STRUCTURAL BIOLOGY OF THE FIBRES OF THE COLLAGENOUS AND ELASTIC SYSTEMS

The different types of fibres of the collagenous and elastic systems can be demonstrated specifically in tissue sections by comparing the typical ultrastructural picture of each of the fibre types with studies using selective staining techniques for light microscopy. A practicalmodus operandi, which includes the recommended staining procedures and interpretation of the results, is presented. Micrographs and tables are provided to summarize the differential procedures. Reticulin fibres display a distinct argyrophilia when studied by means of silver impregnation techniques, and show up as a thin meshwork of weakly birefringent, greenish fibres when examined with the aid of the Picrosirius-polarization method. In addition, electron-microscopic studies showed that reticulin fibres are composed of a small number of thin collagen fibrils, contrasting with the very many thicker fibrils that could be localized ultrastructurally to the sites where non-argyrophilic, coarse collagen fibres had been characterized by the histochemical methods used. The three different fibre types of the elastic system belong to a continuous series: oxytalan—elaunin—elastic (all of the fibre types comprising collections of microfibrils with, in the given sequence, increasing amounts of elastin). The three distinct types of elastic system fibres have different staining characteristics and ultrastructural patterns. Ultrastructurally, a characteristic elastic fibre consists of two morphologically different components: a centrally located solid cylinder of amorphous and homogeneous elastin surrounded by tubular microfibrils. An oxytalan fibre is composed of a bundle of microfibrils, identical to the elastic fibre microfibrils, without amorphous material. In elaunin fibres, dispersed amorphous material (elastin) is intermingled among the microfibrils.     

Host and cell type affect the mode of degradation by Meripilus giganteus

Wood degradation by the white-rot basidiomycete Meripilus giganteus (Pers.: Pers.) Karst. was studied in naturally infected and artificially inoculated wood of beech ( Fagus sylvatica L.) and large-leaved lime ( Tilia platyphyllos Scop.). Semi-thin sections revealed that the secondary walls of most fibres contained internal cavities. Three distinct types of cavity formation, which differed not only between hosts, but also between cell type and location in the annual ring, were identified.
Within discoloured wood of naturally infected beech, the structure of the cavities and their formation by the associated hyphae were reminiscent of a soft-rot. By contrast, cavity formation in artificially inoculated beech and large-leaved lime wood differed from a soft-rot mode of attack as extensive delignification always preceded cavity formation, and neither T-branching, L-bending, nor hyphal growth were found within cell walls. The formation of half-moon shaped cavities in beech wood was present only in tension-wood fibres. From large diameter hyphae, growing within the fibre lumen, numerous fine perforation hyphae extended transversely via helical cracks into the cell wall. Subsequent degradation of cellulose within concentric layers of the tension-wood fibres commenced from the apices of perforation hyphae.
Sections stained with ruthenium red and hydroxlamine-ferric chloride, revealed that M. giganteus preferentially degrades pectin-rich regions of the middle lamellae in xylary ray cells. In large-leaved lime, such regions were uniformly located in the middle lamellae of axial and ray parenchyma. In beech wood, degradation of pectin-rich middle lamellae regions commenced after the delignification of secondary walls and resulted in a conspicuous hollowing of multiseriate xylem rays. Plasticity in wood degradation modes by M. giganteus in large-leaved lime and beech wood reflects variations in cell wall structure and/or prevailing wood conditions.     

The arrangement of neurosecretory and catecholamine fibres in relation to the pituitary intermedia cells of the skate,Raja radiata

Summary The innervation pattern of the intermediate lobe of the skate (Raja radiata) was studied with histological and fluorescence histochemical methods. Neurosecretory fibres, stained with i.a. pseudo-iso-cyanine, were found running in bundles in the central parts of the cell cords. They terminated partly around the perinuclear parts of the intermedia cells, partly around the apices of the cells close to the vascular walls.A catecholamine innervation of the intermedia was also established. Catecholaminecontaining fibres with the appearance of nerve terminals were found around the intermedia cell apices close to the vessels. In some specimens, catecholamine fibres also seemed to terminate at the perinuclear parts of the cells.Thus it is possible, judging solely from structural relations, that both the cell body (the synthesis pole) and the cell apex (the release pole) receive a dual innervation. Recent experimental evidence indicates that the release of MSH from the pars intermedia is controlled by catecholamine fibres, but as yet there is only structural evidence for a special control of hormone synthesis.This study was supported by grants from the Swedish Natural Science Research Council (No. 99-35 and 2126-2) and was carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B70-14X-712-05 and B70-14X-56-06).     

