Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin

Summary Sections of human skin were processed according to the indirect immunofluorescence technique with a rabbit antiserum against human protein gene product 9.5 (PGP 9.5). Immunoreactivity was detected in intraepidermal and dermal nerve fibres and cells. The intraepidermal nerves were varicose or smooth with different diameters, running as single processes or branched, straight or bent, projecting in various directions and terminating in the stratum basale, spinosum or granulosum. The density of the intraepidermal nerves varied between the different skin areas investigated. PGP 9.5-containing axons of the lower dermis were found in large bundles. They separated into smaller axon bundles within the upper dermis, entering this portion of the skin perpendicular to the surface. Then they branched into fibres mainly arranged parallel to the epidermal-dermal junctional zone. However, the fibres en route to the epidermis traversed the upper dermis more or less perpendicularly. Furthermore, immunoreactive dermal nerve fibres were found in the Meissner corpuscles, the arrector pili muscles, hair follicles, around the eccrine and apocrine sweat glands and around certain blood vessels. Such fibres were also observed around most subcutaneous blood vessels, sometimes heavily innervating these structures. Numerous weakly-to-strongly PGP 9.5-immunoreactive cells were found both in the epidermis and in the dermis.     

On the Polarization and Innervation of the Pars Inferior Sensory Epithelia of the Herring Labyrinth

The macula sacculi and the macula lagenae of the herring, Clupea harengus L., were examined by light microscopy, the macula lagenae is large compared to what is normal among non-ostariophysan fishes, the morphological polarization of the hair cells in the inferior maculae shows a pattern which is similar to that usually seen in teleost fishes. The fibres in the nerves supplying the macula sacculi and the macula lagenae were counted and their diameters measured. The ramulus saccularis is divided in two separate ramuli innervating populations of hair cells with different morphological polarization. The saccular rostral nerve trunk contains 1800–2300 fibres, with 1300–1800 fibres in the caudal nerve trunk. The lagenar nerve is composed of 2100–4000 fibres. The fibre diameters are 1–14 μm in all ramuli. Silver staining of the nerve axoplasm reveals a unique differentiation of the maculae, which can be divided into a central area surrounded by a peripheral part. The hair cells in the central area are innervated by thick nerve fibres (5–14 μm diameter) as well as a few thin nerve fibres (about 1 μm diameter), while the receptor cells in the peripheral area are exclusively innervated by thin fibres having diameters of 2 μm or less.     

Fibronectin organization under and near cells

Polymerization of soluble fibronectin molecules results in fibres that are visible as networks using fluorescently labelled fibronectin protomers or by antibody labelling. Displacement of fibres composed of modified protomers in living cells provides information regarding matrix structure, organization, and movement. A static analysis of fibronectin structures and patterns of organization provide insight into their reorganization during adhesion and motility. Confocal microscopy and atomic force microscopy (AFM) reveal fibronectin-containing networks aligned in arrays perpendicular to the retracting cell edge and in apparently disordered networks of fibres under the cell. The change in patterns suggests a reorganization of fibronectin from disordered arrays used for adhesion into ordered arrays during movement of the cell. Comparison of confocal images with corresponding AFM images confirms that the fibres left on the surface as the cell moves away do contain fibronectin. The orientation of these fibres relative to the tail (uropod) and the receding edges of the cell leads us to propose that cells generate a force on the fibres that exceeds the adhesion force of the fibres to the surface causing them to pull fibronectin fibres into straight arrays. However, when the fibres are parallel to the direction of pull, the fibres remain attached to the surface. The data supports the hypothesis that disorganized, linear fibres are the product of Fn polymerization induced by the cell beneath it and serve to adhere the cell to the substrate as the cell spreads, whereas arrays of fibres found outside the cell are formed as existing fibrils and reorganize during cell motility.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Lipid order in hepatocyte plasma membrane blebs during ATP depletion measured by digitized video fluorescence polarization microscopy

