Oncospheral hook morphogenesis in the davaineid cestode Inermicapsifer madagascariensis (Davaine, 1870) Baer, 1956

The fine structural characteristics of oncospheral hook morphogenesis in the davaineid cestode Inermicapsifer madagascariensis are described. The primordia of the hooks appear in the oncoblasts of embryos in the advanced stage of development, in which there is a greatly reduced number of blastomeres that exhibit a bilaterally symmetrical pattern in their organization.

The hook primordium, adjacent to the invaginated part of the nuclear envelope, is surrounded by an abundance of free ribosomes, mitochondrial aggregations and extended Golgi regions. Simultaneously with its elongation and transformation into a blade, the hook primordium material becomes differentiated to form an electron dense cortex and a less dense, inner, crystal-like core. At the beginning of shank formation, the blade of the hook protrudes outside the oncoblast. The membrane-enclosed point of exit of the blade is surrounded by a cytoplasmic sheath which later forms a circular, septate desmosome.

With oncoblast degeneration, muscle fibres attach directly to the collar and the base of the hook.     

Functional ultrastructure of the hexacanth larvae in the bothriocephalidean cestode Eubothrium salvelini (Schrank, 1790) and its phylogenetic implications

Functional ultrastructure and its phylogenic implications in the bothriocephalid cestode Eubothrium salvelini (Schrank, 1790) are described and discussed. The infective hexacanth shows bilateral symmetry in cellular organization. The mature hexacanth is armed with three pairs of oncospheral hooks of a heterogeneous electron density. It is covered by a thin layer of the oncospheral tegument, possessing characteristic bubble-like processes at the surface. Within the infective hexacanth larva five cell types were distinguished: (1) a binucleated subtegumental cell; (2) the U-shaped, tetranucleated penetration gland; (3) two nerve cells; (4) three types of somatic cells represented by: i) myocytons of both somatic and hook musculature, ii) numerous degenerating micromeres with pycnotic nuclei and iii) a new oncospheral cell type, the interstitial cell, that has never been observed in any other hexacanth; and (5) large germinative cells with characteristic prominent nucleoli in their large spherical nuclei. Functions of all the cell types are described on the basis of the obtained ultrastructural characteristics and previously published reports. The mode of the penetration gland secretion is classified as apocrine. Flame cells have never been observed within the hexacanth of E. salvelini. The results of the present study, comparing the functional aspects of the ultrastructure of the hexacanths of E. salvelini with literature data on the oncospheres of other bothriocephallideans and diphyllobothriideans, suggest potential phylogenetic and evolutionary criteria for determining relationships among these groups of tapeworms.     

INCREASE IN OSMIOPHILIA OF AXONAL MEMBRANES OF CRAYFISH AS A RESULT OF ELECTRICAL STIMULATION, ASPHYXIA, OR TREATMENT WITH REDUCING AGENTS

Certain axonal membranes of crayfish abdominal nerve cord display ultrastructural changes if the axons are fixed, during electrical stimulation, by aldehydes followed by osmium. Such changes are characterized by an increase in electron opacity and thickness of the unit membranes' dense strata in the axon surface, endoplasmic reticulum, and outer mitochondrial membranes. The electron opacity completely disappears if the sections are treated with hydrogen peroxide solutions. This suggests that the changes represent an increase in the membranes' reactivity for osmium. An unmasking of SH groups could explain such increased osmiophilia, since SH groups are very reactive with osmium, while disulfide bonds are considerably less reactive. This hypothesis was tested by treating control, glutaraldehydefixed nerve cords with disulfide reducing agents. In these preparations an increase in electron opacity and thickness was observed to be localized in the same axonal membranes which reacted as a result of electrical stimulation. The phenomenon did not appear if the SH groups were blocked by maleimide or N-ethylmaleimide before treatment with osmium. These findings seem to suggest that certain axonal membranes of crayfish contain proteins rich in sulfur whose SH groups are unmasked as a result of electrical stimulation. In preliminary experiments an increase in osmiophilia localized in the same membranes with the same characteristics and distribution was observed also in axons from nerve cords asphyxiated either in vitro or in the living animal.     

