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Biomedical subjects

W K Stell

Publications and source records attributed to W K Stell.

At least 37 records · Page 2Linked to original sources
PubMed1994

Basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF-beta) act as stop and go signals to modulate postnatal ocular growth in the chick.

Occlusion of the eye (form deprivation) during post-natal development leads to ocular enlargement and myopia. The chick model of form deprivation myopia (FDM) has been used to identify candidate factors that underly the control of ocular growth. The major biochemical change associated with eye enlargement is an increase in scleral cartilage proteoglycan production (Rada et al., 1991) which is reduced in recovering eyes. Thus increasing evidence suggests that in the chicken eye, two different signals are involved, one for stop (occluder off) and one for go (occluder on). Because transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) are known to act in a push-pull manner in regulating extracellular matrix, their possible roles in FDM were tested. Chicks were occluded monocularly for 8 days, after which axial dimensions were assessed using A-scan ultrasonography, and refractive errors using streak retinoscopy. Under light halothane anesthesia, the control group received daily vehicle injections into both eyes whereas the experimental groups were treated with growth factors in the occluded eye and vehicle in the unoccluded eye. It was shown that: (1) bFGF reduced FDM in a dose-dependent manner, with a 50% effective dose (ED50) of 1.05 and 1.67 ng for subconjunctival and intravitreal delivery, respectively. Both axial eye length and refraction were similarly affected. The effects were mainly confined to a decrease in vitreous chamber depth. The anterior chamber was less deep but only after intravitreal injections, whereas lens thickness was not affected at all. At maximum effect, after subconjunctival applications the bFGF-treated occluded eyes were only 0.09 +/- 0.16 mm longer than controls, which corresponded to a refractive error of -0.67 +/- 0.82 diopters (D), whereas after intravitreal applications the difference in axial eye length was -0.07 +/- 0.19 mm, corresponding to -0.3 +/- 0.52 D. (2) This effect could be mimicked by aFGF, but with a potency approximately 160 times less than that of bFGF. The aFGF rescue effect could only be demonstrated for subconjunctival delivery; high intravitreal doses (> or = 300 ng per injection) induced retinal detachment and photoreceptor degeneration, while doses of aFGF close to the ED50 for bFGF (3 ng per injection) were completely ineffective. (3) TGF-beta 1 was not found to induce myopia in unoccluded eyes, or to increase myopia in occluded eyes. It was, however, a potent inhibitor of the bFGF rescue effect, if administered together with bFGF in the subconjunctival space.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Quantitative analyses by radioimmunoassay of proliferating cell nuclear antigen (PCNA/cyclin) in ocular tissues.

A radioimmunoassay (RIA) was developed for a proliferating cell nuclear antigen, PCNA/cyclin, which is a protein synthesized in phase with DNA as a marker for cell replication. This antigen was used in rats to detect amounts of PCNA and to estimate rates of cell replication in ocular tissues (cornea, lens epithelium, retina-choroid and optic nerve) and in nonocular tissues (skin, skeletal muscle, brain, thymus, small intestine and liver). Antisera were raised in rabbits to the oligopeptide fragment of human PCNA (hPCNA): the C-terminal fragment [Tyr254]-hPCNA (254-261). For the RIA, synthetic peptides were used as the standard, and the radioiodinated [125I-Tyr254]-hPCNA (254-261) was used as the radioligand. The IC50 was 30-40 fmole/100 microliters with a sensitivity of 0.5-1.0 fmole/100 microliters for this assay. Tissue extracts in 1 N acetic acid were digested with trypsin to release the fragment (254-261) from whole PCNA for assay. The highest amounts of immunoreactive PCNA were found in lens epithelium and skin, while the lowest amounts were found in optic nerve and muscle. The cornea showed a higher value than the retina-plus-choroid or the optic nerve. These results suggest a high rate of cell replication in lens epithelium.

Amino Acid Sequence↗
PubMed1993

Localization of D2 dopamine receptors in vertebrate retinae with anti-peptide antibodies.