Auditory interneurones in the mesothoracic ganglion of crickets

Auditory interneurone responses in the mesothoracic ganglion of the cricket Gryllus bimaculatus were investigated with special regard to temporal features of the calling song. Units representing five response types were found. One type codes verse syllables and intensity. The second codes syllables of highfrequency verses. The third responds as a pulse marker. The fourth shows adaptation and the response pattern depends on the verse frequency. The fifth fires a burst at verse onset.Responses of mesothoracic units recorded in two other cricket species do not differ markedly from those of Gryllus bimaculatus. Particularly, no tuning is found to species-specific differences in their calling songs.The stimulus direction can affect the threshold in different ways: dependence at all frequencies, dependence only between 3 and 6 kHz, and independence are found. The dependence is mainly expressed by a higher threshold for contralateral sounds.The mesothoracic branching of a few neurones was demonstrated by extracellular CoS-staining. These cells pass through the ganglion as connective fibres giving off small branches into the ventro-medial and dorso-medial neuropiles.     

Fibre dimorphism: cell type diversification as an evolutionary strategy in angiosperm woods

Dimorphic fibres in angiosperm woods are designated when zones of two different kinds of fibres can be distinguished in transverse sections. The usage of most authors contrasts wider, thinner‐walled, shorter (sometimes storied) fibres with narrower, thicker‐walled fibres that have narrower lumina. The wider fibres can be distinguished in longitudinal sections from axial parenchyma, which usually consists of strands of two or more cells each surrounded by secondary walls (and thus different from septate fibres). This phenomenon occurs in some Araliaceae, Asteraceae, Fabaceae, Myrtales (notably Lythraceae), Sapindales (especially Sapindaceae), Urticales and even some Gnetales. Additional instances can doubtless be found, especially if instances of wide latewood fibres together with narrow earlywood fibres are included. There is little physiological evidence on differential functions of dimorphic fibres, except in Acer, in which hydrolysis of starch in the wide fibres is known to result in transfer of sugar into vessels early in the growing season. Starch storage in axial parenchyma may, in a complementary way, serve for embolism reversal and prevention and thus for maintenance of the water columns. Crystalliferous fibres (Myrtales, Sapindales) can be considered a form of fibre dimorphism that deters predation. Gelatinous fibres, often equated with tension wood, can also be considered as a form of fibre dimorphism. The evolutionary significance of fibre dimorphism is that a few small changes in fibre structure can result in the accomplishment of diversified functions. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 44–67.     

A synchrotron radiation X-ray diffraction study of the crystallinity of tendon fibres

The equatorial diffraction pattern of tendon collagen fibres was measured during short successive exposures at different lengths using a double focusing X-ray synchrotron radiation camera with film and with an area detector. Similarly, patterns from thin fibres from premature rats were recorded. The patterns unambiguously illustrate the relationship between fibre crystallinity and the age of the animal. Further, the results indicate that in the initial part of the linear region of the stiffness-versus-length curve, the collagen fibres are characterized by a quasihexagonal arrangement of collagen molecules, whereas at the end of this region, the molecular arrangement becomes hexagonal.     

Control of skeletal muscle fibres and adipose cells size in the flesh of rainbow trout

