Adjacent Filaments Research Articles

Regulation of neurofilament interactions in vitro by natural and synthetic polypeptides sharing Lys-Ser-Pro sequences with the heavy neurofilament subunit NF-H: neurofilament crossbridging by antiparallel sidearm overlapping.

Neurofilaments are organised into parallel bundles in axons through crossbridges formed by lateral projections of neurofilament subunits. Pure neurofilaments form gels in vitro, consisting of interconnected parallel arrays of filaments regulated by the phosphorylation level of neurofilament subunits. Neurofilament-associated polypeptides sharing phosphorylated epitopes with the repetitive lysine-serine-proline (Lys-Ser-Pro) motifs of the neurofilament heavy subunit sidearm are characterised: they regulate in vitro the neurofilament gelation kinetics in a concentration- and phosphorylation-dependent manner. Studies with synthetic peptides show that interactions between neurofilaments involve both acid and base amino acid residues of neurofilament sidearms and demonstrate the opposite effects of peptides containing either one (inhibition) or two (activation) Lys-Ser-Pro motifs. Electron microscopy reveals an organised network of native neurofilament sidearms, regulated by the phosphorylation level of neurofilament subunits, suggesting a structural transition between intra- and inter-neurofilament sidearm interactions. These results favour the hypothesis of a mechanism of neurofilament crossbridging through the variable antiparallel overlapping of the phosphorylable Lys-Ser-Pro domains of neurofilament sidearms from adjacent filaments, following an equilibrium regulated by neurofilament-associated proteins, bivalent cations and the phosphorylation level of Lys-Ser-Pro motifs from both neurofilament sidearms and neurofilament-associated proteins.

Giardia agilis: Ultrastructure of the Trophozoites in the Frog Intestine

Intestine samples of Bufo sp. tadpoles with parasitism confirmed for Giardia agilis were studied by transmission electron microscopy. The G. agilis trophozoites were long and thin. The plasma membrane was sometimes undulated and the cytoplasm, adjacent to the dorsal and ventral regions, showed numerous vacuoles. The two nuclei presented prominent nucleoli. The cytoplasm was electron-dense with free ribosomes, glycogen and rough endoplasmic reticulum-like structures. Polyhedral inclusions were observed in the cytoplasm and outside the protozoan; some of these inclusions exhibited membrane disruption. The flagella ultrastructure is typical, with the caudal pair accompanied by the funis. Next to the anterior pair, osmiophilic material was noticed. The ventro-lateral flange was short and thick, supported by the marginal plates that penetrated into its distal extremity; only its distal portion had adjacent osmiophilic filament. The G. agilis trophozoites showed the general subcellular feature of the genus. However, the ventro-lateral flange ultrastructure was an intermediate type between G. muris and G. duodenalis.

Major binding sites for the nuclear import receptor are the internal nucleoporin Nup153 and the adjacent nuclear filament protein Tpr.

A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin alpha and beta in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin alpha, beta, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin beta binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin beta and to importin alpha/beta heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin beta is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant beta or beta45-462 fragment freely exchanges with the endogenous importin beta/Nup153 complex, but cannot displace endogenous importin beta from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153- and Tpr-importin beta complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153- and Tpr-importin beta complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin beta. Models for the roles of these interactions are discussed.

Remarkable nectaries: structure, ecology, organophyletic perspectives I. Substitutive nectaries

