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Development of transient acetylcholinesterase staining in cells and permanent staining in fibers in cortex of rat brain.

The development of the acetylcholinesterase (AChE) texture of the cortex of the rat brain was studied during the first three weeks of life. The Tago technique enables visualization of both AChE+ cells and fibers with both shown in exquisite detail making quantification possible. At each age--0 (birth), 7, 14, 21 and 60 days (adult)--four brain areas were studied (cingulate, dorsal neocortex, lateral neocortex and olfactory) at each of three coronal planes in the brain (anterior, intermediate, posterior). Fiber density reached adult levels by Day 21 in cingulate cortex in intermediate and posterior planes. In other areas fiber density reached adult levels by Day 14 indicating a high rate of fiber growth during the first two weeks of life since at birth rat cortex is innervated only by a sparse AChE+ fiber invasion into neocortex in the anterior plane. Fiber density did not regress after adult levels were reached, however, cell staining showed a different pattern. At birth many lightly stained cells were seen in the olfactory cortex in all three planes, but other areas were devoid of cells. In all areas there was a peak at Day 7 in number of cells stained and in intensity of cells staining with a gradual decline in cell staining until by Day 21 very few stained cells were seen in the cortex (typical adult pattern).

Acetylcholinesterase↗

Alteration of serotonergic innervation in the suprachiasmatic nucleus of the rat following removal of input fibers from retina and lateral geniculate nucleus.

To examine the influence of afferent input to the suprachiasmatic nucleus (SCN) on the development of serotonergic fibers in the SCN, afferent fibers from the retina and lateral geniculate nucleus (LGN) were eliminated in neonatal rats. Eight weeks after lesion, the distribution pattern of serotonergic fibers in the SCN was examined immunohistochemically. Neither bilateral enucleation nor LGN ablation altered the serotonergic fiber distribution in the SCN as compared to the normal adult rat. However, following combined lesions of bilateral enucleation and bilateral LGN ablation, the density of serotonergic fibers decreased throughout the SCN. The present results indicate that both retino-hypothalamic and geniculo-hypothalamic fibers may play an important role in the development of serotonergic innervation in the SCN in vivo.

Animals↗

Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays.

We have developed a randomly ordered fiber-optic gene array for rapid, parallel detection of unlabeled DNA targets with surface immobilized molecular beacons (MB) that undergo a conformational change accompanied by a fluorescence change in the presence of a complementary DNA target. Microarrays are prepared by randomly distributing MB-functionalized 3-microm diameter microspheres in an array of wells etched in a 500-microm diameter optical imaging fiber. Using several MBs, each designed to recognize a different target, we demonstrate the selective detection of genomic cystic fibrosis related targets. Positional registration and fluorescence response monitoring of the microspheres was performed using an optical encoding scheme and an imaging fluorescence microscope system.

Base Sequence↗

Trans-4-stilbenemethanol-doped photosensitive polymer fibers and gratings.

We report the development of photosensitive polymeric optical fibers for gratings. The fiber is made of a copolymer of methyl methacrylate and butyl methacrylate, and its core is made of a copolymer of methyl methacrylate, ethyl methacrylate, and benzyl methacrylate (90/4/6 molar ratio) doped with trans-4-stilbenemethanol. Ultraviolet absorption and photoinduced refractive-index change of the doped core system are presented, and its photosensitivity is demonstrated by fabrication of Bragg gratings in the core of the fiber by use of a pulse laser at 325 nm.

Journal Article↗

[Quantification of the diameter of muscular fibres in the course of the development of the quadriceps].

Muscle biopsies were taken from the medium portion of the vastus lateralis of a hundred fetuses (aged between 10 and 40 weeks), children, adolescents and adults without any neuro-muscular disease. Diameters of the muscle fibers were measured with a Leitz ASM 68K on frozen sections which had been stained for myofibrillar ATPase. The results have been expressed as average diameters and histograms. The results demonstrated a gradual increase in diameter of the fibers during fetal development. At the birth, the average diameter of the type I and II fibers is 10-15 microns. Growth continues during postnatal development up until puberty, when the fibers reach their final diameter of 60-65 microns. Analysis of the histograms shows that before 18 weeks of gestation, there is a large dispersion in the diameter of the fibers. The first type I fibers are larger than the others and correspond to Wohlfart type b fibers. This is followed by the appearance of a second population of smaller type I fibers. These fibers gradually increase in diameter and one observes a homogenous population of type I fibers at the birth.

