressed in zebrafish embryos under a ubiquitous promoter, and the fusion protein was capable of assembling into intermediate filaments only in the epithelia that normally expressed krt8 mRNAs, indicating the specificity of keratin assembly in vivo.

Amino Acid Sequence↗

Ectoderm gene activation in sea urchin embryos mediated by the CCAAT-binding factor.

Transcriptional enhancers are short stretches of DNA that function to achieve highly specific patterns of gene expression. To identify the mechanisms by which enhancers achieve their specificity, we made use of an enhancer from the aboral ectoderm-specific spec2a gene of the sea urchin Strongylocentrotus purpuratus. The spec2a enhancer contains five cis-regulatory elements within 78 base pairs that interact with five distinct DNA-binding proteins to confer aboral ectoderm expression. Here, we present an analysis of the sea urchin CCAAT binding factor (CBF), which binds to a CCAAT motif within the spec2a enhancer. S. purpuratus CBF and SpOtx, a ubiquitously expressed factor, act together at closely placed cis-regulatory elements to mediate spec2a transcription in the ectoderm. SpCBF was the sole factor that bound to the spec2a CCAAT element, and two of the three subunits that make up the CBF holoprotein were cloned and shown to have high sequence conservation with their vertebrate orthologs. Based on its involvement in the regulation of several other sea urchin genes, SpCBF appears to be a major transcription factor in the sea urchin embryo for positive regulation of ectoderm gene expression. In addition to its role in vertebrate cell growth and proliferation, our results indicate that CBF also functions at the early stages of germ layer formation, namely ectoderm differentiation.

Amino Acid Sequence↗

[Isolation of germinal cells from the secondary cysts of Echinococcus granulosus harbored in mice].

The secondary cyst tissues derived from mice infected with protoscoleces of Echinococcus granulosus for 8-10 months were digested with 0.25% trypsin at 37 degrees C for 30 min. The separation of different cells in the remaining suspension was achieved by discontinuous gradient centrifugation. The germinal cells were washed 3 times with ice-cold HBSS, and then cultivated in the medium of RPMI 1640 supplemented with 20% of calf serum. The cells were kept in an incubator at 37 degrees C in an atmosphere of 95% air-5% CO2. After incubation for 5-7 days, the germinal cells began to multiply accompanied by the enlargement of cells as compared with those before incubation. The surface of both isolated and/or cultured cells showed smooth appearance examined by scanning electron microscopy. Immunofluorescence assay and enzyme-linked immunosorbent assay had been used for examining the specific antigenicity of the cells. The results showed that antigen components of E. granulosus were detected either on cell surface or in soluble proteins of the cells. Furthermore, 120 NIH female mice were inoculated intraperitoneally with 1-5 x 10(7) cultured germinal cells and sacrificed 1-3 months after inoculation. Only 2 cystic materials had been detected in two mice. Of which, one located in the liver and the other in peritoneal cavity of the animals. Histological examination noted that the cystic materials consisted of germinal layer and cyst fluid, but no laminated layer was observed. The above mentioned evidence demonstrated that the cells isolated from the cysts of E. granulosus were germinal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Descending projections to the inferior colliculus from the posterior thalamus and the auditory cortex in rat, cat, and monkey.

Projections from the posterior thalamus and medial geniculate body were labeled retrogradely with wheat germ agglutinin conjugated to horseradish peroxidase injected into the rat, cat, and squirrel monkey inferior colliculus. Neurons were found ipsilaterally in the (1) medial division of the medial geniculate body, (2) central gray, (3) posterior limitans nucleus, and the (4) reticular part of the substantia nigra. Bilateral projections involved the (5) peripeduncular/suprapeduncular nucleus, (6) subparafascicular and posterior intralaminar nuclei, (7) nucleus of the brachium of the inferior colliculus, (8) lateral tegmental/lateral mesencephalic areas, and (9) deep layers of the superior colliculus. The medial geniculate projection was concentrated in the caudal one-third of the thalamus; in contrast, the labeling in the subparafascicular nucleus, substantia nigra, and central gray continued much further rostrally. Robust anterograde labeling corresponded to known patterns of tectothalamic projection. Biotinylated dextran amine deposits in the rat inferior colliculus revealed that (1) many thalamotectal cells were elongated multipolar neurons with long, sparsely branched dendrites, resembling neurons in the posterior intralaminar system, and that other labeled cells were more typical of thalamic relay neurons; (2) some cells have reciprocal projections. Similar results were seen in the cat and squirrel monkey. The widespread origins of descending thalamic influences on the inferior colliculus may represent a phylogenetically ancient feedback system onto the acoustic tectum, one that predates the corticocollicular system and modulates nonauditory centers and brainstem autonomic nuclei. Besides their role in normal hearing such pathways may influence behaviors ranging from the startle reflex to the genesis of sound-induced seizures.