The distributions of the diameters of skeletal muscle fibres and adipocytes were studied in rainbow trout. The cellularity of perivisceral adipose tissues and subcutaneous ventral and dorsal adipose tissues were characterized more specifically. In these tissues, a population of small adipocytes was distinguishable from larger adipocytes. The same was observed in white muscle. The effects of extrinsic factors (dietary lipid in two different thermal conditions) and intrinsic factors (strains in two different saline conditions, growth hormone) on the long-term response of the cellularity of both muscle and adipose tissues were studied. The effects of thermal environment were tested on fish fed the same ration and the effects of saline environment on fish fed ad libitum. The mean size of white muscle fibres was relatively unaffected by the different treatments tested: genetic origin and dietary lipid in different environmental conditions. There were significant differences in growth rate due to genetic origin and saline environment. The possible involvement of hyperplasia in response to these different factors is discussed. Growth hormone supplementation enhanced the percentage of small diameter fibres indicating a role of this hormone in the control of muscle hyperplastic growth. The mean size of adipose cells was affected only slightly by the different treatments tested. An increase in adipose cell size with aging and lipid content was observed. The percentage of small adipocytes also increased with aging. Thus, it is proposed that the development of adipose tissues, and thus fat retention, both result from the recruitment of new adipocytes and from the increase in size of existing adipocytes. The hyperplastic process contributed significantly to the differences in fat retention due to different treatments tested (strains, thermal and saline environments). When partially substituting fish oils for corn oils in the diet, a large increase in the ventral adipose cell size was seen indicating a potential negative effect of n-6 fatty acids on cell proliferation. Growth hormone treatment, on the contrary, induced a decrease in the size of perivisceral adipocytes. Thus, diet and hormonal status affect adipose cells size through two different metabolic pathways: lipogenesis and lipolysis respectively.     

Secretory tissues in the East African shrubby aloes

Examination of Aloe leaf sections revealed the presence of three types of cells at the phloem pole of the vascular bundles, aloin cells, outer bundle sheath cells and fibres. Three species contain fibres alone and produce a sparse exudate with few components staining purple with Fast Blue B on thin-layer chromatograms. The majority of species have aloin cells of various sizes and in the tetraploid species these produce a copious exudate containing anthraquinone and chromone derivatives. It is suggested that the aloin cells act as storage tissue and that the compounds are synthesized in the surrounding layer of cells of smaller diameter, many of which can be seen to contain globules of unknown constitution. Anatomical observations support the idea that the shrubby tetraploid Aloe species are derived from a form similar to the diploid Aloe morijensis. Two forms of this species have been described, one with fibres only and few exudate components and another with both fibres and aloin cells and an exudate containing compounds of the tetraploid species. A line of diploid plants similar in anatomy and chemistry to the first form is represented by A. fibrosa and A. babatiensis while a line of tetraploids with affinities to the second form is represented by A. nyeriensis, A. cheranganiensis, A. elgonica, A. dawei and A.yavellana.     

Unusual histology and morphology of the ribs of mosasaurs (Squamata)

We report the presence of two previously unrecognized features in the dorsal ribs of mosasaurs: first, the presence of extremely dense, pervasive extrinsic fibres (anchoring soft tissue to bone, sometimes called Sharpey's fibres); and second, high intraspecific variation in costal bone compactness. Extensive extrinsic fibres are developed in the dorsal ribs of the mosasaurs Tylosaurus proriger and Eonatator sternbergi. The dorsal ribs of these mosasaurs are also characterized by a longitudinally ridged texture that almost completely covers the bone. Pervasive extrinsic fibres and ridged textures are absent in the mosasaur Selmasaurus russelli as well as the dorsal ribs of extant semi‐aquatic reptiles (e.g. crocodyliforms) and mosasaurs' close extant relatives and analogues (e.g. snakes and varanids). Similar ridged textures characterize the dorsal ribs of several other mosasaur taxa but are developed to a lesser extent (e.g. Mosasaurus, Clidastes, Platecarpus and Ectenosaurus), but in no other taxa have pervasive extrinsic fibres been reported. We interpret these osteohistological features in T. proriger and E. sternbergi as evidence of tendinous attachment of extensive and highly differentiated axial musculature capable of producing great stresses, most likely related to stabilization of the trunk relative to contralateral movements of the tail during carangiform locomotion. We also report the compactness indices (percentage of space occupied by bone rather than cavities) for these large mosasaur ribs, which are much higher than previously reported. This suggests high intraspecific variation in bone compactness that complicates its use in reconstructing mosasaur palaeoecology.     

Decrease in light diffraction intensity of contracting muscle fibres

Single fibres from the semitendinosus muscle of frog were illuminated normally with a He–Ne laser. The intensity transient and fine structure pattern of light diffracted from the fibre undergoing isometric twitches were measured. During fibre shortening, the intensity decreased rapidly and the fine structure pattern preserved its shape and moved swiftly away from the undiffracted laser beam. The fine structure patterns of the contracting and resting fibre were nearly identical. The ratio of intensities of the contracting and resting fibre of the same sarcomere length was determined as a function of the time elapsed after fibre stimulation. The time-resolved intensity ratio increased with sarcomere length and became unity when sarcomere length was between 3.5 m and 3.7 m. A diffraction theory based on the sarcomere unit was developed. It contained a parameter describing the strength of filament interaction. The comparison between the theory and data shows that the initial intensity drop during contraction is primarily due to filament interactions. At a later stage of contraction, sarcomere disorder becomes the major component causing the intensity to decrease. Diffraction models which use the Debye-Waller formalism to explain the intensity decrease are discussed. The sarcomere-unit diffraction model is applied to previously reported intensity measurements from active fibres.     

