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Proliferation of skeletal muscle satellite cells after castration and administration of testosterone propionate.

Thirty-six neonatal pigs were randomly assigned to the following treatment groups: sham implanted gonadally intact males (B), sham-implanted castrated males (C), or castrated males implanted with testosterone propionate (C + TP). Four pigs from each group were sacrificed at 7, 14, or 21 days of age after a 6-hr continuous infusion of [3H]thymidine. Myofibers isolated from the triceps brachii were prepared for satellite cell enumeration by light microscope autoradiography. A developmental decline in labeled myofiber nuclei occurred in all groups, however, the greatest decline occurred in C (P less than 0.01). A treatment-by-age interaction was observed for percentage of labeled nuclei. Castration reduced total and labeled nuclei per millimeter myofiber (P less than 0.05), and C + TP had a higher percentage of labeled nuclei than C (2.8 vs 2.2%; P less than 0.05). Since triceps brachii muscles from 21 day B and C + TP were 120% (P less than 0.05) of C, the results indicate that postnatal growth of skeletal muscle is dependent on satellite cell mitotic activity and that testosterone enhances this activity in neonatal pigs.

Animals↗

Ultrastructure of the Eimer's organ of the star-nosed mole.

The nose of the star-nosed mole consists of 22 fleshy appendages that fan out from around the nostrils and are covered with specialized epidermal sensory receptors called Eimer's organs. The Eimer's organs of the star-nosed mole are domed epidermal papillae approximately 40 to 50 microns in diameter. The center of each papilla contains a column of stacked circular epidermal cells closely associated with five to ten neural processes that originate from three myelinated fibers in the underlying dermis. At middle and lower levels in the cell column, a single nerve fiber is located in the center of the column, enclosed by the epidermal cells which wrap around the fiber and form desmosomes between their own adjacent plasma membranes. An additional five to ten fibers travel up the sides of the column ensheathed in the margins of the epidermal cells. At the top of the cell column, the nerve fibers produce a repeated series of terminal swellings. These terminal swellings converge in the center of the column, where a single epidermal cell completely encapsulates each circular arrangement of nerve terminals. No synapses or other cellular junctions were observed between the nerve terminals where they converge within the cell column. There is a single Merkel-like ending at the base of the cell column, and a single encapsulated corpuscle beneath the cell column, in the connective tissue of the dermis. The structure of Eimer's organs is consistent with a mechanoreceptive function.

Animals↗

The role of fine needle aspiration cytology in the diagnosis of primary skin tumors.

This study presents the results of 544 fine needle aspirations of primary skin tumors, (275 basal cell carcinomas, 186 squamous cell carcinomas, 41 melanomas, 5 Kaposi sarcomas, 1 sebaceous epithelioma, 1 Merkel tumor, 1 glomangiosarcoma and 34 benign tumors). The purpose of the study was to investigate the accuracy of fine needle aspiration cytology for the diagnosis of skin tumors and to assess its clinical value. 21-27 Gauge needles used and the specimens were stained by MGG and Pap methods. Immunoperoxidase stains were applied in cases with a problem of differential diagnosis. The histological examination of 382 of the cases revealed one false positive cytological result. Persuaded by our results we believe that fine needle aspiration can give highly reliable information concerning the histological type of primary skin tumors with an equivocal clinical examination. Furthermore, by detecting relapses of previously treated lesions, this method is particularly useful in the management of a great number of patients.

Adult↗

Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.

Adhesion and cytoskeletal structure are intimately related in biological cell function. Even with the vast amount of biological and biochemical data that exist, little is known at the molecular level about physical mechanisms involved in attachments between cells or about consequences of adhesion on the material structure. To expose physical actions at soft biological interfaces, we have combined an ultrasensitive transducer and reflection interference microscopy to image submicroscopic displacements of probe contact with a test surface under minuscule forces. The transducer is a cell-size membrane capsule pressurized by micropipette suction where displacement normal to the membrane under tension is proportional to the applied force. Pressure control of the tension tunes the sensitivity in operation over four orders of magnitude through a range of force from 0.01 pN up to the strength of covalent bonds (approximately 1000 pN)! As the surface probe, a microscopic bead is biochemically glued to the transducer with a densely-bound ligand that is indifferent to the test surface. Movements of the probe under applied force are resolved down to an accuracy of approximately 5 nm from the interference fringe pattern created by light reflected from the bead. With this arrangement, we show that local mechanical compliance of a cell surface can be measured at a displacement resolution set by structural fluctuations. When desired, a second ligand is bound sparsely to the probe for focal adhesion to specific receptors in the test surface. We demonstrate that monitoring fluctuations in probe position at low transducer stiffness enhances detection of molecular adhesion and activation of cytoskeletal structure. Subsequent loading of an attachment tests mechanical response of the receptor-substrate linkage throughout the force-driven process of detachment.

