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The differentiation of repeated epilation (Er/Er) mouse mutant skin in organ culture and in grafts.

The homozygous repeated epilation (Er/Er) mouse mutant dies at birth and shows a variety of malformations, one of which is a skin defect. The developmental abilities of skin fragments from these Er/Er mouse embryos were studied in organ culture and in grafts performed either under the renal capsule of young mouse hosts or under the skin of mouse fetuses. In organ culture, the skin fragments differentiated in accordance with their genetic origin. The most characteristic feature was the abnormally thickened spinous layer and the formation of numerous epidermal nodules in Er/Er skin pieces removed from 13- to 16-day-old embryos, and cultured for 4 to 6 days, whereas the normal skin showed a constant layered organization. As in normal skin, keratin fibers developed within 4 to 6 days of culture. However, in contrast to normal skin, where keratin sheaths developed all over the surface of the epidermis, the Er/Er skin exhibited keratin masses inside the nodules. Combinations of mutant Er/Er epidermis with normal dermis resulted in abnormal skin differentiation, with formation of nodules similar to those observed for unseparated Er/Er skin fragments, whereas the reciprocal combination (normal epidermis with Er/Er dermis) produced normal skin differentiation. Cornified layers developed in both types of explants. Grafts of Er/Er and, for comparison, of normal skin fragments under the renal capsule or under a fetus's skin showed that the development of Er/Er skin in a normal or Er/+ host was similar to that of a normal skin. Thus, if isolated from the mutant organism, and inserted into a normal environment, the skin recovered within 6 to 7 days after transplantation and then developed normally. Our experiments suggest that the abnormal skin development of the Er/Er mutant might be caused by environmental influences.

Animals↗

A histopathological study of the percutaneous implantation of polyester fibers.

A histopathological study was made on scalp biopsies at 1 day, 1 week, 2 weeks, 1 month, 2 months, 5 months, and 20 months following implantation of synthetic fibers developed as artificial hair in Japan, in order to determine the histological conditions of its fixation over an extended period. In addition, the operative technique and materials for implantation were explored.

Biocompatible Materials↗

GAP-43 Immunoreactivity in the brain of the developing and adult wallaby ( Macropus eugenii).

We have examined the distribution of immunoreactivity for GAP-43 in the developing and adult brain of a diprotodontid metatherian, the tammar wallaby ( Macropus eugenii). The distribution of GAP-43 immunoreactivity in the neonatal wallaby brain was strikingly heterogeneous, in contrast to that reported for the newborn polyprotodontid opossum. Immunoreactivity for GAP-43 in the developing wallaby brain showed a caudal-to-rostral spatiotemporal gradient, with the brainstem well in advance of the telencephalon throughout the first 100 days of postnatal life. In many regions examined, GAP-43 immunoreactivity passed through the following phases: 1. intense immunoreactivity in developing fiber tracts and occasional somata; 2. diffuse homogeneous immunoreactivity; 3. selective loss of immunoreactivity in particular nuclei or cortical regions. In the isocortex, selective loss of GAP-43 immunoreactivity in the somatosensory and visual cortex (at postnatal day 115) coincided with the maturation of the laminar distribution of terminal thalamocortical axonal fields. Within adult cortical regions, GAP-43 immunoreactivity was highest in layer I of all regions, lower layers (V and VI) of primary somatosensory and visual cortices, layers II/III of motor and cingulate cortex, and layer IV of entorhinal cortex. Our findings suggest that, while patterning of GAP-43 immunoreactivity in the mature brain is similar across meta- and eutheria, there may be early developmental differences in the distribution of GAP-43 immunoreactivity between poly- and diprotodontid metatheria.

Animals↗

Isolation of the promoter of a cotton beta-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants.

Beta-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) beta-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the beta-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, sequence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.

Base Sequence↗

Collagen accumulation can continue with decreased prolyl hydroxylase activity in the liver.

A single dose of dimethylnitrosamine dose-relatedly increased total hepatic hydroxyproline content in rats 14 days after the dosing. In cases of 35 mg/kg of dimethylnitrosamine, it increased rapidly to 1.7 times the normal level within 14 days. This increase persisted thereafter until 84 days. Hepatic collagen prolyl hydroxylase activity was 1.8 times the normal level by the fourth day after the dosing but normalized within 14 days. It decreased further to levels significantly lower than those in normal rats at 28, 56 and 84 days. Serum glutamic pyruvic transaminase activity was about 13 times the normal level at 2 days and normalized after 7 days. On histology, fiber developed in necrotic areas around the central veins after 7 days and remained after 28 days when the necrosis had already disappeared. These results suggest that abnormal collagen can continue to increase in the state of decreased collagen prolyl hydroxylase activity in the liver.