Low-light digitized video fluorescence polarization microscopy was used to measure lipid order parameters in plasma membrane blebs of single, cultured rat hepatocytes during ATP depletion with the metabolic inhibitors cyanide and iodoacetic acid. Hepatocytes were labeled on the microscope stage with the plasma membrane probe trimethylammoniumdiphenylhexatriene at successive stages of cell injury. A pair of fluorescence polarization ratio images was obtained from a series of four fluorescence images recorded with a polarizer in the emission path oriented first parallel and then perpendicular to each of two orthogonal excitation light polarization directions. From the polarization ratio images, the lipid order parameter S was determined in individual plasma membrane blebs. Results indicate that the plasma membrane becomes uniformly rigid within a few minutes of the addition of metabolic inhibitors when small surface blebs have formed and ATP levels have fallen by greater than 95%. The measured order parameter of S approximately 0.95 in plasma membrane blebs, compared with S approximately 0.75 in normoxic cell plasma membranes, remained unchanged throughout the course of bleb development and ultimate cell death. These findings demonstrate that significant alteration in hepatocyte plasma membrane structure occurs early in hypoxic cell injury.     

Membrane potential of muscle fibers exposed to corticotropin and hydrocortisone]

The effect of a single intraperitoneal administration of corticotropin (1 unit) and hydrocortisone (5 mg) per 100 g of a body weight on the membrane potential (MP) as well as on the response rate of miniature end plate potentials (RRMEPP) of musculus soleus fibres of various polarization levels has been investigated in rats. It is shown that administration of corticotropin does not change the MP value, while that of hydrocortisone elicits its increase at the low initial polarization level of the muscle fibre membrane and its decrease at the high level. Hydrocortisone administration does not change the MP value at normal levels of fibre polarization. Corticotropin having been administered, RRMEPP of fibres both with high MP levels and with low ones has increased. Fibres with normal polarization also show a tendency to increase. Administration of hydrocortisone has induced a substantial increase of RRMEPP in fibres with high polarization levels within 45 min, while PRMEPP of fibres with normal polarization levels increased within first 5 min., and that of fibres with low levels of polarization remained unchanged.     

Scanning electron microscopy of the shell membranes of the hen's egg

Summary Scanning and transmission electron microscopy have been used to study the structure of the hen's shell membranes and their relationship to the shell and to the chorioallantoic membrane.We have confirmed previous observations that the fibres of the outer shell membrane are of larger diameter than those of the inner shell membrane, and that the fibres of the outer shell membrane extend into the mammillary knobs of the shell.The shell membrane fibres are arranged in layers parallel to the surface of the egg and there is no interweaving between the layers. Individual fibres are randomly orientated and may extend for distances of at least 25 m. It is suggested that relative movement between the oviduct and the developing membrane is random in direction and location.Each fibre consists of a core with a covering cortex, but the idea that the core may consist of keratin is criticised. A limiting membrane separating the surface of the albumen from the fibres of the shell membrane is also formed from this cortex. During incubation the chorioallantoic membrane becomes pressed against this inner limiting membrane.No correlation could be found between the positioning of the mammillary knobs and the patterning of the shell membrane fibres. It is suggested that the positioning of the mammillary knobs reflects the pattern of certain secretory cells in the genital tract of the hen.No significant changes in structure of thickness of the shell membrane could be found during incubation. The tips of the mammillary knobs, however, become detached from the shell and remain adherent to the shell membrane.The Cambridge Scientific InstrumentsStereoscan scanning electron microscope was provided by the Science Research Council (UK).We should like to thank Mr.R. F. Moss and Mr.P. S. Reynolds for technical assistance, and Mrs.Jeanne Mills for secretarial assistance.     

Gating currents in the node of Ranvier: voltage and time dependence.

Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmanns law (midpoint potential minus 33.7 mV; saturation value 17200 charges/mum-2). The time course of the asymmetry current and the voltage dependence of its time constant are consistent with the notion that due to a sudden change in membrane potential the charges undergo a first order transition between two configurations. Size and voltage dependence of the time constant are similar to those of the activation of the sodium conductance assuming m-2h kinetics. The results suggest that the presence of ten times more sodium channels (5000/mum-2) in the node of Ranvier than in the squid giant axon with similar sodium conductance per channel (2-3 pS).     