Adolescent↗

Testosterone induces testicular development but reduces GnRH-I fiber density in the brain of the House Finch, Carpodacus mexicanus.

Testosterone (T) in male birds generally inhibits the activity of the hypothalamo-pituitary-gonadal axis, but the androgen can also be gonadostimulatory. The mechanisms responsible for this stimulation are unknown. To address this question, adult male House Finches, Carpodacus mexicanus, held on short day length to inhibit reproductive function, received 20-mm long T-filled Silastic capsules either for 74 days (T+; n=5; Day 1-74) or for 66 days followed by eight days without capsules (T-; n=5), or empty capsules for 74 days (controls, C, n=10). At sacrifice on Day 74, plasma T concentrations were elevated in T+ males and low, and similar in T- and C males. Testes were enlarged in T+ males (136.5+/-57.2mg), small in T- males (16.2+/-9.5 mg), and undeveloped in C males (5.4+/-3.6 mg). The three groups had similar numbers and sizes of hypothalamic gonadotropin-releasing hormone-I immunoreactive (GnRH-I ir) perikarya, but perikaryon GnRH-I ir labelling was lighter in T+ than C males. T+ males also had lower density of GnRH-immunolabelled fibers in the preoptic area and lower intensity of GnRH immunolabelling in the median eminence than C males, suggesting an inhibition of GnRH-I neuronal activity. Plasma luteinizing hormone in the three experimental groups was uniformly low and did not differ. Thus, T administration-induced testicular development was associated with an inhibition of the reproductive neuroendocrine system, demonstrating a direct stimulatory effect of T on testes. Stimulation of testicular function by endogenous T in seasonal breeders may contribute to the rapid gonadal maturation that takes place in response to photostimulation.

Animals↗

Development of a bioartificial liver: properties and function of a hollow-fiber module inoculated with liver cells.

We have developed a bioartificial liver support system utilizing hollow-fiber bioreactor, plasmapheresis and microcarrier cell culture technologies. Liver cells were obtained through portal vein perfusion with ethylenediaminetetraacetate or ethylenediaminetetraacetate/collagenase. A mathematical model of mass transport in a hollow-fiber module, at various plasma flow velocities and system configurations, was developed. The bioartificial liver's ability to carry out specific differentiated metabolic liver functions was tested in vitro and in vivo. A reproducible large-animal model of acute ischemic liver failure was developed. Most major first-generation cyclosporine and 19-norterstosterone metabolites were isolated after substrate addition to the bioartificial liver in vitro. After bioartificial liver treatment for 6 hr (with dog or pig liver cells), dogs with acute liver failure had significantly lower serum ammonia and lactate levels and significantly higher serum glucose levels than did control animals treated with a bioartificial liver system inoculated with microcarriers alone. In addition, bioartificial liver-treated animals had significantly higher mean systolic blood pressures than did controls. Liver cell viability at the end of the 6-hr in vivo experiment was greater than 90%.

Animals↗

Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx.

We have shown that nonvoltage-operated Ca(2+) entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca(2+) influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca(2+) channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca(2+) influx, as stimulated by 0. 5 microM thapsigargin or 0.1 microM ionomycin, yielding faster development of actin stress fibers. Ca(2+) influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca(2+) influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P(2)<0. 05), suggesting Ca(2+) influx is needed for RhoA activation during spreading on type IV collagen; no Ca(2+) regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca(2+) influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca(2+) influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.

ADP Ribose Transferases↗

Initiation of acellular extrinsic fiber cementum on human teeth. A light- and electron-microscopic study.