Animals↗

Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells.

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.

3T3 Cells↗

The restrictive effect of early exposure to lithium upon body pattern in Xenopus development, studied by quantitative anatomy and immunofluorescence.

We have carried out an anatomical study of Xenopus larval and gastrula stages resulting from treatment of synchronous early blastulae for brief periods with Li+. We confirm the proposal that such treatment causes a particular transformation, and partial elimination, of the normal body pattern. Coordinated restriction of pattern, without appreciable loss of cell number, is seen in all three germ layers. The distortion has been investigated by quantitative study of mesoderms at a standard stage, in relation to the normal fate map for mesoderm, and with the help of immunofluorescence on sections for somitic muscle and for blood. In the extreme syndrome, mesoderm arises from all around the blastula as usual, but is symmetrical and corresponds to that arising near the dorsal/anterior meridian of the normally specified egg or embryo with a large posterior subset of the normal pattern values thus missing. The effect is independent of any inhibition of archenteron formation or mesoderm migration (i.e. the cell mechanics of gastrulation) incurred by the treatment. It is also quite separate from a syndrome caused by more prolonged exposure to Li+ during gastrulation. A small, but distinctive, anterior pattern region is also not expressed and, anomalously in relation to their general nature, these forms differentiate considerable blood tissue. We consider the implications of some details of the pattern restriction for our understanding of interaction in the normal development and propose that the Li+ embryo is likely to be useful as a specific 'differential screen', in relation to the normal, during the search for those gene products that mediate initial regionalization of the body.

Animals↗

The role of UV-B radiation in aquatic and terrestrial ecosystems--an experimental and functional analysis of the evolution of UV-absorbing compounds.