TRANSVERSE ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON

The impedance of the excised giant axon from hindmost stellar nerve of Loligo pealii has been measured over the frequency range from 1 to 2500 kilocycles per second. The measurements have been made with the current flow perpendicular to the axis of the axon to permit a relatively simple analysis of the data. It has been found that the axon membrane has a polarization impedance with an average phase angle of 76° and an average capacity of 1.1µf./cm² at 1 kilocycle. The direct current resistance of the membrane could not be measured, but was greater than 3 ohm cm.² and the average internal specific resistance was four times that of sea water. There was no detectable change in the membrane impedance when the axon lost excitability, but some time later it decreased to zero.     

A model for the extracellular release of PAF: the influence of plasma membrane phospholipid asymmetry.

Recent studies suggesting that cellular activation leads to enhanced transbilayer movement of phospholipids and loss of plasma membrane phospholipid asymmetry lead us to hypothesize that such events may govern the release of PAF, a potent, but variably release, lipid mediator synthesized by numerous inflammatory cells. To model these membrane events, we studied the transbilayer movement of PAF across the human erythrocyte and erythrocyte ghost plasma membrane, membranes with documented phospholipid asymmetry which can be deliberately manipulated. Utilizing albumin to extract outer leaflet PAF, transbilayer movement of PAF was shown to be significantly enhanced in erythrocytes and ghosts altered to lose membrane asymmetry when compared to movement in those with native membrane asymmetry. Verification of membrane changes was demonstrated using merocyanine 540 (MC540), a dye which preferentially stains loosely packed or hydrophobic membranes, and acceleration of the modified Russell's viper venom clotting assay by externalized anionic phospholipids. Utilizing the erythrocyte ghost loaded with PAF in either the outer or the inner leaflet, enhanced transbilayer movement to the opposite leaflet was seen to accompany loss of membrane asymmetry. Studies utilizing ghosts loaded with albumin intracellularly demonstrated that 'acceptor' molecules binding PAF further influence the disposition of PAF across the plasma membrane. Taken together, these findings suggest that the net release of PAF from activated inflammatory cells will depend on localization of PAF to the plasma membrane, transbilayer movement, which is facilitated by alteration of membrane phospholipid asymmetry, and removal from the membrane by extracellular and intracellular 'acceptor' molecules.     

Fluorescence anisotropy and myelin structure. II. Azimuth characteristic of surviving nerve fibers]

The dependence of fluorescence polarization of stained nerve fibres on the angle between the fibre axis and electrical vector of exciting light (azimuth characteristics) has been considered. Evidence is provided that the azimuth characteristics of stained nerve fibres depends on dye molecules adsorbed on the myelin sheath membranes. From the previous calculations it may be concluded that part of the dye molecules are oriented at a small angle to the geometrical axis of the nerve fibre.     

Substance P- and CGRP-immunoreactive nerves in bone

The present study demonstrates the occurrence of substance P (SP)- and calcitonin gene related peptide (CGRP)-immunoreactive nerve fibres in bone, bone marrow, periosteum, synovial membrane and soft tissues adjacent to the bone. The distribution pattern of the two types of nerves was similar, although the CGRP-positive fibres generally were more numerous. Both types of nerves were particularly abundant near the epiphyseal plate, in the bone marrow of patella and epiphyses, and in the periosteum. Many SP- and CGRP-immunoreactive fibres were also observed around blood vessels.     

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k_ET > 3 · 10¹² s^?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q_y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.     