Dopamine plays an important role in modulating various aspects of retinal signal processing. The morphology of dopaminergic neurons and its physiological effects are well characterized. Two classes of receptor molecules (D1 and D2) were shown pharmacologically to mediate specific actions, with differences between individual groups of vertebrates. In an attempt to better understand dopaminergic mechanisms at the cellular level, we used antisera against D2 receptors and investigated the localization of the dopamine D2 receptor in the retinae of rat, rabbit, cow, chick, turtle, frog, and two fish species with immunofluorescence techniques. Antisera were raised in rabbits to two oligopeptides predicted from rat D2 receptor cDNA; one specific for the splice-variant insertion in the third cytoplasmic loop and the other directed towards the extracellular amino terminal region shared by both short and long isoforms. Preadsorption with the synthetic peptide resulted in a significant reduction of label, indicating the presence of specific binding in all species except turtle and goldfish. The pattern of labelling produced by the two antisera was essentially identical; however, the staining obtained with antiserum to the extracellular motif was always more intense. Specific staining was present in photoreceptor inner and outer segments, and in the outer and inner plexiform layers of all species. In mammals and chick, strongly fluorescent perikarya were observed in the ganglion cell layer and at the proximal margin of the inner nuclear layer. Label may be present in the pigment epithelium but could not be established beyond doubt. This pattern of labelling is in accordance with previous observations on D2 receptor localization by means of radioactive ligand binding and in situ hybridization techniques. It suggests that retinal dopamine acts as a neuromodulator as well as a transmitter. In the distal retina, it may reach its targets via diffusion over considerable distances, even crossing the outer limiting membrane; in the inner and outer plexiform layers, conventional synaptic transmission seems to coexist with paracrine addressing of more distant targets, and D2 receptors are expressed by both amacrine and ganglion cells.

Amphibians↗
PubMed1992

Effects of lesions of the optic nerve, optic tectum and nervus terminalis on rod precursor proliferation in the goldfish retina.

Teleost retinas grow throughout life by proliferation of neuroblasts at the retinal margin and dedicated rod precursors in the outer nuclear layer. Mechanisms regulating this proliferation are largely unknown. Previous investigators observed that rod precursor replication, as detected by incorporation of radioactive thymidine into cells of the outer nuclear layer, is enhanced after optic nerve crush. We attempted to determine whether this was due to severing of the retinopetal (nervus terminalis, n.t.) or retinofugal (retinal ganglion cell) axons in the optic nerve of the goldfish, Carassius auratus. In the first series of experiments, we ablated unilaterally the optic nerve, olfactory bulb (containing n.t. ganglia), or optic tectum (containing retinal ganglion cell axons and n.t. collaterals). Rod precursor proliferation increased dramatically in both retinas as soon as 5 days after surgery; in addition, the numbers of dividing cells were greater in the ipsilateral retina 10-15 days after optic nerve crush or tectal ablation and in the contralateral retina 20-25 days after olfactory bulb ablation. These observations are not accounted for by the known projections of retinal ganglion cells, but are consistent with the projections of the n.t. In the second series of experiments, n.t. projections to the brain and retina were severed bilaterally 7-8 weeks before the unilateral optic nerve crush or hemitectal ablation. Rod precursor proliferation increased as before, but the quantities of dividing cells were always equal in both retinas. We conclude that the n.t. may modulate rod proliferation locally and that injury to (some) brain regions may cause release of mitogens that affect rod precursors in both retinas.

Animals↗
PubMed1992

Partial characterization of four forms of immunoreactive gonadotropin-releasing hormone in the brain and terminal nerve of the spiny dogfish (Elasmobranchii; Squalus acanthias).

Four forms of immunoreactive GnRH have been detected in tissue extracts of both whole brains and terminal nerves from the spiny dogfish (Squalus acanthias). The GnRH forms were characterized using reverse-phase high pressure liquid chromatography (HPLC) and immunological recognition with four different antisera. Three of these forms possess immunological and chromatographic properties consistent with known forms of GnRH: mammalian GnRH, chicken GnRH-II and salmon GnRH. An additional form, with an HPLC elution position intermediate between chicken GnRH-II and salmon GnRH appears to be a new structure of GnRH. The presence of all four GnRH forms in the terminal nerve suggests a lack of regional specificity of the expressed forms of GnRH in the brain.

Animals↗
PubMed1992

[Immunochemical detection of the proliferating cell nuclear antigen (PCNA/cyclin) in ocular tissues].