We analysed and compared the functioning of UV-B screening pigments in plants from marine, fresh water and terrestrial ecosystems, along the evolutionary line of cyanobacteria, unicellular algae, primitive multicellular algae, charophycean algae, lichens, mosses and higher plants, including amphibious macrophytes. Lichens were also included in the study. We were interested in the following key aspects: (a) does the water column function effectively as an 'external UV-B filter'?; (b) do aquatic plants need less 'internal UV-B screening' than terrestrial plants?; (c) what role does UV screening play in protecting the various plant groups from UV-B damage, such as the formation of thymine dimers?; and (d) since early land 'plants' (such as the predecessors of present-day cyanobacteria, lichens and mosses) experienced higher UV-B fluxes than higher plants, which evolved later, are primitive aquatic and land organisms (cyanobacteria, algae, lichens, mosses) better adapted to present-day levels of UV-B than higher plants? Furthermore, polychromatic action spectra for the induction of UV screening pigments of aquatic organisms have been determined. This is relevant for translating 'physical' radiation measurements of solar UV-B into 'biological' and 'ecological' effects. From the action spectra, radiation amplification factors (RAFs) have been calculated. These action spectra allow us to determine any mitigating or antagonistic effects in the ecosystems and therefore qualify the damage prediction for the ecosystems under study. We summarize and discuss the main results based on three years of research of four European research groups. The central theme of the work was the investigation of the effectiveness of the various screening compounds from the different species studied in order to gain some perspective of the evolutionary adaptations from lower to higher plant forms. The induction of mycosporine-like amino acids (MAAs) was studied in the marine dinoflagellate Gyrodinium dorsum, the green algal species Prasiola stipitata and in the cyanobacterium Anabaena sp. While visible (400-700 nm) and long wavelength UV-A (315-400 nm) showed only a slight effect, MAAs were effectively induced by UV-B (280-315 nm). The growth of the lower land organisms studied, i.e. the lichens Cladina portentosa, Cladina foliacaea and Cladonia arbuscula, and the club moss Lycopodiumannotinum, was not significantly reduced when grown under elevated UV-B radiation (simulating 15% ozone depletion). The growth in length of the moss Tortula ruralis was reduced under elevated UV-B. Of the aquatic plants investigated the charophytes Chara aspera showed decreased longitudinal growth under elevated UV-B. In the 'aquatic higher plants' studied, Ceratophyllum demersum, Batrachium trichophyllum and Potamogeton alpinus, there was no such depressed growth with enhanced UV-B. In Chara aspera, neither MAAs nor flavonoids could be detected. Of the terrestrial higher plants studied, Fagopyrum esculentum, Deschampsia antarctica, Vicia faba, Calamagrostis epigejos and Carex arenaria, the growth of the first species was depressed with enhanced UV-B, in the second species length growth was decreased, but the shoot number was increased, and in the latter two species of a dune grassland there was no reduced growth with enhanced UV-B. In the dune grassland species studied outdoors, at least five different flavonoids appeared in shoot tissue. Some of the flavonoids in the monocot species, which were identified and quantified with HPLC, included orientin, luteolin, tricin and apigenin. A greenhouse study with Vicia faba showed that two flavonoids (aglycones) respond particularly to enhanced UV-B. Of these, quercetin is UV-B inducible and mainly located in epidermal cells, while kaempferol occurs constitutively. In addition to its UV-screening function, quercetin may also act as an antioxidant. Polychromatic action spectra were determined for induction of the UV-absorbing pigments in three photosynthetic organisms, representing very different taxonomic groups and different habitats. In ultraviolet photobiology, action spectra mainly serve two purposes: (1) identification of the molecular species involved in light absorption; and (2) calculation of radiation amplification factors for assessing the effect of ozone depletion. Radiation amplification factors (RAFs) were calculated from the action spectra. In a somewhat simplified way, RAF can be defined as the percent increase of radiation damage for a 1% depletion of the ozone layer. Central European summer conditions were used in the calculations, but it has been shown that RAF values are not critically dependent on latitude or season. If only the ultraviolet spectral region is considered, the RAF values obtained are 0.7 for the green alga Prasiola stipitata, 0.4 for the dinoflagellate Gyrodinium dorsum, and 1.0 for the cyanobacterium Anabaena sp. In the case of P. stipitata, however, the effect of visible light (PAR, photosynthetically active radiation, 400-700 nm) is sufficient to lower the RAF to about 0.4, while the PAR effect for G. dorsum is negligible. RAFs for some damage processes, such as for DNA damage (RAF=2.1 if protective effects or photorepair are not considered [1]), are higher than those above. Our interpretation of this is that if the ozone layer is depleted, increased damaging radiation could overrule increased synthesis of protective pigments. In addition to investigating the functional effectiveness of the different screening compounds, direct UV effects on a number of key processes were also studied in order to gain further insight into the ability of the organisms to withstand enhanced UV-B radiation. To this end, the temperature-dependent repair of cyclobutane dimers (CPD) and (6-4) photoproducts induced by enhanced UV-B was studied in Nicotiana tabacum, and the UV-B induction of CPD was studied in the lichen Cladonia arbuscula. Also, photosynthesis and motility were monitored and the response related to the potential function of the screening compounds of the specific organism.

Biological Evolution↗

Msx1 controls inductive signaling in mammalian tooth morphogenesis.

