Immunohistochemical Differentiation of Fiber Types in Human Skeletal Muscle Using Monoclonal Antibodies to Slow and Fast Isoforms of Troponin I Subunit

The cDNA sequence of troponin I (TnI), one of the subunits of the skeletal muscle regulatory protein, differs between slow-twitch muscle and fast-twitch muscle. We prepared monoclonal antibodies td the slow and fast isoforms of human TnI for the purpose of differentiating muscle fiber types in human neuromuscular disorders. Slow TnI antibody was labeled with tetramethylrhodamine isothiocyanate while fast TnI antibody was labeled with fluorescein isothiocyanate; then these two antibodies were mixed. This mixture was then used to stain biopsied muscle from patients with neuromuscular disorders. It was possible to differentiate muscle fibers into slow, fast and intermediate fibers having various contents of slow and fast TnI. In tissue composed of small muscle fibers, this method facilitated differentiation of types of muscle fibers by allowing staining of only a single section. The usefulness of our technique using slow and fast TnI antibodies is discussed in comparison with ATPase staining. Because our staining method can distinguish slow and fast fiber components, it is useful for clinical application.     

High resolution respirometry of permeabilized skeletal muscle fibers in the diagnosis of neuromuscular disorders

High resolution respirometry in combination with the skinned fiber technique offers the possibility to study mitochondrial function routinely in small amounts of human muscle. During a period of 2 years, we investigated mitochondrial function in skeletal muscle tissue of 13 patients (average age = 5.8 years). In all of them, an open muscle biopsy was performed for diagnosis of their neuromuscular disorder. Mitochondrial oxidation rates were measured with a highly sensitive respirometer. Multiple substrate-inhibitor titration was applied for investigation of mitochondrial function. About 50 mg fibers were sufficient to obtain maximal respiratory rates for seven different substrates (pyruvate/malate, glutamate/malate, octanoylcarnitine/malate, palmitoylcarnitine /malate, succinate, durochinol and ascorbate/TMPD). Decreased respiration rates with reference to the wet weight of the permeabilized fiber could immediately be detected during the course of measurements.In 4 patients with mitochondrial encephalomyopathy (MEM) the respiration pattern indicated a specific mitochondrial enzyme defect, which was confirmed in every patient by measurements of the individual enzymes (one patient with PDHC deficiency, one with complex I deficiency and two patients with combined complex I and IV deficiency). In the 6 patients with spinal muscular atrophy (SMA) oxidation rates were found to be decreased to 23 ± 5% of controls. The normalized respiration pattern was comparable to that of the controls indicating a decreased content of mitochondria in SMA muscle with normal functional properties. Also in the 3 patients with Duchenne muscular dystrophy (DMD) decreased oxidation rates (42 ± 5%) were detected. In addition a low RCI (1.2) indicated a loose coupling of oxidative phosphorylation in the mitochondria of these patients.It is concluded that investigation of mitochondrial function in saponin skinned muscle fibers using high resolution respirometry in combination with multiple substrate titration offers a valuable tool for evaluation of mitochondrial alterations in muscle biopsies of children suffering from neuromuscular disorders. (Mol Cell Biochem 174: 71–78, 1997)     

脊髓背角痛觉传递和调制的一些化学解剖学观察

本实验研究了脊髓背角内Ｃ纤维末梢的分布和突触学特征及其一些神经递质化学构筑;定量观察了急性痛引起背角的递质变化;显示了初级传入Ｃ纤维,抑制性中间神经元和背角伤害性感受神经元三者之间的突触关系,并探讨它们在痛觉信息传递和调制中的作用。     

Participation of Syndecan 2 in the Induction of Stress Fiber Formation in Cooperation with Integrin α5β1: Structural Characteristics of Heparan Sulfate Chains with Avidity to COOH-Terminal Heparin-Binding Domain of Fibronectin

The present study provides direct evidence that syndecan 2 participates selectively in the induction of stress fiber formation in cooperation with integrin α5β1 through specific binding of its heparan sulfate side chains to the fibronectin substrate. Our previous study with Lewis lung carcinoma-derived P29 cells demonstrated that the cell surface heparan sulfate proteoglycan, which binds to fibronectin, is syndecan 2 (N. Itano et al., 1996, Biochem. J. 315, 925–930). We here report that in vitro treatment of the cells by antisense oligonucleotide for syndecan 2 resulted in a failure to form stress fibers on fibronectin substrate in association with specific suppression of its cell surface expression. Instead, localization of actin filaments in the cytoplasmic cortex occurred. A similar response of the cells was observed when the cells were treated to eliminate functions of cell surface heparan sulfates, including exogenous addition of heparin and pretreatment with anti-heparan sulfate antibody, F58-10E4, and with proteinase-free heparitinase I. Size- and structure-defined oligosaccharides prepared from heparin and chemically modified heparins were utilized as competitive inhibitors to examine the structural characteristics of the cell surface heparan sulfates involved in organization of the actin cytoskeleton. Their affinity chromatography on a column linked with a recombinant H-271 peptide containing a C-terminal heparin-binding domain of fibronectin demonstrated that 2-O-sulfated iduronates were essential for the binding. Inhibition studies revealed that a heparin-derived dodecasaccharide sample enriched with an IdoA(2OS)–GlcNS(6OS) disaccharide completely blocked binding of the syndecan 2 ectodomain to immobilized H-271 peptide. Finally, the dodecasaccharide sample was shown to inhibit stress fiber formation, triggered by adhesion of P29 cells to a CH-271 polypeptide consisting of both the RGD cell-binding and the C-terminal heparin-binding domains of fibronectin in a fused form. All these results consistently suggest that syndecan 2 proteoglycan interacts with the C-terminal heparin-binding domain of fibronectin at the highly sulfated cluster(s), such as [IdoA(2OS)–GlcNS(6OS)]₆ present in its heparan sulfate chains, to result in the induction of stress fiber formation in cooperation with integrin α5β1.     