The authors developed radioimmunoassay (RIA) for proliferating cell nuclear antigen (PCNA/cyclin), which a protein synthesizes in phase with DNA, as a marker for cell replication. We used it to detect the amounts of PCNA and estimated rates of cell replication in ocular tissues (cornea, lens epithelium, retina-plus-choroid and optic nerve) and in non-ocular tissues (skin, skeletal muscle, brain, thymus, small intestine and liver) in rats. Antisera were raised in rabbits to oligopeptide fragment of human PCNA (hPCNA): the C-terminal fragment [Tyr254]-hPCNA (254-261). For RIA the synthetic peptides were used as standards and radioiodinated [125I-Tyr254]-hPCNA (254-261) as radioligands. The IC50 was 30-40 f mole/tube with a sensitivity 0.5-1.0 f mole/tube for this assay. Tissue extracts in 1 M acetic acid were digested with trypsin to release fragment (255-261) from whole PCNA and were assayed by this method. The highest amounts of immunoreactive PCNA were found in lens epithelium and skin, while the lowest amounts were found in optic nerve and muscle. The cornea showed a higher value than retina-plus-choroid or optic nerve. A high cell replication in the lens epithelium was revealed.

Animals↗
PubMed1991

Induction of proliferating cell nuclear antigen (PCNA)-immunoreactive cells in goldfish retina following intravitreal injection with 6-hydroxydopamine.

1. The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was injected intravitreally into the eyes of juvenile (5- to 6-cm) goldfish. 2. Proliferation of rod neuroblasts caused by 6-OHDA (2 micrograms in 2 microliters saline) was detected in retinal wholemounts by immunofluorescence for proliferating cell nuclear antigen (PCNA) 3, 7, 14, 20, or 30 days after injection. 3. The injected dose of 6-OHDA was sufficient to cause permanent loss of dopaminergic interplexiform and serotonergic amacrine cells in the injected eye but not in the contralateral control eye. 4. 6-OHDA increased the density (mm-2) of PCNA-ir cells in the outer nuclear layer (ONL) of the injected eye to 2.65 times the initial density 20-30 days after injection, and it increased the density of PCNA-ir cells in the ONL of the contralateral, untreated eye, equally but after a delay of less than or equal to 7 days with respect to the injected eye. 5. 6-OHDA also increased the density of PCNA-ir cells in the inner nuclear layer (INL) to greater than 20 times the initial density 7 days after injection, followed by a rapid decline almost to control levels by 14 days after injection. 6. The sequence of responses to 6-OHDA, with PCNA-ir cells first scattered in the ONL and then clustered in the INL, suggests that neuroblasts from the ONL migrate to the INL to compensate for toxin-induced cell loss. 7. Double staining for 5-bromodeoxyuridine (BrUdR; a thymidine analogue) and PCNA, carried out on 7 days after intravitreal injection with 6-OHDA, showed that 77% of all PCNA-ir cells in the outer nuclear layer had been in S phase during the previous 24 hr. 8. Immunoreactivity for PCNA was found to be a valid marker for rod neuroblasts which have entered S phase within 1-2 days before sampling and was shown to be especially convenient for investigating the distribution of proliferating cells in whole mounts. 9. In controls injected unilaterally with saline or saline plus 1% dimethyl sulfoxide (DMSO), the differences in densities of PCNA-ir rod precursor nuclei 2-30 days after injection vs. day 0 (uninjected) were statistically insignificant in both injected and uninjected eyes (Negishi et al., 1991). Therefore the local effect of injecting 6-OHDA was due to 6-OHDA itself, not to mechanical damage or nonspecific actions of foreign substances.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Early histogenesis of the teleostean retina: studies using a novel immunochemical marker, proliferating cell nuclear antigen (PCNA/cyclin).

Immunoreactive proliferating cell nuclear antigen or cyclin (ir-PCNA), detected by immunofluorescence using a human autoantibody, was used as a marker for proliferating neuroblasts during early development of the teleostean retina. In a killifish (Medaka) and a cyprinid (goldfish), ir-PCNA was present in all retinal cells at the monolayer stage (embryonic day 2 in Medaka, days 3-4 in goldfish). Subsequently ir-PCNA was lost, as neuroblasts became postmitotic in a centre-to-periphery and proximal-to-distal (ganglion cells first, photoreceptors last) sequence. In late larvae and adults, neuronal ir-PCNA was confined to the ring of proliferating neuroblasts at the retinal margin and scattered rod precursors in the outer nuclear layer. The changes in localization of ir-PCNA during development parallel closely the changes in localization of [3H]thymidine incorporation, reported previously.

Animals↗
PubMed1990

Emergence and development of immunoreactive cells in teleostean retinas during the perinatal period.