Members of the Msx homeobox family are thought to play important roles in inductive tissue interactions during vertebrate organogenesis, but their precise developmental function has been unclear. Mice deficient for Msx1 exhibit defects in craniofacial development and a failure of tooth morphogenesis, with an arrest in molar tooth development at the E13.5 bud stage. Because of its potential for experimental manipulation, the murine molar tooth germ provides a powerful system for studying the role of Msx genes in inductive signaling during organogenesis. To further analyze the role of Msx1 in regulating epithelial-mesenchymal interactions during tooth morphogenesis, we have examined the expression of several potential Msx1 downstream genes in Msx1 mutant tooth germs and we have performed functional experiments designed to order these genes into a pathway. Our results show that expression of Bone Morphogenetic Protein 4 (BMP4), the HMG box gene Lef1 and the heparan sulfate proteoglycan syndecan-1 is specifically reduced in Msx1 mutant dental mesenchyme, while expression of the extracellular matrix protein tenascin is unaffected. BMP4 soaked beads can induce Bmp4 and Lef1 expression in explanted wild-type dental mesenchymes, but only Lef1 expression in Msx1 mutant dental mesenchyme. We thus conclude that epithelial BMP4 induces its own expression in dental mesenchyme in a manner that requires Msx1. In turn, we show that addition of BMP4 to Msx1 deficient tooth germs bypasses the requirement for Msx1 and rescues epithelial development from the bud stage to the E14.5 cap stage. Lastly, we show that FGFs induce syndecan-1 expression in dental mesenchyme in a manner that also requires Msx-1. These results integrate Msx1 into a regulatory hierarchy in early tooth morphogenesis and demonstrate that Msx1 is not only expressed in dental mesenchyme in response to epithelial signals, but also in turn regulates the reciprocal expression of inductive signals in the mesenchyme which then act back upon the dental epithelium. We propose that Msx genes function repetitively during vertebrate organogenesis to permit inductive signaling to occur back and forth between tissue layers.

Animals↗

Tubule formation and elemental detection in developing opossum enamel.

Most marsupials and some placental mammals possess enamel characterized by the presence of tubules, and the cellular origin of these structures has been the subject of a number of previous studies (See, for example, Lester, 1970; Azevedo and Goldberg, 1987). In the present report, tooth germs of the American opossum were examined to determine the structure and composition of enamel tubules during development and to analyze the enamel matrix relative to that of placental mammals with atubular enamel. For this purpose, tissues prepared by aqueous (decalcified and undecalcified) and anhydrous (undecalcified) methods were investigated by conventional transmission (TEM) and high voltage electron microscopy (HVEM), as well as by electron probe x-ray microanalysis (EPMA), selected-area electron diffraction (SAED), and electron spectroscopic imaging (ESI). Results indicate that most enamel tubules in the opossum begin as cytoplasmic remnants of Tomes' processes of ameloblasts. During development of the matrix, some of the tubules do not appear to be continuous throughout the prismatic layer. Sulfur is detectable around the lumen of the tubule in decalcified sections by EPMA and in and around the tubule by ESI. Calcium/phosphorus (Ca/P) molar ratios of the mineralizing matrix are generally higher than those found in enamel of other mammals and appear to decrease rather than increase with enamel maturation. The summary of data indicates the presence of sulfated glycoproteins or proteoglycans in this tissue, specifically around enamel tubules. Calcium and phosphorus are also present within the tubules, with the sulfated groups possibly binding calcium to prevent mineralization of the enamel tubules themselves.

Ameloblasts↗

Investigating the secretory pathway of the baculovirus-insect cell system using a secretory green fluorescent protein.

The secretory pathway is important in actively transporting proteins into the extracellular environment of eucaryotic cells. In this study a green fluorescent protein (GFP) mutant engineered to contain a secretion signal was used as a model protein in order to visualize the secretion process inside insect cells. Fluorescent microscopy indicated that significant amounts of secreted green fluorescent protein (sGFP) accumulated in High-Five, Trichoplusia ni, cells following infection with a baculovirus vector containing the gene under the polyhedrin promoter. Laser scanning confocal microscopy was used to reconstruct whole cell images of the infected High-Five cells at multiple days postinfection. While the protein was widely distributed at 2 days postinfection, certain intracellular regions appeared to contain higher or lower concentrations of the sGFP. A layer by layer examination indicated pockets in which sGFP was absent, and these appear to be vesicles that have recently released the sGFP or are not yet accumulating sGFP. By 3 days postinfection, the sGFP in some cells was concentrated in a number of widely dispersed globules, which may represent the vesicle remnants of a deteriorating secretory pathway. In contrast, nonsecreted GFP was more uniformly distributed in the cells than sGFP and did not accumulate in vesicles. In addition to GFP, the lectins wheat germ agglutinin (WGA) and concanavalin A (ConA), which have affinities for sugar residues, were used to examine the secretory pathway. The WGA, which is a Golgi marker, was distributed around the nucleus prior to infection but then was found to be polarized in one region of the cell following the baculovirus infection. The expansion of other cellular compartments following the baculovirus infection may have caused a change in intracellular distribution of the Golgi. While some of the sGFP was found to colocalize with the WGA label, much of the sGFP was outside this Golgi region. In contrast, ConA labeling, which was not as specific as WGA, was found throughout the cell both before and after infection similar to the sGFP distribution. These studies demonstrate that confocal visualization of fluorescent proteins can be used as an in vivo tool for examining secretory processing in insect cells.