Biochemical aspects of the visual process. XXIII. Sulfhydryl groups and rhodopsin photolysis

1. The number of exposed sulfhydryl groups in cattle rod photoreceptor membranes has been determined in suspension and after solubilization in various detergents both before and after illumination.2. In suspensions, two sulfhydryl groups are modified per mole of rhodopsin, both by Ellman's reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, while no extra SH groups are uncovered upon illumination. Neither reagent affects the spectral integrity of rhodopsin at 500 nm and the recombination capacity is retained upon modification of both rhodopsin and opsin.3. However, in detergents (digitonin, Triton X-100 and cetyltrimethylammonium bromide (CTAB)) 2–3 additional sulfhydryl groups appear upon illumination, in agreement with earlier reports.4. A total number of six sulfhydryl groups and two disulfide bridges are found in rod photoreceptor membranes, expressed per mole of rhodopsin.5. DTNB reacts somewhat faster with membrane suspensions after than before illumination. The less reactive sulfhydryl modifying agents O-methylisourea and methyl-p-nitrobenzene sulfonate show a similar behavior.6. It is concluded that illumination of rhodopsin in vivo will not uncover additional SH groups, although the reactivity of one exposed SH group may increase somewhat. These findings also exclude a role of SH groups in the covalent binding of the chromophore.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

Ultrastructure of oncospheral envelopes in Hymenolepidids (Cestoda) with aquatic life cycles

The ultrastructure of the oncospheral envelopes of five species of hymenolepidid cestodes, namely, Fimbriaria fasciolaris, Dicranotaenia coronula, Sobolevicanthus gracilis, Diorchis elisae and Diorchis ovofurcata are described. These cestodes are parasites of aquatic hosts and utilise aquatic invertebrates as intermediate hosts. The ultrastructure of the oncospheral envelopes was essentially similar among these species. However, the structure of the components of the inner oncospheral envelopes displayed significant structural variation among the five species examined. The small eggs of D. coronula and S. gracilis have a thin embryophore and oncospheral membrane. The eggs of these species settle at the bottom of the water body and are eaten by benthic crustaceans. Eggs of F. fasciolaris, D. elisae and D. ovofurcata have thick multilayered embryophores with specific ornamentation. Moreover, in Diorchis species the eggs are elongate, and the oncospheral membrane is thick and striated. Zones of electron-dense aggregates penetrate the polar filaments in these species. Eggs of F. fasciolaris float passively in water whereas those of Diorchis species are attached by long filaments to aquatic plants and are eaten by crustaceans in the littoral zone. The interrelationships among the ultrastructure of the oncospheral envelopes, cestode life cycles and habitat of crustacean intermediate hosts are drawn and discussed.     

Echinococcus granulosus: Hook-muscle systems and cellular organisation of infective oncospheres

The morphology of the oncospheres of Echinococcus granulosus has been reconstructed from thin and semi-thin serial sections. Four major types of oncospheral cells have been identified. These consist of: (1) a bi-nucleate medullary center, (2) glandular regions (eight nuclei) subdivided into three types of oncospheral glands, (3) ten germinative cells, and (4) 34 muscle cells, of which 16 are somatic and 18 are hook muscle cells. The hook muscle cells of each hook are organized functionally into the three following systems: (1) the protraction system, for hook extension, (2) the abduction system for drawing the hooks together toward the median plane of bilateral symmetry, and (3) the retraction system for pulling the hooks back into the body. The interconnections observed between different muscle fibers provide a structural basis for coordinated hook action.     