We have used light-microscopical immunohistochemistry to investigate developmental changes of several neurochemical indicators in retinas of perinatal killifish and goldfish. Immunoreactive proliferating cell nuclear antigen (ir-PCNA/cyclin, a marker for replicating cells) was present in nuclei of all neuroblasts in the early monolayer stage, but was lost progressively in central-to-peripheral and proximal-to-distal order as the layers and cells of the mature retina appeared. The loss of ir-PCNA was slightly prior to the appearance of ir-TH (tyrosine hydroxylase), GAD (glutamic acid decarboxylase) and GS (glutamine synthetase) at the 4th embryonic day (E4) in both fish. Since hatching was earlier in goldfish (E5) than in killifish (E7), neurochemical maturation was evident at 2-3 days before hatching in killifish but not until around hatching in goldfish. Two markers, ir-somatostatin and protein kinase C, were detected by the 1st postnatal day (H1) in goldfish, but not in perinatal or adult killifish retinas. Thus the course of development of killifish and goldfish retinas is similar, but not identical. The validity of ir-PCNA as a marker for proliferating cells is confirmed by the coincidence of its disappearance with the appearance of neurochemical markers for mature, postmitotic retinal cells.

Aging↗
PubMed1990

Electron microscopic study of immunocytochemically labeled centrifugal fibers in the goldfish retina.

The centrifugal fibers innervating the goldfish retina were studied quantitatively by light and electron microscopy. These fibers originating from cell bodies in the olfactory bulb were labeled by antiserum to the tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide). The number of FMRFamide-immunoreactive (ir) centrifugal fibers in each eye of the adult goldfish (body length: 12-15 cm) was 65 +/- 14 (mean +/- S.D., n = 7). All of these fibers in the optic nerve and the retina were unmyelinated. Each FMRFamide-ir centrifugal fiber runs along the optic fiber layer and gives several terminal arborizations in the outermost layer (layer 1) of the inner plexiform layer. Layer 1 is, therefore, densely covered by a plexus of terminal arborizations. Along these terminal arborizations, we found output synapses characterized by a cluster of small clear vesicles (40 nm in diameter) at the presynaptic site and a thickened membrane in the apposed retinal cell processes. In a sample area of 2,000 microns 2, such synapses occurred at a density of one per 105 microns 2, or about 13,000 per centrifugal fiber. Thus, the FMRFamide-ir centrifugal fibers are likely to modulate retinal cell activity through an estimated total of 840,000 output synapses per retina.

Animals↗
PubMed1990

Immunohistochemical identification of calcitonin gene-related peptide and substance P in nerves of the bovine parathyroid gland.

Although peptide neurotransmitters have been shown to modulate hormone secretion in many glands, there are very few studies of neurotransmitters in the parathyroid gland. Bovine parathyroid glands were collected at a local abattoir, fixed with paraformaldehyde, sectioned using a cryostat, and stained by indirect immunohistochemistry for calcitonin gene-related peptide and substance P. We were able to positively identify both neuropeptides. Nerve fibres containing calcitonin gene-related peptide and substance P were identified in contact with the tunica media of arteries and arterioles and dispersed throughout the stroma of the gland. While many of the fibres encircled parenchymal lobules, no intimate contact with the peripheral chief cells was observed. All immunoreactive fibres were found to contain both neuropeptides. Since calcitonin gene-related peptide and substance P are vasodilators, they may increase blood flow within the gland. In addition, the neuropeptides may diffuse from perilobular nerve fibres into the parenchyma, thereby modulating secretion of parathyroid hormone.

Animals↗
PubMed1990

Detection of synenkephalin, the amino-terminal portion of proenkephalin, by antisera directed against its carboxyl terminus.

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.

Adrenal Gland Neoplasms↗
PubMed1988

Immunocytochemical and morphological evidence for a retinopetal projection in anuran amphibians.

The centrifugal projection to the retina in anuran amphibians (Rana catesbeiana and Xenopus laevis) has been investigated by immunocytochemistry, HRP transport, and optic nerve lesionings. FMRFamide- and N-terminal substance P-immunoreactive (Fa-ir and SP(3-7)-ir) fibers were abundant in the normal retina and optic nerve but almost absent distal to an optic nerve section or crush after 7-14 days survival. Fa-ir and SP(3-7)-ir fibers were traced to the optic nerve from the lamina terminalis (or the septopreoptic junctional area), where there are many Fa-ir and SP(3-7)-ir perikarya. After application of HRP to the optic nerve and survival for 9-10 days, retrogradely labeled neurons were observed in the lamina terminalis. Conversely, following HRP injection into the septal and preoptic area, labeled fibers were observed in the optic nerve. These results suggest that Fa-ir and SP(3-7)-ir efferent fibers project from the lamina terminalis to the retina. But in anurans, unlike teleosts, these fibers are not gonadotropin-releasing-hormone (GnRH)-ir. The morphological relations of this retinopetal pathway with the GnRH-ir nervus terminalis are discussed.