In the present, first part of a series of studies on floral and extrafloral nectaries (N, Ns) that deserve interest from the structural, taxonomic, evolutionary and ecological points of view, the nuptial Ns found in members of five families are examined. They are considered to be substitutive, having evolved secondarily in subgroups that have lost another, non-homologous type of N common in their intrafamilial affinity, or that occur within an alliance that lacks floral Ns, but is presumably derived from an ancestry bearing a non-homologous type of N. The subtribe Genistinae (Fabaceae) is characterized by a closed filament tube and suppression of the nectariferous intrastaminal disk. In consequence, typically only pollen serves as a reward for pollinators. Chamaecytisus, Petteria, and Retama, to which Erinacea and Spartocytisus are added, are known to be exceptions. Field data, including behaviour of pollinators (mainly bumblebee queens), prove that they possess true nectar flowers. The site of nectar production was unknown. In the two cases examined, the liquid was found to flow out of two lateral external non-stomatic pore fields of the filament tube. As no glandular tissue is developed, the nectar probably represents phloem sap from leaking adjacent filament bundles. The taxa involved apparently regained nectar production in this distinctive manner in partly independent lineages. The perigynous disk of non-related Stylosanthes (Fabaceae) is obliterated owing to extreme elongation of the receptacle, which forms an empty “exposition tube”. Compensating this deficiency, apparently non-homologous nectar glands are seated at the tube's distal end in several species, accessible for short-tongued bees. Other species of the genus and species of related Arachis (with receptacular tubes up to 8 cm long) have closed filament tubes and no nectar. Complementing the author's previous results, atypical Ns occurring in the Primulaceae are described. In apetalous Glaux, single glandular trichomes scattered over the ovary produce the nectar, conforming to the tribe Lysimachieae, which displays (besides elaoiphores) only trichomatous Ns derived from hydathodes. Dionysia and Coris, on the other hand, share the mesenchymatous carpellary gland type of tribe Primuleae, a presumably more recent acquisition. Contrary to former belief, nectariferous Myrsinaceae do exist (Maesa). Further examples of trichomatous Ns, replacing the stomatal mesenchymatous Ns predominating in the Cucurbitaceae, are shown in species of Sicyos, Microsechium and Sechium (tribe Sicyeae), Echinocystis, Elateriopsis (Cyclanthereae), and six genera of the subfamily Zanonioideae. Trichomatous Ns, anatomically homologous to hydathode hairs, prove to be a constant feature of the three clades. While retaining a primitive level in the zanonioid members, Ns of the two other taxa exhibit parallel trends towards enlargement of glandular heads, concentration to dense carpets and internalization leading to euphily of the flowers. Concealment reaches its extreme in sect. Frantzia of Sechium, with nectariferous carpets enclosed inside ten chambers of the inflated torus. Although probably rooting in an ancestry bearing receptacular Ns, the Melastomataceae typically possess dry pollen flowers. However, as known from Neotropical members of five tribes, a sugary solution may be discharged through spontaneous ruptures of the filaments. Since nectarial tissue is absent, the fluid stems from the leaking filament bundle rich in phloem. This unique manner of nectar production was apparently archieved in independent, parallel progression lines, based on a common propensity. In addition to previous reports, novel cases are examined in species of Brachyotum, Tibouchina, Centronia and Miconia. Nectariferous fissures in most cases develop ventrally, loosely connected with the filament's prior curvature (geniculum) in the bud stage, but are independent and dorsal in Centronia. Nectaring of the petal tips in the flower bud of Medinilla magnifica, extranuptial in function, is unrelated to the foregoing situation. In this case, it rests on stomatal Ns with a pattern homologous to hydathodes seated on the margin of the foliage leaves. Some species of Sambucus, a genus basically devoid of nuptial Ns, have secondarily achieved this attractant in two different ways. Apart from the better known case of S. ebulus with a nectaring stigma, in S. javanica and relatives, numerous large cuplike nectariferous glands are distributed between the (dry) florets of the corymb, where they are exploited by insects. Hitherto taken for transformed flowers, the glands are evidently homologous to leaf-borne extranuptial Ns that occur in the same species and are widespread in the genus.

Functional Organization of Intrinsic Gill Muscles in Zebra Mussels, Dreissena polymorpha (Mollusca: Bivalvia), and Response to Transmitters in Vitro

Functional Organization of Intrinsic Gill Muscles in Zebra Mussels, Dreissena polymorpha (Mollusca: Bivalvia), and Response to Transmitters in Vitro

Novel Gill-Derived Gland in the Male Swordtail Characin, Corynopoma riisei (Teleostei: Characidae: Glandulocaudinae)

early stages of maturation. The gland appears to develop from the anterior 813 gill filaments of the first gill arches. The gill secondary lamellae shorten such that their only remnants in most of the mature gill gland are capillaries at the bases of the columnar cells that line the chambers. The edges of adjacent gill filaments must fuse to form the chambers that open into the gill cavity through antero-ventrally oriented apertures. Skeletal muscle and cartilage from the original gill filaments remain within the chamber walls. Degree of expansion and secretory activity of the glands varied among the 11 mature males. Based on histological characteristics of the gland, its location, and the complex courtship behavior of this species, we hypothesize that the gill gland is producing some type of female attractant (pheromone).