Enhanced stiffness of silk‐like fibers by loop formation in the corona leads to stronger gels

We study the self‐assembly of protein polymers consisting of a silk‐like block flanked by two hydrophilic blocks, with a cysteine residue attached to the C‐terminal end. The silk blocks self‐assemble to form fibers while the hydrophilic blocks form a stabilizing corona. Entanglement of the fibers leads to the formation of hydrogels. Under oxidizing conditions the cysteine residues form disulfide bridges, effectively connecting two corona chains at their ends to form a loop. We find that this leads to a significant increase in the elastic modulus of the gels. Using atomic force microscopy, we show that this stiffening is due to an increase of the persistence length of the fibers. Self‐consistent‐field calculations indicate a slight decrease of the lateral pressure in the corona upon loop formation. We argue that this small decrease in the repulsive interactions affects the stacking of the silk‐like blocks in the core, resulting in a more rigid fiber.     

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.     

Ultrastructural changes in granule cell somata and mossy fibers of the rat hippocampus during picrotoxin-induced convulsions

Summary Ultrastructural changes in hippocampal granule cells, mossy fibers and mossy fiber boutons were examined following the administration of picrotoxin in adult rats. Generalized seizures occurred within 5–10 min after the intraperitoneal injection of picrotoxin. The electron-microscopic examination of hippocampal tissues from rats that had been perfused with fixative during the seizure revealed that the large dense-core vesicles increased in number and accumulated on the presynaptic membranes of mossy fiber boutons; some of these vesicles appeared to be fused with the membranes, and omega-shaped exocytotic profiles were frequently seen. Furthermore, greatly increased numbers of coated vesicles (60–90 nm in diameter) were observed on the maturing faces of Golgi fields of granule cells. Thus, our study not only indicates an increased incidence of exocytosis of large dense-core vesicles during picrotoxin-induced seizures, but also suggests that these vesicles are replaced in excess from the perikaryon of the granule cell.     

Fluoreszenzmikroskopischer Nachweis biogener Monoamine in der Epiphysis cerebri von Rana esculenta und Rana pipiens

Zusammenfassung Die Epiphysis cerebri von Rana pipiens und Rana esculenta wurde fluoreszenzmikroskopisch auf das Vorhandensein bestimmter biogener Amine untersucht. Unter normalen Bedingungen lassen sich im Parenchym der Froschepiphyse keine Amine fluoreszenzmikroskopisch darstellen. Nach Vorbehandlung der Tiere mit dem Monoaminoxydase-Hemmer Nialamid ist aber eine intensive, durch Formaldehyd-Behandlung induzierte Gelbfluoreszenz in den Sinnes- und Stützzellen zu beobachten. Mikrospektrofluorometrische Messungen zeigen, daß das Fluorophor mit 5-Hydroxytryptamin identisch ist, obwohl das Vorkommen von anderen verwandten Indolen — z.B. 5-Hydroxytryptophan und Melatonin — nicht ausgeschlossen werden kann. Grünfluoreszierende adrenerge Nervenfasern sind im meningealen Hüllbindegewebe der Epiphyse zu erkennen; einige dieser Fasern scheinen auch in die Epiphyse einzudringen.Der Nachweis von 5-Hydroxytryptamin wird im Zusammenhang mit der Frage einer Melatoninsynthese diskutiert. Das 5-Hydroxytryptamin könnte außerdem in einer funktionellen Beziehung zu einem bisher noch unbekannten Protein- der Polypeptid-Hormon des Epiphysenparenchyms stehen.

---

Fluorescence microscopic studies of biogenic amines in the pineal organ of Rana esculenta and Rana pipiens

Summary The pineal organ of Rana pipiens and Rana esculenta was studied by fluorescence microscopy for the histochemical demonstration of certain biogenic monoamines. Under normal conditions, no fluorogenic amines were visible in the organ. After pretreatment of the animal with a monoamine-oxidase inhibitor, nialamide, an intense yellow formaldehyde-induced fluorescence appeared both in the sensory cells and in the supporting cells. Microspectrofluorometric analysis indicated that the fluorophore is identical with 5-hydroxytryptamine; the presence of other closely related indoles, such as 5-hydroxytryptophan and melatonin, however, cannot be excluded. Fluorescent adrenergic nerves were found in the connective tissues surrounding the pineal organ; fluorescent fibers were observed also in the pineal parenchyma.The presence of 5-hydroxytryptamine in the anuran pineal organ is discussed with regard to the role that the amine plays in melatonin synthesis and with regard to a possible functional relation to some as yet unidentified protein- or polypeptid-hormone within the pineal parenchyma.