Alanine Transaminase↗

Post-fixation horseradish peroxidase tracing in rat fetus and pups.

Horseradish peroxidase (HRP) has been shown to be applicable to fixed tissue in tracing projections in adult animals. This technique has several advantages in studying short projections in difficult attainable brain areas, especially with fetuses in utero. We have used this method to trace developing fibers (e.g., vestibulo-cerebellar connections) in rat fetuses and pups. It has been possible to make discrete and precise injections in any location. The quality of labeling by this method is distinctly related to the grade of fixation. Fixation grade determines how well HRP is anchored inside fibers and cell bodies. In optimally fixed preparations fibers and cell bodies are intensively stained with HRP up to over 3 mm from the center of an injection site. HRP is probably only taken up by damaged fibers so that the amount of fibers labeled is determined by the extent of the lesion caused by the injection pipette.

Animals↗

Expression of cotton GhMYB109 complements non-hair cell fate in the root epidermis of the Arabidopsis thaliana werewolf mutant.

Root cell fate and patterning in plants are orchestrated by the expression of cell-type-specific genes, including WEREWOLF (WER). Phylogenetic analysis of WER, functional WER homologs in Type III species (Rhodiola rosea and Boehmeria nivea), and related R2R3 MYB proteins identified in the cotton genome revealed that GhMYB109-a known regulator of fiber development in cotton ovules-clustered in a clade with Arabidopsis thaliana WER. To determine whether GhMYB109 is a functional homolog of WER, we expressed GhMYB109 under the control of the CaMV 35S promoter in the Arabidopsis thaliana wer-1 mutant and analyzed root epidermal cell patterning by counting root hairs. GhMYB109 expression significantly decreased the percentage of root hairs at both H and N positions. We found that most epidermal cells in the cotton root develop into root hairs (Type I pattern) although the cotton genome contains a functional WER homolog, GhMYB109. Additionally, GhMYB109 has been reported not to be expressed in cotton roots. These support the idea that GhMYB109 is a functional homolog of WER and that Arabidopsis thaliana and cotton diverged in root epidermal morphology through modifications in cis-regulatory elements rather than a functional divergence of their WER-like R2R3 MYB transcription factors.

Arabidopsis thaliana↗

Isolation of a cotton reversibly glycosylated polypeptide (GhRGP1) promoter and its expression activity in transgenic tobacco.

Reversibly glycosylated polypeptides (RGPs) are thought to be involved in polysaccharide metabolism. A cDNA of the cotton (Gossypium hirsutum) RGP gene, designated GhRGP1, has previously been characterized, and is preferentially expressed in fiber cells. In order to investigate its temporal and spatial control, we isolated a 624bp fragment upstream of the GhRGP1 coding sequence using a polymerase chain reaction (PCR)-based genomic walking method, transcriptionally fused the 624bp promoter sequence to the beta-glucuronidase (GUS) gene, and analyzed the stable gene expression in tobacco (Nicotiana tabacum). In 4-week-old transgenic tobacco plants, the highest expression level was observed in roots, and the GUS activity was 1.13- and 6.65-fold higher than that in stems and leaves, respectively. In the reproductive growth stage, the GUS expression level was highest in the pistils and the GUS activity in the stigmas and styles were 17.6-fold higher than that in the ovaries. High GUS activity was also detected in the anthers. In addition, histochemical staining for GUS activity on transgenic tobacco plants further indicated a higher expression in the trichomes, seeds and vascular tissues of stems. Abiotic stress treatments on transgenic tobacco plants showed that wounding and dehydration induced GUS expression. These results demonstrated the spatial and temporal regulation of a cotton RGP promoter in a model plant, and provided an important insight into the factors that control the fiber development and stress responses of the gene.

Base Sequence↗

Specific antibodies modulate the interactions of adenovirus type 5 with dendritic cells.