Secretoneurin: A new peptide in the human enteric nervous system

Secretoneurin is a functional neuropeptide derived from secretogranin II (chromogranin C). This proprotein is processed to varying degrees in neuroendocrine tissues. In the present study we established by gel filtration high performance liquid chromatography that in human intestinal wall and mucosa an antiserum against secretoneurin detects as the major immunoreactive moiety the free peptide secretoneurin. In the mucosa some larger immunoreactive peptides were also present, however, a significant amount of the intact proprotein secretogranin II could not be detected. By immunohistochemistry we studied the distribution of secretoneurin within the gut. Antibodies to protein gene product 9.5 and chromogranin A were used to identify all neurons and endocrine cells, respectively, whilst those to the peptides substance P. CGRP and somatostatin were used for the further characterization of individual secretoneurin-positive structures. Secretoneurin immunoreactivity was found in nerve fibres in all layers of the gut wall. In both myenteric and submucous plexuses, nerve fibres and the majority of ganglion cells were secretoneurin-immunoreactive. In the mucosa, some secretoneurin-positive nerve processes ran parallel to the basal membrane of epithelial cells, occasionally invading the epithelial layer. Secretoneurin immunoreactivity was found in endocrine cells, mostly D cells, in the following regions in descending order of density: stomach/duodenum; rectum; colon; ileum. Thus, secretoneurin is a new major peptide within the human enteric neuroendocrine system. Its presence in abundant myenteric ganglion cells may imply a role in the modulation of gastrointestinal motility. The chemotactic properties of secretoneurin and its possible localization in sensory fibres suggest that this peptide may be involved in the genesis of intestinal inflammation.     

Linearly polarized excitation enhances signals from fluorescent voltage indicators

Voltage imaging in cells requires high-speed recording of small fluorescent signals, often leading to low signal/noise ratios. Because voltage indicators are membrane bound, their orientations are partially constrained by the plane of the membrane. We explored whether tuning the linear polarization of excitation light could enhance voltage indicator fluorescence. We tested a panel of dye- and protein-based voltage indicators in mammalian cells. The dye BeRST1 showed a 73% increase in brightness between the least and most favorable polarizations. The protein-based reporter ASAP1 showed a 22% increase in brightness, and QuasAr3 showed a 14% increase in brightness. In very thin neurites expressing QuasAr3, improvements were anomalously large, with a 170% increase in brightness between polarization parallel versus perpendicular to the dendrite. Signal/noise ratios of optically recorded action potentials were increased by up to 50% in neurites expressing QuasAr3. These results demonstrate that polarization control can be a facile means to enhance signals from fluorescent voltage indicators, particularly in thin neurites or in high-background environments.     

The structure of the nerve fibre layer and neurocord in the enteropneusts

Summary The nerve fibre layer and the neurocord of the Enteropneusts Saccoglossus horsti, Harrimania kupfferi and Ptychodera flava have been examined with the electron microscope. The nerve fibres vary in diameter between 0.15 to 10 m. The majority of the fibres are of the smaller diameters. The nerve fibre layer is intraepidermal, and is divided by processes running radially from the epithelial cells to the basement membrane that separates the nerve fibre layer from the muscle cells.The cells of origin of these nerve fibres are situated mainly in the innermost layers of the epidermal cells. The nerve fibre profiles contain numerous vesicles of very varied diameter and contents, together with larger granular inclusions that are also found in the nerve cell bodies.Morphologically recognisable synapses are rare, but the majority of fibres are in intimate contact with one another. Sometimes the mass of fibres is divided into bundles by the epithelial cell processes. The majority of giant fibres are situated near to the basement membrane of the neurocord. The giant fibres also have a varied content of vesicles as well as neurofilaments and neurotubules.The central canal in Ptychodera flava and the remnants of the central canal in Saccoglossus horsti are both lined by columnar cells that bear microvilli as well as cilia with the typical 9 + 2 pattern of tubules. Scattered amongst these cells are mucus secreting cells which open into the cavity of the canal.I (P.N.D.) should like to thank Professor J. Z. Young, F. B. S. for much advice and encouragement. Dr. R. Bellairs generously provided the electron microscope facilities, and Dr. R. Newell kindly collected and identified the Saccoglossus specimens. Mr. R. Moss, Mrs. J. Hamilton and Mr. A. Aldrich gave excellent technical and photographic assistance.     