Animals↗

Helicobacter pylori gastric infection in gnotobiotic beagle dogs.

Establishment of infection with Helicobacter pylori and gastritis in nonhuman species is currently only successful in gnotobiotic piglets. This study was designed to determine whether H. pylori will colonize the gastrointestinal tract of gnotobiotic dogs. Gnotobiotic beagle pups were derived by standard methods. Group A (five dogs) was orally challenged with 3 x 10(8) H. pylori at 7 days of age. Group B (two dogs) received only peptone water but was contact-exposed beginning on day 23 postinfection (p.i.). Necropsy was performed on dogs on day 30 p.i. H. pylori colonized the stomach of all dogs (groups A and B). Urease map analysis correlated with the microbiologic findings and indicated that the density of colonization was less than that observed in human tissue. Organisms were also recovered from the pharynx, esophagus, duodenum, and rectum of 1, 2, 2, and 1 dog, respectively. All group A and one group B dog developed serum immunoglobulin G specific for H. pylori by day 30 p.i. Gross lesions were restricted to the stomach and consisted of small (less than 1 mm) lymphoid follicles. Microscopically, there were focal to diffuse lymphoplasmacytic infiltrates with follicle formation and mild to moderate infiltration of neutrophils and eosinophils in the gastric lamina propria. With the Warthin-Starry silver stain, organisms were seen on the surface of the gastric epithelial cells, beneath the mucus layer. We conclude that H. pylori colonizes the stomachs of gnotobiotic dogs for at least 1 month and the lesions resemble those seen in humans. H. pylori is transmissible by contact from infected to noninfected dogs.

Animals↗

Excitatory and inhibitory circuitry in the superficial gray layer of the superior colliculus.

Stratum griseum superficiale (SGS) of the superior colliculus receives a dense cholinergic input from the parabigeminal nucleus. In this study, we examined in vitro the modulatory influence of acetylcholine (ACh) on the responses of SGS neurons that project to the visual thalamus in the rat. We used whole-cell patch-clamp recording to measure the responses of these projection neurons to electrical stimulation of their afferents in the stratum opticum (SO) before and during local pressure injections of ACh. These colliculothalamic projection neurons (CTNs) were identified during the in vitro experiments by prelabeling them from the thalamus with the retrograde axonal tracer wheat germ agglutinin-apo-HRP-gold. In a group of cells that included the prelabeled neurons, EPSCs evoked by SO stimulation were significantly reduced by the application of ACh, whereas IPSC amplitudes were significantly enhanced. Similar effects were observed when the nicotinic ACh receptor agonist lobeline was used. Application of the selective GABA(B) receptor antagonist 3-[[(3,4-dichlorophenyl)-methyl]amino]propyl](diethoxymethyl)phosphinic acid blocked ACh-induced reduction in the evoked response. In contrast, the ACh-induced reduction was insensitive to application of the GABA(A) receptor antagonist bicuculline. The ACh-induced reduction was also diminished by bath application of muscimol at the low concentrations that selectively activate GABA(C) receptors. Because GABA(C) receptors may be specifically expressed by GABAergic SGS interneurons (Schmidt et al., 2001), our results support the hypothesis that ACh reduces CTN activity by nicotinic receptor-mediated excitation of local GABAergic interneurons. These interneurons in turn use GABA(B) receptors to inhibit the CTNs.

Acetylcholine↗

Lectin-mediated drug delivery: the second generation of bioadhesives.