Adenovirus type 5 (Ad5) is able to induce an efficient CD8+ T lymphocyte (CTL) response against a transgene product, a property thought to be linked to its ability to transduce dendritic cells (DCs). Little, however, is known about the capacity of Ad5 to interact with DCs in the presence of specific antibodies, although most people test positive for antibodies directed against Ad5. In the present study, we found that in the presence of Ad5 antibodies, a large fraction of Ad5 binds very efficiently to DCs, and that this binding is FcgammaRII/FcgammaRIII dependent. Nevertheless, in the presence of high levels of antibodies against the whole virion, Ad5 entry was inhibited. Increased binding led to increased entry in DCs in the presence of fiber-specific antibodies or in the presence of low amounts of a whole antiserum raised against whole virions, showing that the relative concentration of antibodies directed against fiber and penton base plays a major role in entry efficacy. Nevertheless, mice previously immunized with virions or purified fiber developed a lower transgene-specific CD8+ T cell response than naive mice, although their serum appeared to increase virus entry into DCs in vitro.

Adenoviruses, Human↗

Stiffness and force in activated frog skeletal muscle fibers.

Single fibers, isolated intact from frog skeletal muscles, were held firmly very near to each end by stiff metal clasps fastened to the tendons. The fibers were then placed horizontally between two steel hooks inserted in eyelets of the tendon clasps. One hook was attached to a capacitance gauge force transducer (resonance frequency up to approximately 50 kHz) and the other was attached to a moving-coil length changer. This allowed us to impose small, rapid releases (complete in less than 0.15 ms) and high frequency oscillations (up to 13 kHz) to one end of a resting or contracting fiber and measure the consequences at the other end with fast time resolution at 4 to 6 degrees C. The stiffness of short fibers (1.8-2.6 mm) was determined directly from the ratio of force to length variations produced by the length changer. The resonance frequency of short fibers was so high (approximately 40 kHz) that intrinsic oscillations were not detectably excited. The stiffness of long fibers, on the other hand, was calculated from measurement of the mechanical resonance frequency of a fiber. Using both short and long fibers, we measured the sinusoids of force at one end of a contracting fiber that were produced by relatively small sinusoidal length changes at the other end. The amplitudes of the sinusoidal length changes were small compared with the size of step changes that produce nonlinear force-extension relations. The sinusoids of force from long fibers changed amplitude and shifted phase with changes in oscillation frequency in a manner expected of a transmission line composed of mass, compliance, and viscosity, similar to that modelled by (Ford, L. E., A. F. Huxley, and R. M. Simmons, 1981, J. Physiol. (Lond.), 311:219-249). A rapid release during the plateau of tetanic tension in short fibers caused a fall in force and stiffness, a relative change in stiffness that putatively was much smaller than that of force. Our results are, for the most part, consistent with the cross-bridge model of force generation proposed by Huxley, A. F., and R. M. Simmons (1971, Nature (Lond.), 213:533-538). However, stiffness in short fibers developed markedly faster than force during the tetanus rise. Thus our findings show the presence of one or more noteworthy cross-bridge states at the onset and during the rise of active tension towards a plateau in that attachment apparently is followed by a relatively long delay before force generation occurs. A set of equations is given in the Appendix that describes the frequency dependence of the applied sinusoid and its response. This model predicts that frequency dependent changes can be used as a measure of a change in stiffness.

Animals↗

Pathobiology of neuropathic pain.