Tensile fatigue behaviour of articular cartilage

Although fatigue has been implicated in cartilage failure there are only two studies by the same author, and in both of which cartilage was tested in the direction parallel to the collagen orientation in the surface layer. In the present work articular cartilage was tested also along the perpendicular direction, being the direction in which cartilage possesses lower tensile strength.Specimens were tested under cyclic tensile load. Number of cycles at failure was recorded as well as elongation of the specimen. To date 72 specimens have been tested all from one knee joint.The number of cycles to failure ranged between two and 1.5 million. The surface and deep layers have better fatigue properties whether tested in the parallel or the perpendicular direction, while the middle layer was far weaker. Better fatigue behaviour was observed with specimens tested in parallel than in perpendicular direction to the fibres.     

Effect of vitamin E-deficiency on regeneration of the sciatic nerve

The regeneration of the sciatic nerve fibres was studied in both normal and vitamin E-deficient rats at 30 and 60 days after crush. The vitamin E is involved in one of the most important mechanisms of protection against peroxidation of plasma membrane lipids; the plasma membrane plays certainly a role in nerve regeneration. Both the diameter and the total number of myelinated nerve fibres was calculated at different times. The number of myelinated fibres in the undenervated deficient animals was lower than that found in the undenervated normals animals. Following the nerve crush, in normal animals after two months the number of myelinated fibres exceeded the number found in undenervated normal animals, whereas in the deficient rat nerves it was significantly lower than in the corresponding controls and moreover it did not even reach the number found in the nerves of undenervated deficient rats. Finally, the caliber distribution of myelinated fibres in undenervated and denervated deficient rats shows a relative percent increase in the number of greatest axons and a decrease in smaller axons. This result confirm the vitamin E to be an important factor of the normal process of nerve regeneration.     

The fin musculature of cuttlefish and squid (Mollusca, Cephalopoda): morphology and mechanics

The lateral fins of cuttlefish and squid consist of a tightly packed three-dimensional array of musculature that lacks bony skeletal support or fluid-filled cavities for hydrostatic skeletal support. During swimming and manoeuvring, the fins are bent upward and downward in undulatory waves. The fin musculature is arranged in three mutually perpendicular planes. Transverse muscle bundles extend parallel to the fin surface from the base of the fin to the fin margin. Dorso-ventral muscle bundles extend from dorsal and ventral connective tissue fasciae to a median connective tissue fascia. A layer of longitudinal muscle bundles is situated adjacent to both the dorsal and ventral surface of the median fascia. The muscle fibres are obliquely striated and include a core of mitochondria. A zone of muscle fibres with a more extensive core of mitochondria is present in both the dorsal and the ventral transverse muscle bundles. It is hypothesized that these muscle masses include two fibre types with different aerobic capacity. A network of connective tissue fibres is present in the transverse and dorso-ventral muscle masses. These fibres, probably collagen, are oriented at 45 to the long axes of the transverse and dorsoventral muscle fibres in transverse planes.
A biomechanicayl analysis of the morphology suggests that support for fin movements is provided by simultaneous contractile activity of muscles of specific orientations in a manner similar to that proposed for other 'muscular-hydrostats'. The musculature therefore provides both the force and support for movement. Connective tissue fibres may aid in providing support and may also serve for elastic energy storage.     

The depolarising action of capsaicin on rat isolated sciatic nerve

The effect of capsaicin on the isolated sciatic nerve of rat was studied by extracellular recording of membrane polarisation. Capsaicin depolarised the sciatic nerve, but desensitization occurred rapidly upon repeated administration. Several other neuroactive substances, including substance P, were inactive. The depolarisation was reduced in nerves depleted of unmyelinated fibres by neonatal capsaicin treatment, suggesting that it occurs mainly in C-fibres. This depolarising action of capsaicin could explain the irritant and acute antinociceptive properties of capsaicin.