Cytoskeletal differences between stereocilia of the human sperm passageway and microvilli/stereocilia in other locations.

Stereocilia of the human ductus epididymidis and ductus deferens display unique features in that they arise from an apical cell protrusion (hillock) and contain thick stem portions which are interconnected by cytoplasmic bridges. The molecular basis for this unique fusion and branching pattern is hitherto unknown. These morphologic specialties led us to study the cytoskeleton of male spermway stereocilia with respect to the major proteins that constitute the supportive cytoskeleton of intestinal microvilli and inner ear stereocilia. Samples of the human epididymidis and ductus deferens were studied by immunoblotting and immunocytochemistry at the light and electron microscope levels. Spermway stereocilia are supported by an internal actin filament bundle crosslinked by fimbrin and associated with the membrane linker molecule ezrin. The stem portions and hillock area are supplied with the crossbridge forming molecule alpha-actinin. Spermway stereocilia differ from brush border microvilli of the intestine, kidney, and ductuli efferentes by the lack of the second bundling protein villin and the unusual expression of alpha-actinin in the stem region. They resemble inner ear stereocilia by the presence of fimbrin and absence of villin, but differ from them by expression of ezrin and alpha-actinin. Thus, the main molecular difference between spermway stereocilia and stereocilia/microvilli of other locations is the presence of alpha-actinin in their stem portion and the hillock area. Since alpha-actinin can form crossbridges between adjacent actin filaments (bundles) at longer distances than the other crosslinker of the stereocilium core bundle, fimbrin, we assume that alpha-actinin is essential for both the formation of the stem portions of spermway stereocilia and for the generation of their striking branching pattern. A developmentally regulated temporal sequence of expression of fimbrin and alpha-actinin might control the unique architecture of spermway stereocilia.

F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling.

Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product).

Immunocytochemistry of the muscle cell cytoskeleton

The muscle cell cytoskeleton is defined for this review as any structure or protein primarily involved in linking or connecting protein filaments to each other or to anchoring sites. In striated muscle, the M line connects thick filaments at their centers to adjacent thick filaments. Titin forms elastic filaments that extend from the M line to the Z line and may contribute to the resting tension properties of striated muscle. Nebulin forms inextensible filaments in skeletal muscle that are closely associated with thin filaments and that may provide a length template for thin filaments. Z lines anchor thin filaments from adjacent sarcomeres via the actin-binding function of alpha-actinin. Other proteins located at the Z line include Cap Z, Z-nin, Z protein, and zeugmatin. Intermediate filaments connect myofibrils to each other at the level of the Z line and to the sarcolemma at the Z- and possibly the M-line levels. Immunolocalization has identified the adhesion plaque proteins spectrin, vinculin, dystrophin, ankyrin, and talin at subsarcolemmal sites where they may be involved with filament attachment. Smooth muscle cell cytoskeletons are believed to include membrane associated dense bodies (MADBs), intermediate filaments, cytoplasmic dense bodies (CDBs), and perhaps a subset of actin filaments. MADBs contain a menu of attachment plaque proteins and anchor both thin filaments and intermediate filaments to the sarcolemma. CDBs are intracellular analogs of striated muscle Z lines and anchor thin filaments and intermediate filaments.

Nerve terminals of mucous gland electroreceptors in the platypus ( Ornithorhynchus anatinus )

Platypus mucous gland electroreceptors differ from electroreceptors described for fish in that they lack an associated specialized sensory cell. Thus a bare nerve terminal is used to detect electrical stimuli, and also to generate local and action potentials. Previous studies have identified these terminals (an average of 16 per mucous gland), but had not shown whether the terminals have direct contact with the duct of the mucous gland. This poses the problem of how the electrical stimulus reaches the nerve terminals. This study demonstrates the portions of the nerve terminals responsible for electroreception, and shows how these portions use the surrounding epidermal tissue to overcome the combined problems of lacking a sensory cell and making physical contact with the conducting medium in the duct of the gland. A terminal axonal filament is described which accommodates for these problems, the terminal filament provides a low-resistance pathway for the electrical stimuli, and is embedded with its proximal and distal portions in high and low resistance epidermis, respectively. Lateral interactions occur between adjacent terminal filaments via a plexus that is directed circumferentially around the duct from the proximal portion of the terminal filament. These circumferential arbors form an interconnecting ring between all 16 terminal filaments, and may be used to lower the signal-to-noise ratio of the electroreceptor and thus enhance overall sensitivity.