This review deals with physiological and biological mechanisms of neuropathic pain, that is, pain induced by injury or disease of the nervous system. Animal models of neuropathic pain mostly use injury to a peripheral nerve, therefore, our focus is on results from nerve injury models. To make sure that the nerve injury models are related to pain, the behavior was assessed of animals following nerve injury, i.e. partial/total nerve transection/ligation or chronic nerve constriction. The following behaviors observed in such animals are considered to indicate pain: (a) autotomy, i.e. self-attack, assessed by counting the number of wounds implied, (b) hyperalgesia, i.e. strong withdrawal responses to a moderate heat stimulus, (c) allodynia, i.e. withdrawal in response to non-noxious tactile or cold stimuli. These behavioral parameters have been exploited to study the pharmacology and modulation of neuropathic pain. Nerve fibers develop abnormal ectopic excitability at or near the site of nerve injury. The mechanisms include unusual distributions of Na(+) channels, as well as abnormal responses to endogenous pain producing substances and cytokines such as tumor necrosis factor alpha (TNF-alpha). Persistent abnormal excitability of sensory nerve endings in a neuroma is considered a mechanism of stump pain after amputation. Any local nerve injury tends to spread to distant parts of the peripheral and central nervous system. This includes erratic mechano-sensitivity along the injured nerve including the cell bodies in the dorsal root ganglion (DRG) as well as ongoing activity in the dorsal horn. The spread of pathophysiology includes upregulation of nitric oxide synthase (NOS) in axotomized neurons, deafferentation hypersensitivity of spinal neurons following afferent cell death, long-term potentiation (LTP) of spinal synaptic transmission and attenuation of central pain inhibitory mechanisms. In particular, the efficacy of opioids at the spinal level is much decreased following nerve injury. Repeated or prolonged noxious stimulation and the persistent abnormal input following nerve injury activate a number of intracellular second messenger systems, implying phosphorylation by protein kinases, particularly protein kinase C (PKC). Intracellular signal cascades result in immediate early gene (IEG) induction which is considered as the overture of a widespread change in protein synthesis, a general basis for nervous system plasticity. Although these processes of increasing nervous system excitability may be considered as a strategy to compensate functional deficits following nerve injury, its by-product is widespread nervous system sensitization resulting in pain and hyperalgesia. An important sequela of nerve injury and other nervous system diseases such as virus attack is apoptosis of neurons in the peripheral and central nervous system. Apoptosis seems to induce neuronal sensitization and loss of inhibitory systems, and these irreversible processes might be in common to nervous system damage by brain trauma or ischemia as well as neuropathic pain. The cellular pathobiology including apoptosis suggests future strategies against neuropathic pain that emphasize preventive aspects.

Animals↗

Fibrogenic response in murine lungs to asbestos.

Pulmonary fibrogenic response was investigated in mice following intratracheal inoculation of amosite, anthophylite and tremolite varieties of Indian asbestos and studies were made over a period of 150 days. At early periods all the varieties produced acute inflammatory reaction in the lungs. Thick reticulum fibers were encountered at later periods with amosite, while only thin reticulum fibers developed with anthophyllite or tremolite variety. The formation of asbestos bodies did not take place with any of the asbestos varieties even at 150 days. The deviation in the pulmonary fibrogenic response in mice has been attributed to species difference.

Acute Disease↗

Regulation of Limulus skeletal muscle contraction.

Skeletal muscle contraction of Limulus polyphemus, the horseshoe crab, seemed to be regulated in a dual manner, namely Ca2+ binding to the troponin complex as well phosphorylation of the myosin light chains (MLC) by a Ca2+/calmodulin-dependent myosin light chain kinase. We investigated muscle contraction in Limulus skinned fibers in the presence of Ca2+ and of Ca2+/calmodulin to find out which of the two mechanisms prevails in Limulus skeletal muscle contraction. Although skinned fibers revealed high basal MLC mono- and biphosphorylation levels (0.48 mol phosphate/mol 31 kDa MLC; 0.52 mol phosphate/mol 21 kDa MLC), the muscle fibers were fully relaxed at pCa 8. Upon C2+ or Ca2+/calmodulin activation, the fibers developed force (357+/-78.7 mN/mm2; 338+/-69.7 mN/mm2, respectively) while the MLC phosphorylation remained essentially unchanged. We conclude that Ca2+ activation is the dominant regulatory mechanism in Limulus skeletal muscle contraction.

Animals↗

Multi-residue methodology for pesticide screening in wines.

A multi-residue solid-phase microextraction/gas chromatography/electron-capture detection method for pesticide screening (nine organochlorine, eleven organophosphorous and a tiadiazine insecticides, a benzoylurea acaricide and a halogenated sulfamide fungicide) in wine was developed. Fiber coating, extraction time and temperature, exposure of the fiber to the headspace or to the liquid phase and the ethanol effect on pesticides extraction were the parameters studied. The best results were obtained for a 100 microm poly(dimethylsiloxane) fiber, with 30 min immersion, in a 3 ml sample, at 45 degrees C. Pesticides extraction yield from aqueous spiked solutions was studied, for different ethanol concentrations (9%, 12.5% and 20%, v/v), which are representative of the main types of Portuguese wines. Calibration curves for extracted standards gave linear responses for all the pesticides, except dichlorvos, mevinphos, disulfoton and methidathion. Average detection limits were lower than 5 ppb and no significant interference from the matrix was found in the conditions studied, except for buprofezin.