Animals↗
PubMed1987

Urotensin I-like immunoreactivity in amacrine cells of the goldfish retina.

Urotensin I-like immunoreactivity (UILI), in different localization from that of corticotropin releasing factor-like immunoreactivity (CRFLI), in the goldfish retina has been demonstrated by means of radioimmunoassay, high-performance liquid chromatography (HPLC) and immunohistochemistry. Radioimmunoassay showed 350 +/- 40 pg/mg prot. of UILI in goldfish retina extracts. The immunoreactive material present in the retina was also characterized by reversed phase HPLC. Some of the UILI co-eluted with synthetic carp UI, though the HPLC experiments suggested the existence of other UILI substance(s) with less hydrophobicity than synthetic UI. By immunohistochemistry, UILI and CRFLI were seen in different amacrine cells of the goldfish retina. It is suggested that UI may be involved in the fish visual transmission system together with CRF and other neuropeptides.

Animals↗
PubMed1987

Segregation of FMRF amide-immunoreactive efferent fibers from NPY-immunoreactive amacrine cells in goldfish retina.

Immunocytochemical studies were conducted on goldfish to determine whether a retinal efferent fiber system, immunoreactive to the tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), might contain instead a substance similar to one of the 36-amino acid pancreatic polypeptides, the C-terminus of which is similar to FMRFamide. Our results demonstrate the presence of two separate peptidergic systems, one containing FMRFamide-like, and the other pancreatic polypeptide-like peptides. Antisera to FMRF amide reveal the efferent fibers, whose axons exit the optic nerve and terminate in layer 1 of the inner plexiform layer, as previously described. Antisera to porcine neuropeptide Y, and to avian and bovine pancreatic polypeptides label a sparse population of putative amacrine cell bodies and a dense fiber plexus in layers 1, 3, and 5 of the inner plexiform layer. Based on intensity of staining, this amacrine cell peptide appears to be most similar to neuropeptide-Y. Radioimmunoassay and immunocytochemical staining of retinas in which the efferent fiber peptide was depleted by optic nerve crush confirm in large part the observation that the two peptide systems are distinct. However, there is some cross-recognition of the FMRF amide-like tissue antigen by pancreatic polypeptide antibodies. Double-label studies with antisera to tyrosine hydroxylase and neuropeptide-Y indicate that the pancreatic polypeptide antigen is not co-localized with catecholamines.

Animals↗
PubMed1987

Colour vision abnormalities in multiple sclerosis.

A battery of colour vision tests was employed to evaluate visual function in patients with multiple sclerosis (M.S.). Colour deficits were found in 45% of patients tested with the Ishihara plates and 42.5% of patients tested with the FM 100-Hue test. 65% of M.S. patients failed at least one of the tests. The colour vision deficits were not restricted to patients with optic neuritis or with visual evoked potential (VEP) abnormalities and there was no significant correlation between an abnormal VEP latency and a colour vision deficit. Colour vision testing may be a useful option to consider in the investigation of M.S. patients, even if there is no other evidence of visual system involvement.

Adult↗
PubMed1987

Functional-anatomical studies on the terminal nerve projection to the retina of bony fishes.

We have explored the structure and actions of terminal nerve (TN) fibers in the teleostean retina, the most accessible of TN projections. Using immunocytochemistry we have shown that the goldfish TN contains neuropeptides related to the molluscan cardioexcitatory peptide (FMRFamide) as well as luteinizing hormone-releasing hormone (LHRH). Retinal TN terminals were found upon major dendrites in the distal inner plexiform layer and neuronal cell bodies in the amacrine cell layer. Electron-microscopic double-labeling revealed TN terminals applied to the surface of [3H]-dopamine-, glycine-, and gamma-aminobutyric acid (GABA)-accumulating cells. Synthetic LHRH and FMRFamide at less than 1 microM modified spontaneous and light-evoked activity of ganglion cells in isolated superfused goldfish retina, especially during the active breeding season. Salmon(I)-LHRH was 10-30 times as potent as mammalian LHRH and caused rapid, prolonged desensitization. We conclude that LHRH- and FMRFamide-like peptides may be released by retinal TN endings, probably in concert with reproductive activity, and that they act independently through horizontal and/or amacrine cell pathways to modify visual information processing in the retina.

Animals↗