Animals↗

[New morphologico-functional concepts of sensory structures].

The author analyzes the terminology of sensory nerve structures serving mechanoreception, nociception, thermoreception, chemoreception and baroreception which is not only very varied but also inaccurate, as it reflected so far only some morphological or functional phenomena. The author considers particularly inapt to use the term "receptor" for so-called nerve endings, as at present the term receptor stands for the specific binding capacity of protein molecules in particular in cell membranes, the transmission of information into the cell and evoking of a biological response. From the aspect of the contemporary concept the so-called nerve endings must be considered systems formed by a different number of sub-systems which participate in a certain way in the activity of the structure as a whole. This system is formed by the dendritic zone with the ability to form a generating potential, by modified Schwann cells, Grandry's on Merkel's modified fibrocytes, in corpuscles by fluid, a system of cellular lamellae and microfibrils and in Pacini's corpuscles also by a network of capillaries. Every sensory structure is associated metabolically with surrounding tissue. The author defines sensitive nervous formations as basic special sensory systems which serve the receipt of information and their transmission to the CNS and complex sensitive nervous formations which are systems of a higher order with an adaptive graded sensitivity. The author submits then his own classification of sensory nervous formations which respects morphological and functional phenomena which can be essentially summarized in two basic and one transient class.

Animals↗

Parasympathetic postganglionic nerve fibers in the fungiform papillae of the bullfrog, Rana catesbeiana.

An investigation was made of the precise origin of the unmyelinated nerve fibers in the fungiform papillae of the bullfrog's tongue. Some unmyelinated nerve fibers in the fungiform papillae originate from the parasympathetic postganglionic cells in the glossopharyngeal nerve. Axonal enlargements of the parasympathetic nerve fibers were in close contact with the Merkel-like basal or supporting cells in the taste disk. These results seem to provide morphological evidence for the existence of an efferent control system in the taste disk.

Animals↗

Fibroblast growth factor, epidermal growth factor, insulin-like growth factors, and platelet-derived growth factor-BB stimulate proliferation of clonally derived porcine myogenic satellite cells.

Clonally derived cultures of porcine skeletal muscle satellite cells were developed. The mitogenic effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factors (IGF-I and -II), and platelet-derived growth factors (PDGF-AA and -BB) were examined. Individually, bFGF, IGFs, and PDGF-BB stimulated proliferation of porcine satellite cells grown in basal serum-free medium or Minimum Essential Medium containing 2% fetal bovine serum (MEM-2% FBS). EGF stimulated proliferation in MEM-2% FBS, but neither EGF nor PDGF-AA were mitogenic when added to serum-free medium. The interactions among bFGF, EGF, IGF-I, and PDGF-BB were examined in serum-free medium, using growth factor concentrations shown in dose-response experiments to induce maximal proliferative responses (10 ng/ml bFGF, EGF and PDGF-BB, and 50 ng/ml IGF-I). The combination of bFGF and IGF-I dramatically increased proliferation, and IGF-I also synergized with EGF to increase proliferation. EGF, IGF-I, and bFGF interacted with PDGF-BB to stimulate proliferation. With the exception of EGF and bFGF, combinations of two growth factors typically resulted in greater than additive responses. Simultaneous exposure of satellite cells to bFGF, PDGF-BB, EGF, and IGF-I produced a fivefold increase in DNA compared to cells grown in basal serum-free medium. Elimination of EGF did not reduce the mitogenic response, yet removal of IGF-I, bFGF, or PDGF-BB reduced proliferation by approximately 40, 20, and 10%, respectively. These mitogens are likely physiological regulators of porcine satellite cell activity.

Animals↗

Size of the polyadenylic acid region of newly synthesized globin messenger ribonucleic acid.