The development of acellular extrinsic fiber cementum (AEFC) has never before been studied in human teeth. We have therefore examined the initiation of AEFC in the form of a collagenous fiber fringe and its attachment to the underlying dentinal matrix, in precisely selected, erupting human premolars with roots developed to 50%-60% of their final length. Freshly extracted teeth were prefixed in Karnovsky's fixative, decalcified in EDTA and subdivided into about 10 blocks each, cut from the mesial and distal root surfaces, vertical to and along the root axis. The blocks were postfixed in osmium tetroxide, embedded in Epon and cut for light- and electron-microscopic investigation. Starting at the advancing edge of the root, within a region extending about 1 mm coronal to this edge, fibroblast-like cells were seen closely covering the external root surface. Along the first 100 microns from the root edge, these cells extended cytoplasmic processes and contacted the dentinal collagen fibrils. Between these cells and the dentinal matrix, new collagen fibrils and very short collagen fibers gradually developed. Within the second 100 microns from the root edge, this resulted in the formation of a cell-fiber fringe network. Newly formed fibers of the fringe were directly attached to the non-mineralized matrix containing dentinal collagen fibrils and could be distinguished from the latter by differences in fibril orientation. During the process of dentin mineralization, the transitional zone between the fiber-fringe base and the dentinal matrix, i.e., the future dentino-cemental junction, also mineralized. It is suggested that this fiber fringe is the base of AEFC, which later increases in thickness by fiber extension and subsequent mineralization.

Bicuspid↗

Norepinephrine and GTP-gamma-S increase myofilament Ca2+ sensitivity in alpha-toxin permeabilized arterial smooth muscle.

A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using alpha-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After alpha-toxin treatment the fibers developed tension as a function of Ca2+ concentration (EC50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5 X 10(-7) M Ca2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca2+ solution and a transient and maintained contraction in 5 X 10(-7) M Ca2+ solution. GTP-gamma-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca2+ and maintained tension revealed that NE and GTP-gamma-S cause increases in Ca2+ sensitivity of myofilament shifting the EC50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-gamma-S causes an increase in myofilament Ca2+ sensitivity and that G protein may be involved in receptor signal transduction system. alpha-Toxin is a useful tool to permeabilize the smooth muscle tissue to ions and small molecules without any damage of receptor and signal transduction system.

Animals↗

Pituitary-thyroid axis controls the final differentiation of the dorsal skeletal muscle in urodelan amphibians.

A histoenzymological study of the ATPase activity of myosin in the dorsal axis muscle (dorsalis trunci) was carried out on two species of urodelan amphibians: Pleurodeles waltlii, a euthyroid species with spontaneous metamorphosis and Ambystoma mexicanum, a neotenic hypothyroid species. P. waltlii and A. mexicanum underwent an operation after which cytological analysis of the remaining pituitary were carried out in parallel. The muscle phenotype of urodelan amphibians varies according to the thyroid status of the species. In euthyroid adults, IIA fibers are dominant whereas in hypothyroid adults, IIC fibers are dominant. The number of type IIB (fast) and type I fibers (slow) are similar in both species. Physiological or experimental modulation of the concentration of circulating thyroid hormones results in a modification of the muscle fiber type profile pertaining to the considered species. We found that pituitary (TSH) plays a dominant role in the maturation of type IIC fibers in both species. Moreover, it seems to modulate the development of IIA fibers in P. waltlii and that of IIB fibers in A. mexicanum. Its action is thus species specific. Through partial or total hypophysectomy experiments, we have been able to demonstrate the influence of the hypophysothyroidian axis on the appearance of the adult muscle phenotype during metamorphosis.

Adenosine Triphosphatases↗

Neural cell adhesion molecule expression in Xenopus embryos.

The spatiotemporal pattern of expression of the neural cell adhesion molecule NCAM was mapped immunohistochemically in embryos of the frog Xenopus, from blastula to early swimming stages, using a polyclonal antibody that recognizes Xenopus NCAM. The neural plate stage was the earliest at which NCAM could be detected. The initial sites of NCAM immunoreactivity were neural ectoderm, somitic mesoderm, and chordamesoderm. During formation of the neural tube, NCAM immunoreactivity became restricted to the neuroectoderm and its derivatives. During closure of the neural tube and for 2-4 hr thereafter, NCAM was expressed in a distinctive radial pattern in coronal sections of the neural tube. NCAM was observed in neural crest cells before migration and after formation of cranial and spinal ganglia. During the period of initial neurite outgrowth, NCAM became concentrated in the developing central nerve fiber pathways. NCAM was seen on peripheral nerves from the time of their initial outgrowth and it was strongly expressed at neuromuscular junctions during the period of their formation. These results show that NCAM is expressed after neural induction and functions during morphogenesis of the neural plate and tube, some neural crest derivatives, development of nerve fiber tracts, and formation of neuromuscular connections.