Properties of the vertebrate skeletal muscle tubular system as a sealed compartment

Confocal imaging of impermeant fluorescent dyes trapped in the tubular (t-) system of skeletal muscle fibres of rat and cane toad was used to examine changes in the morphology of the t-system upon mechanical skinning, the time course of dye loss from the sealed t-system in mechanically skinned fibres and the influence of rapid application and removal of glycerol on the morphology of the sealed t-system. In contrast to intact fibres, which have a t-system open to the outside, the sealed t-system of toad mechanically skinned fibres consistently displayed local swellings (vesicles). The occurrence of vesicles in the sealed t-system of rat-skinned fibres was infrequent. Application and removal of 200-400 mM glycerol to the sealed t-system did not produce any obvious changes in its morphology. The dyes fluo-3, fura-2 and Oregon green 488 were lost from the sealed t-system of toad fibres at different rates suggesting that the mechanism of organic anion transport across the tubular wall was not by indiscriminate bulk transport. The rate of fluo-3 and fura-2 loss from the sealed t-system of rat fibres was greater in rat than in toad fibres and could be explained by differences in surface area: volume ratio of the t-system in the two fibre types. Based on the results presented here and on other results from this laboratory, an explanation is given for the formation of numerous vesicles in toad-skinned fibres and lack of vesicle formation in rat-skinned fibres. This explanation can also help with better understanding the mechanism responsible for vacuole formation in intact fibres.     

Electron microscopic immunocytochemical demonstration of separate vasotocinergic and mesotocinergic nerve fibres in the median eminence of the frog hypophysis

Summary With the use of the unlabelled antibody peroxidase-antiperoxidase complex (PAP) technique at the electron microscopic level, it was shown that both the internal and the external regions of the median eminence of the frog contain separate vasotocinergic and mesotocinergic nerve fibres. This observation confirms the results of previous immunocytochemical studies at the light microscopic level. The mean size of the neurohypophysial hormone-containing granules in the external region of the median eminence is significantly smaller than that of the neurohypophysial hormone-containing granules in the internal region of the median eminence. No significant difference could be found between the mean granule size of the vasotocinergic and mesotocinergic fibres of either the internal or the external region of the frog median eminence.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

AMP-deaminase in elasmobranch fish: a comparative histochemical and enzymatic study

AMP-deaminase activity was measured in white muscle from a wide range of fish, including one cyclostome, 13 chondrosteans, and one teleost to elucidate the pattern of the AMP-deaminase activity in white muscle of fish. Compared to a mammalian (rat) muscle extract, low enzyme activities are found in the cyclostome and two elasmobranchs from two families (Scyliorhinidae, Hexanchidae). In contrast, higher AMP-deaminase activities, similar to mammals, are expressed in Squalidae, all families of skates, Chimaeridae and in the teleostean fish. We then compared AMP-deaminase activities in red and white muscles from two representative elasmobranch fish, the dogfish (Scyliorhinus canicula) and the thornback ray (Raja clavata). The fibre type composition and distribution of the locomotory musculature were determined in these two elasmobranchs to establish a relationship between the morphology, the type of fibres of the locomotion-implicated muscles and the AMP-deaminase activity. Experimental data are discussed with respect to the layout of fibres in the myotome. In both species, three fibre types were identified. In the two fish myotomes, most of the axial muscles are white fibres while red fibres constitute a thin sheet. Some differences were observed between the two species in the distribution of intermediate fibres: in dogfish, these are located between the red and white fibres; in thornback ray, some are dispersed within the white fibre region, while others form an intermediary layer like in dogfish. These results suggest that in the course of evolution, an amplification of the AMP-deaminase activity in muscle was coupled with increase of complexity of the muscular structure.     