Oligomycin sensitivity of mitochondrial sulfhydryl groups

Dithionitrobenzoate has been used to titrate sulfhydryl groups of rat liver mitochondria in glutamate buffer, pH 7.4.Reaction with oligomycin and different SH reagents preceded the SH titration. Under these conditions it was found that 2-mercaptopropionylglycine and N-ethylmaleimide reacted in an oligomycin-sensitive manner, so that the control values (in the absence of SH reagent) were obtained.Similar concentrations of mersalyl and of N-(N-acetyl-4-sulfamoylphenyl) maleimide, in the presence of oligomycin, enhanced reactivity toward Nbs₂.The concentration range of oligomycin-sensitive SH groups was thus defined between approx. 5 and 9 nmol reagent/mg mitochondrial protein.In this way, a differentiation between SH groups, which are implicated in phosphate transport and those, which react in an oligomycin-sensitive manner, and which are probably connected with the coupling mechanism, was achieved.     

Biochemical aspects of the visual process XXVIII. Classification of sulfhydryl groups in rhodopsin and other photoreceptor membrane proteins

Reaction of isolated bovine rod outer segment membrane with radioactiveN-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule.On the basis of their reactivity towardsp-chloromercuribenzoate andp-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with thep-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and is probably located in the vicinity of the chromophoric center.The difference between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) orN-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of thep-chloromercuri-derivatives.     

Protein-bound sulfhydryl groups and thiolesters in mitochondria and submitochondrial particles and their relationships to oxidative phosphorylation

Procedures are described for measurement of total protein-bound —SH groups and apparent thiolester linkages in mitochondrial preparations. The procedure for total —SH is based on reaction with radioactive p-mercuribenzoate in 6 m guanidine hydrochloride at pH 9 containing 1 m ammonia buffer. The procedure for apparent thiolesters is based on alkylation of available — SH groups in 6 m guanidine hydrochloride, protein precipitation with perchloric acid, and ammonolysis at pH 9 to liberate — SH from any thiolester present.No variations in total —SH groups measured in Submitochondrial particles or in mitochondria were noted with presence or absence of ATP, ADP, substrates, or uncouplers of oxidative phosphorylation. Previous reports of changes in measured — SH groups thus appear to reflect solely changes in the reactivity of — SH groups and not in the total — SH groups present.Submitochondrial particles contained less than 0.5 nmole of apparent thiolester per mg of protein; mitochondria contained 1 to 2 nmoles. No variation of apparent thiolester content was noted with change in conditions affecting oxidative phosphorylation. The data do not, of course, eliminate possible changes in thiolester content below the sensitivity of the detection procedures.     

Antibodies to rat sperm tail polypeptides recognize Sertoli cell secretory proteins

We have previously reported that (a) polyclonal antisera raised against rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein recognize outer dense fiber polypeptides from rat sperm tail, and (b) protein S70 is antigenically related to polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein. We now report that polyclonal antisera generated against three different outer dense fiber polypeptides recognize (a) the putative antigen of the sperm tail and (b) Sertoli cell secretory protein S70 and its antigenically-related polypeptides. Immunogold electron microscopy shows that outer dense fibers of epididymal sperm crossreact with anti-S70 serum as well as with an antiserum raised against the polypeptide D complex of extracted outer dense fibers. Electron microscopy demonstrates that outer dense fibers consist of filamentous, coil-coiled units aligned side-by-side with each other. Results of this study strengthen the antigenic homology between Sertoli cell secretory proteins and outer dense fiber polypeptides of the sperm tail.     

The Structure of Myelin Sheaths in the Central Nervous System of Xenopus laevis (Daudin)

The structure of myelinated nerve fibres has been studied in the spinal cord and optic nerve of the tadpoles of Xenopus laevis. Potassium permanganate-fixed material was examined with the electron microscope. The myelin sheath itself is made up of spirally arranged lamellae in which the intraperiod and dense lines alternate. Inside the myelin sheath an inner cytoplasmic process surrounds the axon and where the external surfaces of its bounding membrane come together an internal mesaxon is formed. The intraperiod line begins within the mesaxon and the dense line usually begins in the same region by apposition of the cytoplasmic surfaces of the membrane. The width of each lamella is 140 A. The outer line in the sheath is the dense line and this terminates in a tongue where the cytoplasmic surfaces of the myelin-forming glial cell separate. Thus, central myelin in Xenopus tadpoles is arranged in the same way as peripheral myelin, the only difference being that in central fibres, cytoplasm on the outside of the sheath is confined to that present in the tongue. For this reason adjacent central sheaths come into apposition without any intervening material being present. When this occurs an intraperiod line is formed between them.     