Animals↗

Experimental approaches for exposure to sized glass fibers.

A number of studies have shown that glass fibers induce both malignant mesothelioma and fibrosis in rats and that these reactions may be primarily a function of the physical properties of the fiber. However, these studies were carried out with fibers having broad size distributions and used methods of administration which bear little resemblance to the way man is exposed. To better characterize the health effects of glass fibers, techniques have been developed to expose rats to glass fibers of defined sizes by intratracheal instillation of aqueous suspensions and by "nose only" inhalation exposure, and to determine the deposition, translocation, and ultimate fate of these fibers in the rat. The fibers have known size distributions with geometric mean diameters of 1.5 micrometers (sigma g = 1.1) and lengths of either 5 micrometers (sigma g = 1.49) or 60 micrometers (sigma g = 3.76). The fibers have been activated with neutron irradiation. Of the several resulting radionuclides, 65Zn appeared to be the most suitable for long-term clearance studies by use of in vivo whole body radioassay techniques. A fluidized bed aerosol generator has been developed to expose rats by "nose only" inhalation to approximately 500 fibers/cm3. The generator and exposure system permits reuse of fibers which pass through the exposure chamber and produces no significant alteration of the fiber size distribution. Rats were exposed by intratracheal instillations to 20 mg of the longer fibers and to equal numbers (2 mg) and equal mass (20 mg) of the shorter fibers. Through approximately 19 weeks little difference was observed in the whole rat clearance rate of long versus short fibers in the initial exposure group. Histopathology, however, showed differences at this time with the short fibers apparently successfully phagocytized by alveolar macrophages and cleared to the lymph nodes, while the long fibers were not.

Aerosols↗

Development and distribution of serotonin in the central nervous system of Manduca sexta during embryogenesis. I. The brain and frontal ganglion.

Development of the serotonergic system in the brain and frontal ganglion of the Manduca embryo between 35 and 100% of development was studied immunocytochemically with an antiserum to serotonin (5-HT). Serotonin immunoreactivity was initially detectable at 40-45% development in short fibers in the head region, prior to differentiation of the brain. Immunoreactive cell bodies were first seen in the brain at 60% development, located in the protocerebrum and tritocerebrum. Thick fiber tracts crossing the midline (commissures) could also be observed at this early stage. As development of the embryo progressed, eight immunoreactive cell groups, containing a total of about 38-40 cells, and four commissures with terminal arborizations appeared successively in the brain. From 75 to 100% development, no obvious changes occurred in the number or distribution of cells, and the brain exhibited the same pattern of 5-HT immunoreactive cells, fiber tracts and arborizations as in last instar larvae of Manduca. However, an increase in the size of the cells in both the brain and frontal ganglion was noted between 75 and 80% development, followed by a decrease by 100% development. The frontal ganglion was found to contain three 5-HT immunoreactive cells, which appeared to send bilateral projections into the frontal connectives and the recurrent nerve. During embryonic development, the dendritic arborizations of these frontal ganglion cells increased, while the amount of 5-HT immunoreactivity in the cell bodies decreased. Thus, the serotonergic system first appears in the Manduca embryo at an early stage of development, similar to the situation in other insects as well as vertebrates. By the end of the embryonic period, the same number of serotonergic neurons are present in the brain as in larval and adult Manduca, suggesting that once formed, these cells persist through postembryonic development and metamorphosis.

Animals↗

Dog model for studying detrusor instability secondary to acutely induced variable bladder outlet obstruction.