Gill ultrastructure and symbiotic bacteria in Codakia orbicularis (Bivalvia, Lucinidae)

The cellular organization of the gill, which harbors symbiotic bacteria, is described in juveniles and adults of Codakia orbicularis, a large tropical Lucinidae. The ciliary zone is similar in every species of Lucinidae described and includes the large clear cell which has been previously described as an intermediary cell. The intermediary zone is composed of a few narrow unciliated cells, which bind adjacent filaments together and constitute channels through which sea water circulates along the abfrontal part of the filaments. The lateral zone is more complex in C. orbicularis than in other Lucinidae, being composed of four cell types and differentiated into two distinct regions. The bacteriocytes and intercalary cells occupy the outermost bacteriocyte zone, while mucocytes and numerous cells crowded with proteinic, cystine-rich granules constitute the innermost secretory zone which has not been described in other species. The newly described granule cells are considered to be a key factor in the storage and metabolic conversion of sulfur compounds.

Mechanism of ATP hydrolysis by polymeric actin

The lag between polymerization of actin and ATP hydrolysis in actin filaments was analyzed in terms of the mechanism of the hydrolysis reaction. Under the experimental conditions (100 mM KCI and 1 mM MgCI 2, or without KCI, 1 mM MgCI 2 and 0.4 mM EGTA, 25°C) ATP hydrolysis lagged behind polymerization by about 100 s independently of the concentration of polymerizing filament ends and of the actin monomer concentration. Three models of ATP hydrolysis were compared to experimental data: (i) Random ATP hydrolysis, ATP is assumed to be hydrolyzed at a rate that is independent of the type of nucleotide bound to adjacent filament subunits. (ii) Cooperative hydrolysis, the rate of ATP hydrolysis is thought to depend on the type of nucleotide bound to adjacent subunits. (iii) Sequential hydrolysis, ATP is assumed to be hydrolyzed only at the interface between ATP-subunits and ADP-subunits. The model of sequential ATP hydrolysis could be excluded. The results were in agreement with random or cooperative ATP hydrolysis. The differences of the rates of ATP hydrolysis by a random or cooperative mechanism are so small that based on the experimental results no distinction between these two mechanisms could be made. All available evidence points towards a mechanism of ATP hydrolysis in which several or perhaps many interfaces between ATP- and ADP-subunits are formed within a filament.

The nuclear pore complex: three-dimensional surface structure revealed by field emission, in-lens scanning electron microscopy, with underlying structure uncovered by proteolysis

The structure of the nuclear pore complex (NPC) has been previously studied by many different electron microscopic techniques. Recently, scanning electron microscopes have been developed that can visualise biologically relevant structural detail at the same level of resolution as transmission electron microscopes and have been used to study NPC structure. We have used such an instrument to visualise directly the structure of both cytoplasmic and nucleoplasmic surfaces of the NPC of manually isolated amphibian oocyte nuclear envelopes that have been spread, fixed, critical point dried and coated with a thin fine-grained film of chromium or tantalum. We present images that directly show features of the NPC that are visible at each surface, including coaxial rings, cytoplasmic particles, plug/spoke complexes and the nucleoplasmic basket or fishtrap. Some cytoplasmic particles are rod-shaped or possibly "T"-shaped, can be quite long structures extending into the cytoplasm and may be joined to the coaxial ring at a position between each subunit. Both coaxial rings, which are proud of the membranes, can be exposed by light proteolytic digestion, revealing eight equal subunits each of which may be bipartite. We have determined that the nucleoplasmic filaments that make up the baskets are attached to the outer periphery of the coaxial ring at a position between each of its subunits. These filaments extend into the nucleoplasm and insert at the distal end to the smaller basket ring. The space left between adjacent basket filaments would exclude particles bigger than about 25 nm, which is consistent with the exclusion limit previously found for NPC-transported molecules.

