Inositol Uptake and Metabolism in Molluscan Neuronal Tissue

Abstract: The uptake of myo -[³H]inositol into neurones from Lymnaea stagnalis has been demonstrated to be a sodium-dependent process, saturable with a K _m of approximately 50 μ M and shown to be linear with time for at least 120 min. The rate of transport of myo -inositol into the cell appears to influence directly its incorporation into neuronal lipids. Using anion-exchange high-performance liquid chromatography, we have demonstrated a high rate of breakdown of phosphatidylinositol 4,5–bisphosphate in Lymnaea nerve under basal conditions. Stimulation with carbamylcholine enhanced production of inositol 1–phosphate, inositol bisphosphate, inositol 1,4,5–trisphosphate, and inositol 1,3,4–trisphosphate. Formation of inositol tetrakisphosphate was not detected. Electrical stimulation also caused an increased formation of inositol phosphates. These results provide evidence for an active myo -inositol transport system in molluscan neurones and suggest that the hydrolysis of inositol lipids may play a role as an intracellular signalling system in this tissue.     

PCR-Based Detection of Angiostrongylus cantonensis in Tissue and Mucus Secretions from Molluscan Hosts

Angiostrongylus cantonensis is a common cause of human eosinophilic meningitis. Recent outbreaks of this infection have shown that there is a need to determine the distribution of this nematode in the environment in order to control transmission. A. cantonensis is generally identified morphologically in the molluscan intermediate host by microscopic examination, which can be labor-intensive. The aim of this study was to develop a PCR-based method to detect A. cantonensis directly from molluscan tissue. A total of 34 Parmarion cf. martensi (Simroth) semislugs, 25 of which were naturally infected with A. cantonensis, were used to develop this assay. Tissue pieces (approximately 25 mg) were digested with pepsin-HCl to recover third-stage larvae for morphological identification or were used for DNA extraction. PCR primers were designed to amplify 1,134 bp from the Angiostrongylus 18S rRNA gene, and the amplicons produced were sequenced for identification at the species level. Both microscopy and the PCR-DNA sequencing analysis indicated that the same 25 semislugs were positive for A. cantonensis, showing that the two methods were equally sensitive and specific for this application. However, morphological detection requires access to living mollusks, whereas molecular analysis can also be performed with frozen tissue. The PCR-DNA sequencing method was further evaluated using tissue from Veronicella cubensis (Pfeiffer) slugs and mucus secretions from infected P. martensi. To our knowledge, this is the first use of a PCR-based method to confirm the presence of A. cantonensis in mollusks collected in the environment.     

Metamorphosis of the Basidiomycota Ustilago maydis: Transformation of yeast-like cells into basidiocarps

Ustilago maydis (DC) Cda., a phytopathogenic Basidiomycota, is the causal agent of corn smut. During its life cycle U. maydis alternates between a yeast-like, haploid nonpathogenic stage, and a filamentous, dikaryotic pathogenic form that invades the plant and induces tumor formation. As all the members of the Subphylum Ustilaginomycotina, U. maydis is unable to form basidiocarps, instead it produces teliospores within the tumors that germinate forming a septate basidium (phragmobasidium). We have now established conditions allowing a completely different developmental program of U. maydis when grown on solid medium containing auxins in dual cultures with maize embryogenic calli. Under these conditions U. maydis forms large hemi-spheroidal structures with all the morphological and structural characteristics of gastroid-type basidiocarps. These basidiocarps are made of three distinct hyphal layers, the most internal of which (hymenium) contains non-septate basidia (holobasidia) from which four basidiospores develop. In basidiocarps meiosis and genetic recombination occur, and meiotic products (basidiospores) segregate in a Mendelian fashion. These results are evidence of sexual cycle completion of an Ustilaginomycotina in vitro, and the demonstration that, besides its quasi-obligate biotrophic pathogenic mode of life, U. maydis possesses the genetic program to form basidiocarps as occurs in saprophytic Basidiomycota species.     

Protein Changes in Regressing Tail Tissue of Xenopus laevis during Natural Metamorphosis

Sodium dodecyl sulfate (SDS) gel electrophoresis was used to study the soluble protein fraction of Xenopus laevis tail tissue during in vivo metamorphosis. Prior to morphological signs of tail regression stage 45, a new subunit protein was resolved. At stage 64 three additional subunit proteins were resolved at the end of tail resorption. Results indicate that the altered balance between protein synthesis and degradation has little effect on the protein subunit population prior to morphological signs of tail regression.     