The size of the polyadenylic acid region of newly synthesized globin mRNA was determined on mRNA isolated from nucleated erythroid spleen cells of anemic mice. The globin mRNA was purified by a combination of affinity chromatography on oligodeoxythymidylate-cellulose (oligo-(dT)-cellulose) and sucrose density gradient centrifugation. The purified RNA was shown to be globin mRNA by virtue of its ability to direct the synthesis of mouse alpha- and beta-globin chains in a cell-free system and by the presence of two bands migrating identically with authentic mouse alpha- and beta-globin mRNA when subjected to electrophoresis in polyacrylamide gels in the presence of formamide. When labeled for 1 hour with [3H]adenosine, the newly synthesized radioactive mRNA also migrated as two bands in these gels but they moved slower than the main bands suggesting that they have higher molecular weights. The polyadenylic acid region of the mRNA was isolated from the T1 and pancreatic RNase digestion mixture by acrylamide sodium dodecyl sulfate gel electrophoresis. The polyadenylic acid region was found to contain approximately 150 adenylate residues. As it is known that globin mRNA isolated from reticulocytes contains only 40 to 60 residues, it follows that at least 150 adenylic acid residues are added to the globin mRNA soon after its synthesis and that some of these are removed during the subsequent maturation of the erythroid cell.

Adenine Nucleotides↗

Effect of growth hormone, testosterone and serum concentration on actin synthesis in cultured satellite cells.

Because most of the DNA in a mature muscle accumulates during postnatal life and is derived from satellite cells, cellular regulation of protein accumulation in muscle fibers originating from satellite cells is an important aspect of muscle growth control. These experiments were designed to study the modulation of a alpha-actin accumulation in satellite cell-derived myotubes by serum and two anabolic hormones commonly assumed to be involved in muscle growth regulation; growth hormone and testosterone. Satellite cells were cultured from rats ranging in age from 5 d to 1 yr. After fusion into myotubes, various levels of pig serum, 3 to 20%, were added to culture medium, and the amount of alpha-actin per myotube nucleus was determined 4 d later. The quantity increased with increasing percentages of serum in the medium. For an assessment of the extent to which serum stimulatory activity was due to growth hormone or testosterone in the serum, similar experiments were conducted with medium containing 10% pig serum plus various levels of porcine growth hormone or testosterone. Neither of these hormones stimulated actin accumulation at concentrations from 10(-9) to 10(-6) M, a range encompassing physiological levels for each hormone. These experiments do not support the premise that either growth hormone and (or) testosterone are the blood-borne agents that interact directly with muscle to stimulate protein accretion, because neither growth hormone nor testosterone acted directly on muscle cells in vitro to stimulate muscle protein accumulation.

Actins↗

Transforming growth factor-beta, basic fibroblast growth factor, and platelet-derived growth factor-BB interact to affect proliferation of clonally derived porcine satellite cells.

Transforming growth factor beta-1 (TGF-beta) stimulated porcine satellite cell proliferation in basal serum-free medium by 25%, but inhibited growth in serum-containing medium by 58%. The effect of TGF-beta on cell proliferation in serum-free medium was examined in combination with the following human recombinant growth factors: platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). TGF-beta inhibited PDGF-stimulated proliferation, enhanced FGF-stimulated proliferation, and had no effect on proliferation stimulated by IGF-I. The response of satellite cells to EGF and TGF-beta in serum-free medium was not different than TGF-beta alone. TGF-beta depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF-I, PDGF and EGF, PDGF and FGF, and IGF-I and EGF. In combination with IGF-I and FGF, TGF-beta did not affect proliferation. TGF-beta inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF-I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM-10% PS) by 13% above control. When the combination of TGF-beta and FGF was added to MEM-10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF-AB (this form neutralizes PDGF-AA, AB, and BB) reduced proliferation in MEM-10% PS by 44%. The combination of TGF-beta and anti-PDGF-AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF-beta interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF-beta on proliferation of clonally derived porcine satellite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF.

Animals↗

Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells.

Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively, on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater in perifused than in static cultures (2034 +/- 160 vs. 387 +/- 33 ng/12 h). A dose-dependent increase in growth hormone secretion in response to challenge with growth hormone-releasing hormone (10(-12) to 10(-8) M) for 1 h was observed in both static and perifusion cultures; however, perifused cells were more responsive to the same concentration of neuropeptide than those in static culture. Concentrations of somatostatin (10(-12) to 10(-8) M) for 1 h did not inhibit basal growth hormone secretion in either static or perifusion cultures. To establish the hypothalamic-pituitary in series-perifusion model, slices of the hypothalamus, immediately adjacent to the sagittal midline, were perifused in series with anterior pituitary cells, and media effluent was assayed for growth hormone concentrations. Release of growth hormone was pulsatile and seemed to mimic the episodic pattern of bovine secretion. Hypothalamic slices were placed in one chamber of the perifusion system, and basal secretion of growth hormone-releasing hormone and somatostatin was pulsatile in media effluent. Tissue viability of hypothalamic slices and anterior pituitary cells was evaluated by KCl depolarization. Tissues were viable for at least 120 h. Thus, this hypothalamo-pituitary dual chamber perifusion system is a valid in vitro model to study regulation of growth hormone secretion.

Animals↗

Sensory nerve endings in the hard palate and papilla incisiva of the goat.

The sensory innervation of the papilla incisiva in the hard palate of the domestic goat was studied with light and electron microscopy, supplemented by electrophysiological studies of free nerve endings. The goat lacks incisor teeth. Grass and leaves are not bitten, but pulled off by pressing them between the tongue and papilla incisiva. Thus, the masticatory mucosa is subject to particularly heavy mechanical loads requiring functional specialization of the horny epithelium in the form of thickening, i.e., the papilla incisiva and 12-14 pairs of rugae palatinae. A thin layer of firm connective tissue (lamina propria) attaches the mucosa to the periost of the hard palate. Sensory nerve fibers were found most abundantly in the papilla incisiva. Their number decreased drastically in aboral direction. A section through the first four rugae palatinae contains only about 10% of the number of free nerve endings found in the same area of mucosa from the papilla incisiva. Four types of sensory nerve endings were found. Free nerve endings were seen ubiquitously in the epithelium and superficial layer of the lamina propria. Merkel nerve endings were found in the bases of the epithelial thickenings in the papilla incisiva and rugae palatinae. Few Ruffini corpuscles were found in the deeper layer of the lamina propria, while lamellated corpuscles were seen just below the basement membrane of the epithelial pegs. Thus, a variety of sensory nerve endings were found in the hard palate, especially in those areas that are in close contact with the tongue during chewing of food. This rich innervation suggests an important role in monitoring the mechanical properties of food. Recordings were made from cell bodies supplying these terminals. Classic low-threshold, slowly adapting responses were observed in Ass afferent populations. This activity was probably mediated by Merkel type endings. Alternately, high-threshold and suprathreshold responses obtained from Adelta category afferents were likely to be nociceptive. In support of this, threshold and suprathreshold sensitization was observed following injection of serotonin into the receptive field of Adelta populations. This activity was likely to be derived from the aforementioned free nerve endings.

Animals↗

Dynamic force spectroscopy to probe adhesion strength of living cells.

We studied the mechanical strength of the adhesion of living cells to model membranes. The latter contained a RGD lipopeptide which is a high affinity binding site for a cell adhesion molecule (integrin alpha(V)beta(3)). Cells adhered specifically to the vesicles. We used micropipette aspiration for breaking this adhesion with well defined forces. Systematic variation of the rate of force application revealed pronounced kinetic effects. The dependence of the detachment forces on the loading rate was well described by a power law (exponent approximately 0.4), in agreement with recent theoretical work.

Binding Sites↗

The touch dome in human skin is supplied by different types of nerve fibers.

Receptor end organs and free-nerve endings in the skin are the peripheral sentinels of the sensorial nervous system encoding for touch, temperature, and pain. Using a novel approach to analyze the outermost nerves of the skin, we visualized for the first time the distinct microanatomical structure of the touch dome of human hairy skin. The dermal nerve fibers of this slowly adapting type 1 mechanoreceptor were embedded in dermal protrusions that could be readily discerned by Laminin-5 staining. Concerning the nerves supplying the touch domes, we found, unexpectedly, that besides Abeta-fibers, Adelta- and C-fibers also were regularly present. The epidermis overlying the nerve convolutes showed a distinctive architecture of the rete ridges clearly demarcated from the surroundings and extending over 0.193 +/- 0.138 mm(2) (mean +/- standard deviation). Within this area, 756 +/- 386 Merkel cells/mm(2) (mean +/- standard deviation) were present compared with less than 50/mm(2) outside the touch dome, demonstrating for the first time a highly discontinuous distribution of these cells in nonglabrous skin. Our findings strongly suggest that the receptive qualities of human touch domes exceed mechanosensation, and that they may serve as multifunctional nerve end organs in human skin.