Evaluation of polysulfone hollow fibres and ceramic suction samplers as devices for the in situ extraction of soil solution

An investigation was carried out to assess the potential of using polysulfone hollow fibres for the extraction of soil solution. In comparison to ceramic suction samplers the fibres were shown to contain very low levels of potential contaminants and a low exchange capacity. Carry over between individual samples was negligible permitting the accurate monitoring of temporal changes in solution chemistry. Their flexible and root sized nature makes them ideal for sampling small soil volumes. Some concern however remains about retention of colloidal Fe at the fibre interface. When comparing the soil solution of samples extracted with polysulfone fibres with those extracted by ceramic suction samplers, no significant differences in solute concentrations were observed. No sample contamination resulting from dissolution of Al or Si from the ceramic cup was detected. Analysis of soil solutions collected over a number of extractions, and during and after a rainfall event showed concentrations of elements in solution to remain relatively constant with time.     

Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis

Summary Immuno-enzyme cytochemical investigations showed that the whole amphibian pars intermedia of the hypophysis is innervated by an intercellular network of peptidergic varicose nerve fibres which contain mesotocin or (and) parts of the mesotocin molecule. The pars intermedia does not contain vasotocinergic fibres. The mesotocinergic fibres are branches of axons leaving the pituitary stalk and the neural lobe. In animals of which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive mesotocinergic fibres of the pars intermedia had totally disappeared. From this result, it is concluded that the mesotocinergic fibres of the pars intermedia of the amphibian hypophysis are axons of neurosecretory perikarya located in the hypothalamic magnocellular neurosecretory preoptic nuclei.Dedicated to Professor Berta Scharrer on the accasion of her 70th birthdayThis investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

The fine structure of striated muscle fibres of tunica muscularis of the intestine in some teleosts

Summary The muscular layer of the fish oesophagus is made up exclusively of striated muscles, which form a circular and a longitudinal layer. The intestinal muscular layer of the tench, which is an exception among the vertebrates in this respect, consists of two layers of striated muscles and a layer of smooth ones. The organization of striated muscle fibres is described from the muscular layer of the alimentary canal in four species: the tench, crucian carp, gudgeon and stone-loach. The fibres of the oesophagus differ from each other in diameter, length of sarcomeres, glycogen content and, in the case of one species, organization of SR. On the basis of the organization of sarcomeres all the fibres examined must be counted among the slow-contracting ones (broad Z line). The sarcoplasmic reticulum is organized according to the Z type except for the fibres of the oesophageal muscles in which the SR represents the A-I type. Two types, en grappe and en plaque, of nerve endings have been observed. The presence of relatively large dense core vesicles seems to indicate their adrenergic origin.This work has been supported by the Polish Academy Sciences.     

The effect of a growth promoting drug,clenbuterol, on fibre frequency and area in hind limb muscles from young male rats

The effect of dietary administration of clenbuterol on soleus and extensor digitorum longus muscles was studied after 4 and 21 days. Both muscles showed an increase in wet weight with no significant change in total fibre number. After 4 days fibre cross-sectional areas were increased in soleus, but not in extensor digitorum longus, and after 21 days there was a change in fibre frequencies in extensor digitorum longus but not soleus muscles.     

On the anisotropy and inhomogeneity of permeability in articular cartilage

Articular cartilage is known to be anisotropic and inhomogeneous because of its microstructure. In particular, its elastic properties are influenced by the arrangement of the collagen fibres, which are orthogonal to the bone-cartilage interface in the deep zone, randomly oriented in the middle zone, and parallel to the surface in the superficial zone. In past studies, cartilage permeability has been related directly to the orientation of the glycosaminoglycan chains attached to the proteoglycans which constitute the tissue matrix. These studies predicted permeability to be isotropic in the undeformed configuration, and anisotropic under compression. They neglected tissue anisotropy caused by the collagen network. However, magnetic resonance studies suggest that fluid flow is "directed" by collagen fibres in biological tissues. Therefore, the aim of this study was to express the permeability of cartilage accounting for the microstructural anisotropy and inhomogeneity caused by the collagen fibres. Permeability is predicted to be anisotropic and inhomogeneous, independent of the state of strain, which is consistent with the morphology of the tissue. Looking at the local anisotropy of permeability, we may infer that the arrangement of the collagen fibre network plays an important role in directing fluid flow to optimise tissue functioning.     

Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway

We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.