Control of Tissue Specificity: The Pattern of Cellular Synthetic Activities in Tissue Transformation

During transformation of the dorsal marginal iris into lenstissue after removal of the lens in the adult newt, the cellsundergo gradual changes in synthetic activities. Autoradiographicdata indicate an enhancement of RNA synthesis in the nucleusof iris cells, which is followed by enhancement of protein synthesisand onset ot DNA synthesis. After discharge of pigment granulesand a period of cellular multiplication accompanied by ribosomeproduction, the cells stop DNA synthesis and start to show detectableamounts of lens-specific proteins (-,ß-, and -crystallins).This time coincides with initiation of differentiation of primarylens fibers. In the later stages of regeneration, - and ß-crystallinsare present in the dividing cells of the lens epithelium, aswell as in the cells of the fiber area, but -crystallins aredetected only in Che cells of the fiber area. The data wereinterpreted as suggesting that the gene utilization patterntypical for the iris is not directly converted into that forthe lens, but goes through intermediate patterns before thetissue transformation is completed.     

Metamorphosis in Tokophrya infusionum; an Electron-Microscope Study

SYNOPSIS. In Tokophrya infusionum metamorphosis from a ciliated swimming embryo to a sessile organism with a stalk, disc, and tentacles lasts only 3 minutes. The remarkable speed of meta-morphosis was clarified by an electron-microscope study of embryos before and during metamorphosis. Ultrathin sections have revealed that the embryo has at the anterior end of the body a number of specialized structures, such as dense bodies containing the precursor material for the disc and stalk, and microtubules which align the dense bodies into rows leading to pit-kite invaginations of the pellicle at the tip of the anterior end. At meta-morphosis the embryo settles down on this end and the precursor material is released thru the pits to the outside. At the same time the body of the embryo invaginates at this end, forming a cavity which becomes deeper and narrower until it acquires the shape of a channel. The 1st drops released from the dense bodies spread out on the substrate, forming the disc. The rest of the material, secreted into the channel, solidifies there to form the stalk. It seems obvious that the channel serves as a mold for the stalk, since after completion of the stalk the channel disappears. The stalk is structureless with no limiting membrane; it is outside the boundaries of the cell. Both the stalk and disc are extra-cellular organelles.
Of the new organelles appearing at metamorphosis, only the stalk and disc are formed de novo. The electron-microscope study disclosed that the embryo has internal parts of tentacles composed of a tube formed of microtubules. At the distal end of the microtubules is a ring of dense material. During metamorphosis the microtubules, together with the dense ring, grow out of the body, and along with them the pellicle and plasma membrane to form the external part of the tentacle.     

An X-Ray Diffraction Study of Contracting Molluscan Smooth Muscle

The living anterior byssus retractor muscle of Mytilus (ABRM), a smooth, “catch” muscle, has been studied by X-ray diffraction while relaxed and while tonically contracted. X-ray reflections were observed from the actin and paramyosin filaments and from the α-helical substructure of the paramyosin filaments. No differences in spacings or relative intensities were observed when the relaxed and contracting muscle patterns were compared. This result is consistent with a sliding filament mechanism involving an interaction between actin and paramyosin filaments.     

Mechanisms of Contraction in Molluscan Muscle

The muscles of molluscs show a wide variety of structural types,a variation that is paralleled by the behavior of the muscles.All these muscles have certain features in common; they containlong filaments of two types, but differ in the length and thicknessof the thick filaments and in the proportion of the protein,paramyosin, in the muscle. Structural and physiological experimentssuggest that the basic contractile mechanism is the same inall molluscan muscles. The muscle-relaxing mechanism, however,may be specialized, particularly in certain smooth muscles whichhave the very slow relaxation characteristic of "catch" muscles.     

Low Energy Defibrillation in Human Cardiac Tissue: A Simulation Study

We aim to assess the effectiveness of feedback-controlled resonant drift pacing as a method for low energy defibrillation. Antitachycardia pacing is the only low energy defibrillation approach to have gained clinical significance, but it is still suboptimal. Low energy defibrillation would avoid adverse side effects associated with high voltage shocks and allow the application of implantable cardioverter defibrillator (ICD) therapy, in cases where such therapy is not tolerated today. We present results of computer simulations of a bidomain model of cardiac tissue with human atrial ionic kinetics. Reentry was initiated and low energy shocks were applied with the same period as the reentry, using feedback to maintain resonance. We demonstrate that such stimulation can move the core of reentrant patterns, in the direction that depends on the location of the electrodes and the time delay in the feedback. Termination of reentry is achieved with shock strength one-order-of-magnitude weaker than in conventional single-shock defibrillation. We conclude that resonant drift pacing can terminate reentry at a fraction of the shock strength currently used for defibrillation and can potentially work where antitachycardia pacing fails, due to the feedback mechanisms. Success depends on a number of details that these numerical simulations have uncovered.     

Sudden Death in Infancy: A Study of Cardiac Specialized Tissue

The hearts from 15 cot death patients have been compared with those of 15 controls. In one of the cot death patients an accessory atrioventricular connexion known to be capable of producing ventricular pre-excitation and sudden death was identified. In all hearts examined additional segments of specialized tissue were found in relation to the atrioventricular orifices which may have been significant in producing pre-excitation. Haemorrhagic lesions were present in the atrial myocardium and conducting tissues of hearts from both groups, but no evidence was found of cellular degeneration in the atrioventricular bundle. There is a need for further studies of conducting tissues in sudden death syndromes.     