Adult↗

[Complications following cell therapy].

So far, the law in the Federal Republic of Germany still allows the injection of fresh-cell preparations from animals as a roborant to increase the vitality of the organism and to strengthen the body's immune defense system. The use of "sicca-cell" preparations was provisionally forbidden in 1987 by the Federal Health Organization (Bundesgesundheitsamt; BGA). Prohibition of fresh-cell injections would have exceeded the authority of this office, although the same serious reservations also applied in the case of this treatment method. Several publications that have appeared since 1955 have reported serious complications of this therapy, some life-threatening and some even lethal. Two further cases are now added: (1) A woman aged 69 had been receiving treatment with cell injections for 9 years. Immediately after an injection of sicca cells she collapsed and was hospitalized; 7 days thereafter she developed an ascending paralysis with increasing inability to swallow or breathe. She died 25 days after the injection as a consequence of central and peripheral respiratory failure. Autopsy revealed the alterations typical for acute Landry-Guillain-Barré-Strohl syndrome. (2) A 76-year-old healthy woman had been receiving treatment with fresh-cell preparations for several years. After an injection of cell suspensions a painful local swelling was observed. The symptoms were interpreted as the consequence of an iatrogenic local hematoma, and repeated punctures were performed to obtain blood. The patient was transferred to a surgical department for further therapy. Two days after the injection she suddenly died with signs of acute cardiovascular failure. Autopsy revealed the signs of a fulminating clostridial infection and also the characteristic signs of Landry-Guillain-Barré syndrome with involvement of the autonomic nervous system. In both cases the development of an inflammatory process in the peripheral nervous system could be interpreted as an immune-mediated allergic disease, related to the repeated injection of heterologous antigenic material containing nervous tissues. This hypothesis would also explain the two other cases already published and would be consistent with the observed perivenous leukoencephalopathy of the central nervous system. The human disease pictures correspond to the well-established animal models of EAEM (experimental allergic encephalomyelitis) and EAN (experimental allergic neuritis). The pathogenesis is discussed; the major role of the central and peripheral nervous system is stressed, with special reference to the risk of acute autonomic failure. The need for specific autopsy techniques for the investigation of the entire nervous system, including spinal cord, roots, spinal ganglia and peripheral nerves with sympathetic chains, is raised.

Adult↗

TrkC kinase expression in distinct subsets of cutaneous trigeminal innervation and nonneuronal cells.

Neurotrophin-activated receptor tyrosine kinases (Trks) regulate sensory neuron survival, differentiation, and function. To permanently mark cells that ever express TrkC-kinase, mice with lacZ and GFP reporters of Cre recombinase activity were crossed with mice having IRES-cre inserted into the kinase-containing exon of the TrkC gene. Prenatal reporter expression matched published locations of TrkC-expression. Postnatally, more trigeminal neurons and types of mystacial pad innervation expressed reporter than immunodetectable TrkC, indicating that some innervation transiently expresses TrkC-kinase. Reporter-tagged neurons include all those that immunolabel for TrkC, a majority for TrkB, and a small proportion for TrkA. TrkA neurons expressing TrkC-reporter range from small to large size and supply well-defined types of mystacial pad innervation. Virtually all small neurons and C-fiber innervation requires TrkA to develop, but TrkC-reporter is present in only a small proportion that uniquely innervates piloneural complexes of guard hairs and inner conical bodies of vibrissa follicle-sinus complexes. TrkC-reporter is expressed in nearly all presumptive Adelta innervation, which is all eliminated in TrkA knockouts and partially eliminated in TrkC knockouts. Many types of Abeta-fiber innervation express TrkC-reporter including all Merkel, spiny, and circumferentially oriented lanceolate endings, and some reticular and longitudinally oriented lanceolate endings. Only Merkel endings require TrkC to develop and survive, whereas the other endings require TrkA and/or TrkB. Thus, TrkC is required for the existence of some types of innervation that express TrkC, but may have different functions in others. Many types of nonneuronal cells affiliated with hair follicles and blood vessels also express TrkC-reporter but lack immunodetectable TrkC.

Animals↗