Organization of Actin Filaments in the Axial Rod of Abalone Sperm Revealed by Quick Freeze Technique: (actin filaments/abalone sperm/quick-freeze deep-etch/freeze-substitution/cytoskeleton).

An axial rod in abalone (Haliotis discus) sperm is a structure composed of a bundle of actin filaments, which elongates anteriorly to form the acrosomal process during the acrosome reaction. The ultrastructure of the actin filament bundle constituting the axial rod was examined using quick freeze technique followed by either freeze-substitution or deep-etch electron microscopy. Thin sections of quick freeze and freeze-substituted sperm revealed that the actin filaments in the axial rod are hexagonally packed in a paracrystalline array through its almost entire length with an average center-to-center spacing of 12 nm. Periodic transverse bands were also observed across the actin filament bundle, which may reflect the cross-bridges interconnecting the adjacent filaments. Quick-freeze deep-etch analysis provided the three-dimensional view of the axial rod. Actin filaments exhibiting 5.5-6 nm spaced striations were observed to run in parallel with each other inside the axial rod. The existence of cross-bridging structures was also displayed between adjacent filaments. These results suggest that the actin filaments in the axial rod are probably held together by regularly spaced cross-bridges to form a well ordered hexagonally packed bundle, and also cross-linked by fibrous structure to the lateral inner acrosomal membrane which closely surrounds the anterior half of the actin filament bundle.

Ultrastructure of Glial Cells and Extracellular Matrix in the Guinea Pig Lamina Cribrosa

The ultrastructure of glial cells and extracellular matrix that support normal guinea pig optic nerve axons at the lamina cribrosa was examined by conventional thin-section transmission electron microscopy and by the quick-freeze, deep-etch, rotary-shadow technique. Glial cell bodies and processes were identified by their characteristic parallel intermediate filaments that were closely packed with reduced cross-linking between adjacent filaments when compared with neighboring axons. Glial processes surrounded lamina cribrosa beams and separated axons from the beams. These beams contained bundles of collagen fibrils. Short regular cross-bridges spanned the spaces between the collagen fibrils much like rungs of a ladder. Surrounding the collagen bundles was a network-like peribeam extracellular matrix. This matrix extended to the basement membrane of the surrounding, stratified processes of glial cells. The glial basement membrane was connected to the glial plasma membrane by numerous orthogonal fibrous strands. In regions within basement membrane there also were cross-bridges from the glial plasma membrane to intermediate filaments within the cell. Apposed glial process membranes appeared to directly contact each other. Unmyelinated optic nerve axon membranes and glial process membranes also directly abutted each other. Overall, the cross-bridging structural organization was dense near collagen beams, and became progressively less dense toward the axon. This organization of the lamina cribrosa may play an important role in the distribution of tissue shear stress associated with intraocular pressure and thus protect the retinal ganglion cell axons as they pass through the lamina cribrosa.

Oxygen distribution in oxidatively stabilized mesophase pitch fiber

The oxygen profile along the radial direction in mesophase pitch fiber of 10 μm diameter, stabilized by heating at a constant rate of 0.5°C/min in air, was measured with secondary ion mass spectrometry (SIMS), by correcting the atomic O C signal ratio according to that obtained by elemental analysis. The oxygen profile was found to be flat along the radial direction in fibers oxidized to 350°C which showed the maximum weight gain among the stabilized fibers, providing the maximum tensile strength and elongation after the carbonization. When stabilization was stopped at a lower temperature of 250°C, the oxygen profile indicated an unoxidized core, from which volatile components may diffuse to the surface, causing sticking of adjacent filaments together in the successive carbonization. The heating rate during stabilization was also found to influence the oxygen distribution, more rapid heating tending to increase the gradient. Such a situation indicates that a balance of oxygen diffusion and oxidation reactivity governs the oxygen distribution in the stabilized fiber. Hence, SIMS can be an appropriate tool to monitor the stabilization step in carbon fiber manufacture.