Molluscan hemocyanin: structure,evolution, and physiology

Most molluscs have blue blood because their respiratory molecule is hemocyanin, a type-3 copper-binding protein that turns blue upon oxygen binding. Molluscan hemocyanins are huge cylindrical multimeric glycoproteins that are found freely dissolved in the hemolymph. With molecular masses ranging from 3.3 to 13.5 MDa, molluscan hemocyanins are among the largest known proteins. They form decamers or multi-decamers of 330- to 550-kDa subunits comprising more than seven paralogous functional units. Based on the organization of functional domains, they assemble to form decamers, di-decamers, and tri-decamers. Their structure has been investigated using a combination of single particle electron cryo-microsopy of the entire structure and high-resolution X-ray crystallography of the functional unit, although, the one exception is squid hemocyanin for which a crystal structure analysis of the entire molecule has been carried out. In this review, we explain the molecular characteristics of molluscan hemocyanin mainly from the structural viewpoint, in which the structure of the functional unit, architecture of the huge cylindrical multimer, relationship between the composition of the functional unit and entire tertiary structure, and possible functions of the carbohydrates are introduced. We also discuss the evolutionary implications and physiological significance of molluscan hemocyanin.     

Molecular Evolution and Functionally Important Structures of Molluscan Dermatopontin: Implications for the Origins of Molluscan Shell Matrix Proteins

A major shell matrix protein originally obtained from a freshwater snail is a molluscan homologue of Dermatopontins, a group of Metazoan proteins also called TRAMP (tyrosine-rich acidic matrix protein). We sequenced and identified 14 molluscan homologues of Dermatopontin from eight snail species belonging to the order Basommatophora and Stylommatophora. The bassommatophoran Dermatopontins fell into three types, one is suggested to be a shell matrix protein and the others are proteins having more general functions based on gene expression analyses. N-glycosylation is inferred to be important for the function involved in shell calcification, because potential N-glycosylation sites were found exclusively in the Dermatopontins considered as shell matrix proteins. The stylommatophoran Dermatopontins fell into two types, also suggested to comprise a shell matrix protein and a protein having a more general function. Phylogenetic analyses using maximum likelihood and Bayesian methods revealed that gene duplication events occurred independently in both basommatophoran and stylommatophoran lineages. These results suggest that the dermatopontin genes were co-opted for molluscan calcification at least twice independently after the divergence of basommatophoran and stylommatophoran lineages, or more recently than we have expected. [Reviewing Editor: Dr. David Pollock]     

Cell Determination and Organogenesis in Molluscan Development: A Reappraisal Based on Deletion Experiments in Ilyanassa

The vegetal region of the Ilyanassa egg, incorporated into thepolar lobe and segregated to the D blastomere, appears to havean organizing influence in development. The results of deletingindividual micromeres of the first three quartets, and the 4dcell, indicate that most of these cells play rather distinctivedevelopmental roles. Both cytoplasmic segregation and embryonicinduction are believed to be involved in determining the fateof cells during development in Ilyanassa. Comparisons are madewith other forms.     

Ontogeny of the Molluscan Shell Field: a Review

In the gastropod, scaphopod, lamellibranch, and cephalopod gastrulae a thickened portion of the posttrochal region is referred to as the embryonic shell field. It invaginates and gives rise to the shell gland. In species with an at least temporarily external shell, the shell gland evaginates and again forms a shell field. In lamellibranchs, the shell field grows into two halves connected by the ligament-secreting isthmus. In polyplacophorans plate fields are produced without invagination. Slugs and endocochleate cephalopods overgrow the embryonic shell field to form an internal shell sac. The calcified part of the shell is secreted by the flattened central region. The periostracum has its origin in the permanently thickened peripheral region of the shell field. In many forms, this region is depressed in a periostracal groove. If the shell is external, the central region of flattened cells, the mantle roof, along with the two or three marginal folds of the free mantle edge and, in species with internal shell, the shell sac are parts of the mantle. The shell field descends from the first somatoblasts. Either of 2 d or 2 c alone is able to form the shell field. There are arguments that the formation of the embryonic shell field is not autonomic, but induced by the entoderm during a period of contact. The shell gland and the shell field grow by mitotic cell divisions. Cells secreting organic material are highly prismatic, have a well developed ergastoplasm and large dictyosornes, and contain much peroxidase. The secretion of calcium manifests itself in very flat cells, rich in alkaline phosphatase and glycogen. The shell gland and the rosette of ectocochleate conchifera together are homologous to the proximal part of the shell sac in slugs and endocochleate cephalopods.