Peptidergic regulation of the Limulus midgut

1. The morphology and innervation of the midgut (intestine) in the horseshoe crab, Limulus polyphemus was investigated. The organization of this tissue was examined with routine histology. Radioimmunoassay, immunohistochemistry and high performance liquid chromatography were employed to detect, localize and identify peptidergic innervation of the midgut. The actions of synthetic and native proctolin-like and FMRFamide-like peptides were compared on the isolated midgut preparation. 2. Levels of proctolin and FMRFamide were determined in extracts of Limulus midgut tissue using radioimmunoassay. High levels of proctolin-like immunoreactivity (69.5 +/- 11.3 ng/g) were detected, while levels of FMRFamide-like immunoreactivity (0.8 +/- 0.2 ng/g) were less. Proctolin levels were equally distributed, while the levels of FMRFamide-like immunoreactivity exhibited an anterior bias. 3. Proctolin- and FMRFamide-like immunoreactivities in the Limulus midgut were localized with immunohistochemistry. Proctolin- and FMRFamide-immunoreactive elements were detected in intestinal nerve branches and individual fibers running along the surface of the midgut in whole-mount preparations. In sectioned tissue, staining for these peptides was observed throughout the midgut, typically associated with muscle bands and fibers. Only a few immunoreactive cell bodies were observed. 4. Proctolin, and several FMRFamide-like peptides produced distinct and opposing actions on the isolated Limulus midgut preparation. Proctolin elicited contracture and rhythmic contractions of this tissue, while FMRFamide and N-terminally extended analogs of FLRFamide relaxed gut tension. FMRFamide-like peptides partially reversed the excitatory actions of proctolin. 5. Proctolin- and FMRFamide-like peptides in Limulus midgut extracts were partially characterized with high performance liquid chromatography. One peak of proctolin-like activity was detected on a linear gradient of 18 to 31.5% acetonitrile. The native proctolin-like peptide produced excitatory actions on the isolated midgut preparation which were indistinguishable from those produced by synthetic proctolin. Several peaks of FMRFamide-like bioactivity (Busycon radula protractor muscle assay) were detected with a linear gradient of 5 to 30% acetonitrile. Fractions from two distinct peaks produced FMRFamide-like inhibitory effects on the isolated Limulus midgut preparation. These findings suggest a role for proctolin-like and FMRFamide-like peptides as regulators of intestinal motility in Limulus.     

Novel DNA structures resulting from dTam3 excision in tobacco

A Tam3 two-element system has been designed by combining an immobilized Tam3 element with a non-autonomous dTam3 element inserted into the HPT gene. The phenotypic assay employed, restored hygromycin resistance, indicated thattrans-activation of the non-autonomous dTam3 element occurred. Molecular analyses of the excision sites revealed that the ends of the dTam3 element remain in the empty donor sites. The predominant consequence of this type of excision appears to be that excised fragments fail to re-integrate into the tobacco genome. Only one case of dTam3 re-integration could be detected. The ends of this element had been degraded upon integration into the tobacco genome. Either the altered structure of the Tam3 derivatives or tobacco host factors are influencing thetrans-activation of a dTam3 element, resulting in aberrant excision.     

Decrease in insulin-containing secretory granules and mitochondrial gene expression in mouse pancreatic islets maintained in culture following streptozotocin exposure

We have previously described a preferential reduction in the secretory response to nutrient secretagogues in pancreatic mouse islets maintained in culture after in vitro exposure to streptozotocin (SZ). This reduction was associated with an impaired substrate metabolism at the mitochondrial level. To further clarify this issue, mouse pancreatic islets were exposed in vitro to 2.2 mM SZ for 30 min. At 4 h after SZ treatment ultrastructural changes were apparent in the endoplasmic reticulum and Golgi areas of the B-cells. However, 2 and 6 days following SZ exposure the B-cells appeared well preserved, except for a marked decrease in the number of insulin-containing secretory granules. A morphometric analysis of the B-cells 6 days after SZ exposure showed a normal B-cell size and a normal volume fraction of B-cell mitochondria. However, there was a decrease in total islet size and a 13% decrease in the volume fraction of B-cells in the islets. These mouse islets exhibited a decreased content of the mitochondrial DNA-encoded cytochrome b mRNA, as evaluated by dot-blot analysis. As a whole, the data obtained indicate that SZ treatment does not induce a decrease in the number of mitochondria or long-lasting ultrastructural damage to this organelle. However, there is a clear decrease in the cytochrome b mRNA, suggesting that SZ can induce damage to the mitochondrial DNA.     