Scanning Electron Microscopy and Pathogenicity ofChilodonella piscicola(Ciliophora) on Juvenile Salmonids

Abstract An ectoparasitic ciliate, Chilodonella piscicola (=C. cyprini), became epizootic and caused chronic mortalities in juvenile masu (cherry) salmon Oncorhynchus masou and pink salmon O. gorbuscha being reared at the Nemuro Hatchery, eastern Hokkaido, Japan, in the spring of 1988. Cumulative mortalities amounted to 20% in a masu salmon population over 5 months and 10% in a pink salmon population over 2 months. Transmission experiments in masu salmon fry confirmed that C. piscicola can cause severe proliferation of the gill epithelium in the absence of other stressors. The infected fish exhibited reduced growth and chronic mortalities after the mean intensity had reached a peak (619 parasites per fish) at week 4 postinfection. The marked epithelial hyperplasia was followed by intense fusion of adjacent gill lamellae and filaments. Respiratory failure due to gill epithelial hyperplasia may have been a primary cause of the deaths.

Scanning electron microscopic evaluation of effects of low pH on gills of Channa punctata (Bloch)

Bioassay of Channa punctata (± 36 g) was carried out in acidic waters of different pH. A 96 h LC50, value was obtained at pH 5.3. Scanning electron microscopy of gills of C. punctata showed fusion of adjacent secondary lamellae. At this low pH, dissociation of epithelium of branchial arches and gill filaments take place. At 360 h the branchial epithelium ruptures developing lesions in the gills exposing the efferent filament vessels. At pH 6.6 the tips of adjacent filaments belonging to both oral and aboral hemibranchs fuse in blocks.

Effects of chronic exposure to soft, acidic water on gill development and chloride cell numbers in embryo-larval brook trout, Salvelinus fontinalis

Recruitment failure is considered to be a major factor contributing to the decline of fish populations in soft, acidic waters; direct mortality of embryo-larval fishes has been postulated as a major cause of the decline. Little is understood of the physiological consequences to embryo-larval fishes of prolonged exposure to soft, acidic waters; however, dysfunction of respiratory and ionoregulatory processes is suspected. In order to evaluate the effects of acid exposure on the respiratory and ionoregulatory systems of developing brook trout, Salvelinus fontinalis, differences in gill morphology and numbers of chloride cells were compared between groups cf developing embryo-larval fish continuously exposed to moderately hard well water (130.0 mg.l−1 as CaCO3, pH 7.94) or to reconstituted soft, acidic water (4.4 mg.l−1 as CaCO3, pH 5.25) designed to mimic acidic waters of northern Wisconsin acidified lakes. Exposures were maintained for up to 48 days (82 days after fertilization) during critical periods of growth and differentiation of branchial structures. The second right gill arch of each fish was examined for changes in the development of filaments and lamellae and for differences in numbers of chloride cells. Gills of fish that developed in soft, acidic water contained greater numbers of normal and degenerating chloride cells, exhibited hyperplasia of primary epithelium and multiple fusions of adjacent filaments and lamellar epithelium than the gills of control fish. Filament and lamellar lengths and numbers of lamellae per filament were significantly less (P

Histone-DNA interactions and their modulation by phosphorylation of -Ser-Pro-X-Lys/Arg- motifs.

The sea urchin sperm-specific histones H1 and H2B are multiply phosphorylated in spermatids, dephosphorylated in the final stages of spermatogenesis to give mature sperm, and rephosphorylated upon fertilization. Phosphorylation in spermatids, and probably at fertilization, occurs at repeated -Ser-Pro-X-Basic-motifs in the distinctive N-terminal basic domains of both histones and at the end of the much longer C-terminal domain of H1. Here we identify the consequences of multiple phosphorylation through comparison of some physical and biochemical properties of spermatid (phosphorylated) and sperm (dephosphorylated) chromatin and histones. Study of the DNA binding properties of the intact histones and isolated basic domains suggests that phosphorylation at three dispersed sites in the C-terminal tail of H1 has little effect on its overall DNA binding affinity, whereas, strikingly, binding of the N-terminal domains of H2B and H1 is abolished by phosphorylation at four or six tandemly repeated sites respectively. Together with the relative timing of events in vivo, this suggests that phosphorylation/dephosphorylation of the N-terminal (and distal end of the C-terminal) tail of H1, and/or the N-terminal tail of H2B, effectively controls intermolecular interactions between adjacent chromatin filaments, and hence chromatin packing in the sperm nucleus.