BACKGROUND: Involuntary detrusor contractions often cause irritative symptoms such as urgency and incontinence. A dog model for acutely induced variable bladder outlet resistance was developed to investigate the possible role of prostatic afferent nerve fibers in the development and maintenance of detrusor instability. METHODS: Fifty-eight mongrel dogs (weight range 19.5-36.5 kg) were divided into five groups: group I (n = 11) had surgically induced bladder outlet obstruction. Group II (n = 14) had urinary obstruction and bilateral sectioning of the lowest branches of the pelvic plexus supplying the prostate. Group III (n = 10) had prostate denervation only. Groups IV (n = 10) and V (n = 13) were sham-operated and controls, respectively. In the obstructed groups (I and II), an artificial urinary sphincter (length 4.5-6.0 cm) was placed around the bladder neck and connected to a reservoir placed subcutaneously to allow postoperative adjustments of urinary resistance. All dogs were evaluated at baseline and postoperatively at 1, 3, and 6 months with uroflowmetry, postvoid residual urine volume, cystometry as well as serum creatinine, and urinalysis. RESULTS: Occurrences of detrusor instability were not associated with prostatic denervation input. The mean peak flow rates decreased significantly in the obstructed groups at all follow-ups, but did not change significantly in the nonobstructed groups. Postoperatively, the mean maximum bladder capacity was significantly decreased for groups I and II only. However, a significant correlation between maximum bladder capacity and maximum detrusor pressure could not be detected at any time point in any of the groups. Mean postvoid residual urine volume varied considerably in all groups over time. Creation of a urinary model of infravesical obstruction was associated with considerable problems. CONCLUSIONS: In our dog model of bladder outlet obstruction, prostatic sensory nerve fibers appear not to be involved in detrusor instability. Surgical induction of a constant model of bladder outlet obstruction was difficult even in a large animal. The observations from the present study raise questions about the validity of obstructive urinary animal models.

Animals↗

Isolation and characterization of an avian slow myosin heavy chain gene expressed during embryonic skeletal muscle fiber formation.

We have isolated and begun characterization of the quail slow myosin heavy chain (MyHC) 3 gene, the first reported avian slow MyHC gene. Expression of slow MyHC 3 in skeletal muscle is restricted to the embryonic period of development, when the fiber pattern of future fast and slow muscle is established. In embryonic hindlimb development, slow MyHC 3 gene expression coincides with slow muscle fiber formation as distinguished by slow MyHC-specific antibody staining. In addition to expression in embryonic appendicular muscle, slow MyHC 3 is expressed continuously in the atria. Transfection of slow MyHC 3 promoter-reporter constructs into embryonic myoblasts that form slow MyHC-expressing fibers identified two regions regulating expression of this gene in skeletal muscle. The proximal promoter, containing potential muscle-specific regulatory motifs, permits expression of a reporter gene in embryonic slow muscle fibers, while a distal element, located greater than 2600 base pairs upstream, further enhances expression 3-fold. The slow muscle fiber-restricted expression of slow MyHC 3 during embryonic development, and expression of slow MyHC 3 promoter-reporter constructs in embryonic muscle fibers in vitro, makes this gene a useful marker to study the mechanism establishing the slow fiber lineage in the embryo.

Amino Acid Sequence↗

HIV type 1 Nef perturbs eye lens development in transgenic mice.

HIV transgenic mice often display lens cataracts as a consequence of viral-specific expression of HIV gene products in the developing lens. Cataractous mouse lines encoding either HIV-1 proviral DNA, HIV delta Gag/Pol] proviral DNA, or the HIV-1 nef gene alone were examined to ascertain the effect of Nef on murine lens development. Ocular disease was characterized by a progressive architectural disorganization within the lens fiber cell compartment developing in 100% of HIV-positive mice in five reported transgenic lines. Late embryonic stage transgenic lenses featured a mild microphthalmia, pyknotic nuclei within the lens fiber department, ballooning lens fiber cells, and elongated lens epithelial cells. Increased DNA fragmentation was evident in transgenic embryonic lenses, suggesting that cell death occurred by apoptosis. As studied in HIV delta Gag/Pol] transgenic mice, HIV transcription was developmentally linked to alpha A- and alpha B-crystallin gene expression, preceded disease development (in E14.5-E16.5 embryos), and persisted for weeks after birth. HIV-1 Nef was the predominant HIV gene product detected in the lens fiber cells of this line and was expressed almost to the exclusion of other HIV gene products. Nef was implicated as a major determinant of disease because (1) cataracts developed in mice transgenic for Nef alone and (2) the expression of other HIV gene products in wild-type HIV provirus transgenic mice occurred without a concomitant change in lens pathology.

Animals↗