This paper reviews some recent developments in the area of bioadhesive drug delivery systems. The area of bioadhesion in drug delivery had started some 20 years ago by using so-called mucoadhesive polymers. Many of these polymers were already used as excipients in pharmaceutical formulations. This has facilitated the development of the first bioadhesive drug products, which are now commercially available. A major disadvantage of the hitherto known mucoadhesives, however, is their non-specificity with respect to the substrate. In particular for gastro-intestinal applications, this may cause some premature inactivation and moreover limits the duration of mucoadhesive bonds to the relatively fast mucus turnover. Nevertheless, for some mucoadhesive polymers other interesting functionalities were discovered, such as their ability to modulate epithelial permeability and to inhibit proteolytic enzymes. In contrast to the mucoadhesive polymers, lectins and some other adhesion molecules specifically recognize receptor-like structures of the cell membrane and therefore bind directly to the epithelial cells themselves ("cytoadhesion") rather than to the mucus gel layer. Furthermore, when bioadhesion is receptor-mediated, it is not only restricted to mere binding, but may subsequently trigger the active transport of large molecules or nanoscalic drug carrier systems by vesicular transport processes (endo-/transcytosis). Rather than only acting as a platform for controlled release systems, the concept of lectin-mediated bioadhesion therefore bears the potential for the controlled delivery of macromolecular biopharmaceuticals at relevant biological barriers, such as the epithelia of the intestinal or respiratory tract.

Adhesives↗

Embryonal "Botryoid" rhabdomyosarcoma of the larynx: a clinicopathologic and immunohistochemical study of two cases.

Two cases of embryonal rhabdomyosarcoma of the larynx are reported. The tumors occurred in a 16-year-old boy and in a 66-year-old man. They manifested clinically with nonspecific symptoms, including voice hoarseness and sense of throat fullness. Treatment consisted of total and partial laryngectomy, respectively. Grossly, both lesions had an exophytic growth pattern and microscopically featured a proliferation of small round to oval cells. Cell cytoplasms were occasionally stainable and fibrillary. Quite often, tumor cellularity was denser beneath the covering mucosa, recalling a "cambium layer" pattern. Tumor cells immunoreacted for desmin, actins, myoglobin, and sarcomeric actin; no immunostaining was noted for epithelial markers. No further antitumoral treatment was administered after surgery. There has been no recurrence of tumor at 2 and 10 years, respectively. Based on our series and the available literature, it seems that rhabdomyosarcoma of the larynx pursues a less-aggressive course than that seen in the homonimic juvenile or adult soft tissue lesion. Surgery alone appears to be a valid treatment option, especially when a polypoid, or "botryoid" gross pattern, coupled with the embryonal small cell histotype is encountered. In light of these findings, it is suggested that botryoid rhabdomyosarcoma of the larynx may deserve a specific consideration among the various laryngeal mesenchymal malignancies.

Adolescent↗

The fps/fes tyrosine kinase is expressed in myeloid, vascular endothelial, epithelial, and neuronal cells and is localized in the trans-golgi network.

The fps/fes proto-oncogene encodes a cytoplasmic protein tyrosine kinase that is thought to participate in signaling pathways involving members of the cytokine receptor superfamily, including those for erythropoietin, granulocyte-macrophage colony-stimulating factor, leukemia inhibitory factor, oncostatin M, ciliary neurotropic factor, and interleukins 3, 4, 6, and 11. Expression of fps/fes has been detected in hematopoietic cells, vascular endothelial cells, and cell types arising from all three germ layers during early development. Here, we describe fps/fes expression in developing and adult tissues from normal mice or from transgenic animals overexpressing wild-type or activated mutant fps/fes alleles. The highest levels of fps/fes expression were seen in angioblasts of early yolk sac blood islands, chondrocytes, vascular endothelial cells, neuronal cells, and several epithelial cell types, including those of the choroid plexus and the uterus. Fps/Fes protein was concentrated in the perinuclear region of cultured neuronal, myeloid, epithelial, and vascular endothelial cells, and a chimeric Fps/Fes-green fluorescence protein colocalized with gamma-adaptin, a marker for the trans-Golgi apparatus. These observations suggest the involvement of Fps/Fes in vesicle transport processes in cells with prominent secretory functions.

Adult↗

Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development.