Chromatography, Gas↗

Biocompatibility of a physiological pressure sensor.

A newly developed fiber optic micropressure sensor was evaluated for biocompatibility using the International Organization for Standardization (ISO) test standard 10993-6. The test material and an inert control (fused silica glass) were tested in New Zealand white rabbits. Four test specimens were implanted in the paravertebral muscles on one side of the spine about 2-5 cm from the mid-line and parallel to the spinal column. Similarly, four control specimens were implanted on the opposite side. The implantation periods were 1, 4, and 12 weeks to ensure a steady state biological tissue response. Four animals were tested at each time period. Macroscopic and microscopic observations were performed to compare the biological reactions between the test and control materials. There was an inflammatory reaction at 1 week which subsided at 4 weeks. There was fibrous tissue growth near the implant that also decreased over time. Most importantly, there was no significant difference in the biological response between the test and control materials. Therefore, we conclude that the pressure microsensor is biocompatible.

Animals↗

Nucleation and growth of macrofibrils in trichocyte (hard-alpha) keratins.

The intermediate filaments (IF) in trichocyte (hard-alpha) keratin form ordered aggregates that are infiltrated by sulfur-rich and tyrosine-rich proteins during fiber development to give a filament-matrix texture, which is stabilized in the later stages by the formation of disulfide linkages. Two polymorphic forms of macrofibril are found in the cortical cells of fine Merino wool. In the first the packing of the IF in the macrofibril is quasi-hexagonal whilst in the second the IF are packed in cylindrical sheets around a central core. In hairs the second type generally predominate. In the present contribution specific models for the mechanisms of nucleation and growth are developed for the two types of macrofibril and their applicability tested by analyzing transmission electron micrographs of wool and hair. Evidence is presented which supports the idea that sheet formation plays an important role in both types of macrofibril assembly and it is suggested that differing intersheet interactions are responsible for the differences between the ortho- and para-types. It is shown that the increase in IF tilt with radius in the ortho-type can be related to the surface lattice of the IF as determined from X-ray diffraction studies. Two possible types of intersheet interaction in the ortho-type are discussed, the first leading to an increase of around 0.5 degrees in IF tilt per layer and the second leading to a much larger tilt of 9.4 degrees per layer. A crude estimate based on the decrease in visibility of the IF with increasing radius in cross-section yielded a value of 0.35 degrees -0.7 degrees.

Animals↗

Emerin.

Emerin encoded by the STA gene is the first nuclear protein linked with a muscular dystrophy. Emerin is a 34 kDa, predominantly hydrophilic protein with a single hydrophobic region supposed to serve as a transmembrane domain. It was classified as a type II integral membrane protein localized at the inner nuclear membrane/nuclear lamina with an ubiquitous tissue distribution. It is speculated that emerin is required for the stability and normal function of rigorously moving nuclei in skeletal muscle and heart. During mitosis, emerin is cell-cycle-dependent phosphorylated and shows stage-dependent changes in distribution and localization suggesting that it plays a role in re-assembly of nuclear membranes. Mutations of the emerin gene have been associated with X-linked Emery-Dreifuss muscular dystrophy clinically defined by early joint contractures, progressive muscle weakness, and cardiomyopathy. Hopefully, identification of the protein defect may promote new therapeutic strategies concerning muscle fiber development and stability.

Animals↗

Tropoelastin massively associates during coacervation to form quantized protein spheres.

Tropoelastin, the precursor of elastin, undergoes a rapid monomer to multimer association in an inverse temperature transition. This association culminates in the rapid formation of stable, optically distinct droplets of tropoelastin. Light scattering and microscope measurements reveal that these droplets are 2-6 microm in diameter. Scanning electron microscopy confirms that the droplets are spherical. Three-dimensional confocal image stacks based on the autofluorescence of tropoelastin reveal that droplets are loaded with hydrated tropoelastin. Droplets are viable intermediates in synthetic elastin macroassembly. Dense clusters of aggregated droplets and partially formed fibers develop when droplets are incubated in the presence of a lysyl oxidase. Lysine-reacting chemical and enzyme-assisted cross-linking conditions generate cross-linked beads due to interactions between multiple, surface-exposed lysine epsilon-amino groups. Droplets represent an efficient mechanism for the bolus delivery during elastogenesis of quantized packages of preaccreted tropoelastin.

Cross-Linking Reagents↗