Sand rhizosheath of an arid zone grass

Summary The root sheath of the arid zone grassZygochloa paradoxa comprises a dense cylinder of sand grains held mainly by short twisted root hairs. The sand sheath and hypodermal sleeve enclose a thick cortex with a sclerenchymatous inner zone and a partially disintegrated aerenchymatous outer zone. The outer tube comprising sheath and hypodermal sleeve is largely impermeable to water and its primary function is probably to insulate the stele against moisture loss during translocation.     

Development of the Embryonic Envelopes of Mesocestoides lineatus (Cestoda: Cyclophyllidea)

Summary

Transmission electron microscopy revealed that the eggs of Mesocestoides lineatus consisted of an oncosphere larva surrounded by various coverings. The outermost of these was the embryonic capsule, which appeared as a thin electron-dense membranous sac. The capsule enclosed inner and outer embryonic envelopes, each of which was syncytial and apparently formed from embryonic blastomeres. The envelopes became increasingly vesiculated during embryogenesis, and were attached to each other by desmosomes by the time the larva was fully formed. An electron-dense intracellular embryophore was produced by the inner envelope; it first appeared under the distal plasma membrane as a series of blocks, which grew and fused to form a thick unbroken layer. Early in development, the proximal plasma membrane of the inner envelope was connected to the larval epithelium by a multilaminate membrane complex that was ultrastructurally similar to a continuous junction. At the end of embryogenesis, this appeared to detach from its formative cells on both sides to form the distinctive oncospheral membrane. Several eggs were bound together in clusters by a cluster capsule that was ultrastructurally identical to the individual embryonic capsules. This type of egg packaging has not been described previously for any cestode. Both the cluster and individual capsules broke down by the end of embryogenesis.     

Reactivity of the sulphydryl groups of the mitochondrial phosphate carrier

The rate of reaction of - SH groups of the mitochondrial phosphate carrier with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) and N-ethylmaleimide (MalNEt) was followed by measuring the inhibition of phosphate transport. The changes in the rate of reaction caused by alterations of the ionic composition of the matrix were compared with changes of the total intramitochondrial phosphate content, the intramitochondrial K+ content and the value of intramitochondrial pH. The ionic composition was manipulated by addition of valinomycin to non-respiring or to respiring mitochondria and by addition of inorganic phosphate to respiring and non-respiring mitochondria. From all these variables it was the changes of the intramitochondrial pH which correlated with the - SH group reactivity. Internal acidification decreased and internal alkalinization increased the rate of reaction of mitochondrial phosphate carrier with both Nbs2 and MalNEt. Nbs2 did not penetrate the inner mitochondrial membrane as assayed by determination of the acid-soluble thiol content of the matrix. From this fact it follows that the Nbs2-reactive SH groups of the carrier were accessible from the outer surface of the inner membrane in our experiments. It is concluded that intramitochondrial pH modifies the reactivity of the externally oriented - SH groups indirectly. A hypothesis is presented according to which protonation and deprotonation of the carrier molecule on the inner side could induce a conformational change of the whole protein altering also the microenvironment of the - SH groups near the opposite surface.     