Transforming growth factor beta (TGFbeta) superfamily signaling has been implicated in patterning of the early Xenopus embryo. Upon ligand stimulation, TGFbeta receptors phosphorylate Smad proteins at carboxy-terminal SS(V/M)S consensus motifs. Smads 1/5/8, activated by bone morphogenetic protein (BMP) signaling, induce ventral mesoderm whereas Smad2, activated by activin-like ligands, induces dorsal mesoderm. Although ectopic expression studies are consistent with roles for TGFbeta signals in early Xenopus embryogenesis, when and where BMP and activin-like signaling pathways are active endogenously has not been directly examined. In this study, we investigate the temporal and spatial activation of TGFbeta superfamily signaling in early Xenopus development by using antibodies specific for the type I receptor-phosphorylated forms of Smad1/5/8 and Smad2. We find that Smad1/5/8 and two distinct isoforms of Smad2, full-length Smad2 and Smad2(delta)exon3, are phosphorylated in early embryos. Both Smad1/5/8 and Smad2/Smad2(delta)exon3 are activated after, but not before, the mid-blastula transition (MBT). Endogenous activation of Smad2/Smad2(delta)exon3 requires zygotic transcription, while Smad1/5/8 activation at MBT appears to involve transcription-independent regulation. We also find that the competence of embryonic cells to respond to TGF(delta) superfamily ligands is temporally regulated and may be a determinant of early patterning. Levels of phospho-Smad1/5/8 and of phospho-Smad2/Smad2(delta)exon3 are asymmetrically distributed across both the animal-vegetal and dorsoventral axes. The timing of the development of these asymmetries differs for phospho-Smad1/5/8 and for phospho-Smad2/Smad2(delta)exon3, and the spatial distribution of phosphorylation of each Smad changes dramatically as gastrulation begins. We discuss the implications of our results for endogenous functions of BMP and activin-like signals as candidate morphogens regulating primary germ layer formation and dorsoventral patterning of the early Xenopus embryo.

Amanitins↗

A role of insular cortex in cardiovascular function.

We sought to determine whether the insular cortex contributes to the regulation of arterial blood pressure (AP). Responses to electrical and chemical stimulation of the cortex were studied in the anesthetized, paralyzed, and artificially ventilated Sprague-Dawley rat. The insular cortex was initially defined, anatomically, by the distributions of retrogradely labeled perikarya following injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the nucleus tractus solitarii (NTS). Injections of WGA-HRP into the insular cortex anterogradely labeled terminals in cardiopulmonary and other divisions of the NTS and confirmed projections revealed by retrograde tracing experiments. Electrical stimulation of the insular cortex elicited elevations of AP (less than or equal to 50 mm Hg) and cardioacceleration (less than or equal to 40 bpm). The locations of the most active pressor sites corresponded closely to the locations of retrogradely labeled cells in layer V of granular and posterior agranular areas of the insular cortex (areas 14 and 13) and the extreme capsule. Maximal pressor responses were obtained at a stimulus intensity of three to five times threshold current of 20-30 microA. Responses elicited mostly with higher-threshold currents were also mapped in areas 2a and 5lb and the claustrum and within the corpus callosum. Unilateral injections into the insular pressor area of the excitatory amino acid monosodium glutamate (L-Glu; 0.05 nmol to 10 nmol) or the rigid structural analogue of L-Glu, kainic acid (KA) (0.4 nmol) (which specifically excite perikarya), caused topographically specific elevations in AP and tachycardia. During the course of the anatomical transport studies, new findings were obtained on the organization and characteristics of the cortical innervation of the NTS and the nucleus reticularis parvocellularis. Topographic relationships between the cortex and the NTS were organized in a more complex manner than previously thought. Cells projecting to caudal cardiopulmonary segments of the NTS were fewer and generally located ventrally and caudally and in a more restricted area than cells projecting rostrally or to the parvicellular reticular formation. Anterograde transport data revealed new presumptive terminal fields in dorsolateral, ventral, periventricular, and commissural regions of the NTS, including an area overlapping the terminal field of the aortic baroreceptor nerve. We conclude that neurons within an area of the insular cortex projecting to multiple brainstem autonomic nuclei, including a region of the NTS innervated by baroreceptor afferents, increase arterial blood pressure and heart rate.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