The duplication in Charcot-Marie-Tooth disease type 1a spans at least 1100 kb on chromosome 17p11.2

Summary Recently, it has been shown that Charcot-Marie-Tooth disease type 1a (CMT1a) is linked with a duplication of a DNA segment that is detected by probe VAW409R3, and that is located on chromosome 17p11.2. Here, we show that this duplication also contains VAW412R3a, but not A10-41 and EW503. Accounting for the duplication in recombination analysis, we found recombinants between CMT1a and EW301 and EW502, but not with A10-41, VAW409R3, and VAW412R3. Using pulsed-field gel electrophoresis analysis, we estimated the minimal size of the duplicated region in CMT1a patients to be 1100 kb.     

Ultrastructure of the frontal organ in Convoluta and Macrostomum spp.: significance for models of the turbellarian archetype

Present models of turbellarian evolution depict the organism with a frontal organ — a complex of glands whose necks emerge at the anterior tip of the body — and therefore imply that this organ is homologous throughout the Turbellaria. However, comparisons of representatives of the Acoela and Macrostomida, two putatively primitive orders of the Turbellaria, show that frontal organs in these two are not similar in ultrastructure or histochemistry. The acoel Convoluta pulchra had a prominent cluster of frontal mucous glands whose necks emerged together in a frontal pore at the exact apical pole of the organism, and an array of smaller glands of at least five other types opened at the anterior end, separately from and ventral to this pore. The frontal organs (Stirndrüsen) of two species of Macrostomum on the other hand, comprised an array of discretely emerging necks of at least two gland types including one with rhabdiform (rhammite) and one with globular mucous secretion granules neither of which emerge at the apical pole. In neither species did the organ appear to be sensory. Our findings indicate a low probability of homology between the frontal glands of the Acoela and Macrostomida.     

Neuropeptides in free-living and parasitic flatworms (Platyhelminthes). An immunocytochemical study

The nervous systems of the turbellarians Microstomum lineare and Polycelis nigra and of the cestodes Diphyllobothrium dendriticum and Schistocephalus solidus were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the neuropeptides FMRF-amide, vasotocin, leu-enkephalin, met-enkephalin, neurotensin, somatostatin, and VIP, and to the bioamine serotonin. Anti-FMRF-amide positive perikarya and fibers occurred in all species, while the occurrence of the vertebrate brain-gut peptides and serotonin varied between the species. Anti-somatostatin and anti-VIP gave a negative result. Anti-FMRF-amide and anti-vasotocin positive immunoreactivity was found in the brain and gut of M. lineare, and in the CNS and the peripheral nerve net of the cestodes. We suggest that the brain-gut peptides of free-living flatworms act on the subtegumental region in the cestodes, which lack a gut but absorb their nutrients directly through the tegument.     

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.     

Immunocytochemical demonstration of neuropeptides and serotonin in the tapeworm Diphyllobothrium dendriticum

Summary The present immunocytochemical study concerns the distribution of four neuropeptides, FMRF-amide, vasotocin, leu-enkephalin and neurotensin, and of the bioamine serotonin in the plerocercoid larva of Diphyllobothrium dendriticum. Anti-FMRF-amide and vasotocin-reactivity occurs in perikarya and nerve fibres in the CNS and PNS of this worm. The peptide-containing fibres surround and seem to innervate the musculature and to terminate beneath the basal lamina of the tegument at the inner surface of the bothridia, suggesting a neurotransmitter function. Antileu-enkephalin reaction occurs in perikarya and fibres in the main nerve cords and in the PNS. Anti-neurotensin reactive fibres were observed in the neuropile of the nerve cords. Serotonin immunoreactivity was found in neurons in the ganglionic commissure of the brain and along the main nerve cords. This study is the first immunocytochemical identification of neuropeptides and serotonin in a parasitic flatworm and the information gained may be of importance for the development of new antihelminthics.     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Boar sperm surface glycoproteins: Isolation,localization, and temporal expression during spermatogenesis

Boar sperm glycoprotein fractions were isolated by Lens culinaris hemagglutinin affinity chromatography of detergent-solubilized ejaculated spermatozoa, followed by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In order to develop methods for further investigations of the sperm proteins, we proceeded with two of the isolated glycoproteins. Antibodies were raised in female rabbits against each of the two sperm glycoproteins. By a combination of immunosorbent chromatography, using the antibodies obtained, and preparative SDS polyacrylamide gel electrophoresis, highly purified sperm proteins were isolated. The sperm proteins were immobilized on Sepharose gel columns and specific immunoglobulin Fab fragments were enriched by affinity chromatography. The specificity of the Fab fragments was ascertained by immunoprecipitation analysis. The Fab fragments were used in indirect immunofluorescence analysis to localize the corresponding antigens on the surface of boar spermatozoa. Both antigens were exclusively confined to the postacrosomal region. Immunohistochemical staining of boar testis sections revealed that both antigens are expressed from the spermatid stage. This technique also revealed that one of the antigens congregated at the Golgi complex-acrosome region during spermatogenesis.