双子叶植物幼苗顶端弯钩发育的分子机制

幼苗顶端弯钩的形成是双子叶植物暗形态建成过程中的一个重要事件。它保护双子叶植物幼嫩的子叶和脆弱的顶端分生组织在幼苗出土时免受机械损伤,进而保证幼苗的出苗率和成活率。幼苗顶端弯钩的形成是由于下胚轴顶端的两个对立面之间细胞分裂和延伸的不对称所引起的。目前,关于双子叶植物幼苗顶端弯钩发育分子机制的研究已有较大进展：发现生长素在顶端弯钩内外侧组织的梯度分布是顶端弯钩两侧细胞差异生长的重要驱动力;乙烯、赤霉素和油菜素内酯促进顶端弯钩的形成和维持;茉莉酸抑制顶端弯钩的形成;而光照则促进弯钩的打开;无弯钩1 (hookless1, HLS1)、乙烯不敏感3以及EIN3 相似蛋白1 (ethylene insensitive 3 /EIN3 like 1, EIN3/EIL1)、DELLA、组成型光形态发生1(constitutive photomorphogenic 1, COP1) 和光敏色素相互作用因子(phytochrome interacting factors, PIFs) 等多种因子已被发现参与顶端弯钩的发育过程,并介导了多种激素之间的互作。本文综述了双子叶植物幼苗顶端弯钩发育过程中的重要作用因子及调控网络,并对该领域的研究前景进行了展望。     

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacteriumGloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1–2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen.Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.     

Succinate and phenylsuccinate as modifiers of sulfhydryl groups of inner mitochondrial membrane protein. Study by EPR

Paramagnetic labels specific for sulfhydryl (SH) groups have been used to study the conformational changes of the inner mitochondrial membrane. The EPR spectra of the SH-groups spin-labeled with maleimide or iodoacetamide show the existence of two populations of sulfhydryl groups, differing in their mobility (one weakly, the other strongly immobilized). The incubation with succinate or phenylsuccinate decreased the binding of these labels of the weakly immobilized sites while the number of total SH groups was the same before and after the incubation. These results suggest that succinate or phenylsuccinate induce a reversible change in protein conformation or in protein arrangement within the inner mitochondrial membrane. This change is concomitant to the protein movement between inner membrane and perimembranal space induced by either of these two molecules.     

Mechanism of cisplatin nephrotoxicity

cis-Diamminedichloroplatinum II (cisplatin) is widely used in cancer treatments. Renal dysfunction is the major toxic effect of this drug. Micropuncture studies suggest that cisplatin reduces single-nephron glomerular filtration rate (GFR) and causes a significant backleak of inulin across the renal tubule. Pathological alterations are localized to the S3 segment of the proximal tubule situated in the outer stripe of the outer medulla. Renal clearance studies in humans demonstrate that the free platinum clearance exceeds the GFR, which suggests that cisplatin or a platinum metabolite is actively secreted by the kidney. Studies with renal cortex slices indicate that platinum is accumulated by renal tissue against a concentration gradient. This uptake is blocked by metabolic inhibitors and the organic base triethanolamine. Heavy metals are thought to produce renal damage because of interaction with renal sulfhydryl (SH) groups. After cisplatin administration to rats, total renal SH groups decreased by 14% owing to a decrease of protein-bound SH groups. The greatest decline of SH groups occurred in the mitochondrial and cytosolic fractions. These fractions also had the highest platinum concentrations. These results suggest that the nephrotoxic effects of cisplatin may be related to depletion of SH groups, but a cause and effect relationship has not been definitively established.     

Phosphate transport protein of rat heart mitochondria: location of its SH-groups and exploration of their environment

(1) The properties of the SH groups of the phosphate transport protein of rat heart mitochondria were investigated on the basis of inhibition caused by SH reagents under different conditions. (2) The essential thiol groups are located near the external surface, as they are accessible to impermeable reagents from the external space. (3) The environment of the sulfhydryl groups influences their reactivity, as alteration of the external pH affects adversely their reactions with ionizable and non-ionizable SH reagents. (4) Intramitochondrial pH exerts a transmembrane effect: alkalinization augments and acidification diminishes the reaction rate of the sulfhydryl groups on the opposite surface of the membrane. (5) Changes of the concentration of the transported substrate occurring exclusively in the extramitochondrial space do not influence the reactivity of the essential SH groups. (6) It is concluded that in transport studies the phosphate transport protein of heart and liver mitochondria show basic similarity. It is suggested that the amino-acid sequence around the NEM-reactive cysteine (i.e., Lys-41 - Cys-42 - Arg-43) does not participate in substrate binding.