Finanzierung eines Studienaufenthaltes an der Universität Lund durch die Deutsche Forschungsgemeinschaft (Forschungsvorhaben — Ok 1/15 — von Prof. Dr. A. Oksche u.a.).     

Zur funktionellen Bedeutung der osmiophilen Granula in Herzorganen niederer Vertebraten

Zusammenfassung Elektronenmikroskopische Untersuchungen am Herzen niederer Vertebraten (Frosch, Forelle, Goldfisch) und am Lymphherzen des Frosches zeigen, daß osmiophile intrazelluläre Granula (800–1300 Å) als Ausdruck sekretorischer Funktionen der Herzmuskelzellen bei verschiedenen poikilothermen Vertebraten vorkommen. — Innerhalb der Granulaansammlungen in Muskelzellen des Froschventrikels werden neben Dictyosomen, Vesikeln und Multivesikulärkörpern besondere Multivesikulärkörper mit Dichtekern gefunden. Übereinstimmende vesikuläre Substruktur dieses Dichtekerns und der spezifischen Granula sowie der Übergangsformen ergeben eine morphogenetische Reihe: Golgiapparat Multivesikulärkörper Multivesikulärkörper mit Dichtekern spezifisches Granulum. — Zwischen den Muskelzellen des Rana-Herzens wird wie bei Cyclostomen ein Zelltyp mit osmiophilen Granula gefunden, die in Größe und Struktur auch den Granula in den terminalen adrenergen Nervenfasern des Froschherzens entsprechen.Vieltägige Isolation des Froschherzens unter physiologischen Bedingungen sowie Inkubierung oder längere Behandlung isolierter Forschherzen mit Reserpin führt zum Abbau der spezifischen Granula. Oxypertin und Acetylcholin haben nur geringen Einfluß auf deren Größe, Struktur und Zahl. L-Dopa und Adrenalin wirken einer isolationsbedingten Reduktion der Granula entgegen. — Fluoreszenzmikroskopisch kann am Rana-Herzen intramuskulär eine auf Catecholamine hindeutende punktartige Gelbgrün-Fluoreszenz nachgewiesen werden. Nach Rückgang des Granulagehaltes durch Reserpin zeigt das isolierte Froschherz eine Sensibilisierung für Acetylcholin und Adrenalin.Es ist wahrscheinlich, daß die spezifischen Granula in den Herzorganen der untersuchten Arten (wie die entsprechenden Strukturen im Cyclostomenherzen) Catecholamine speichern, die für eine gleichbleibende Erregbarkeit der Muskelfasern von Bedeutung sein könnten.

---

Function of the osmiophilic granules in the heart-organs of lower vertebratesA comparative electron microscopic and pharmacological study on the isolated heart of frog and other poikilothermic vertebrates

Summary Electron microscopic studies of the hearts of lower vertebrates (frog, trout, goldfish) and of the lymph-heart (frog) show that osmiophilic intracellular granules probably are related to secretory functions of cardiac muscle cells in poikilothermic vertebrates. — Beside dictyosomes, vesicles, and usual multivesicular bodies, a special type of multivesicular body with a dense core is found among the granules in the muscle cells of the frog's ventricle. The vesicular substructure of this dense core, and the specific granules as well as the intermediate forms suggest the following morphogenetic process: Golgisystem multivesicular body multivesicular body with a dense core specific granule. Between the muscle cells of the heart of Rana, as well as with Cyclostomata, a cell type with osmiophilic granules can be found corresponding in size and structure to the adrenergic terminals of the frog's heart.Many days' isolation of the frog's heart under physiological conditions, as well as incubation, or longer treatment of isolated hearts of frogs with reserpine leads to the dissolution of the specific granules. Oxypertin and acetylcholine have only little influence on the size, structure, and number of granules. L-Dopa and adrenalin appose a reduction of granules caused by isolation. Fluorescence microscopic results show a point-like yellow-green fluorescence in the heart of Rana, pointing to an accumulation of catecholamines intramuscularly.— After reduction of the content of granules by reserpine, the isolated frog's heart shows a higher sensitivity to acetylcholine and adrenalin.It is, therefore, probable that the specific granules store catecholamines in the heart organs of the investigated species in the same way as corresponding structures do in the hearts of Cyclostomata. These catecholamines are possibly important for a continual irritability of the cardiac muscle fibres in poikilothermic vertebrates.

Frau A. Beyerle-v. Wehren sei für ihre wertvolle technische Unterstützung gedankt.     

Scanning electron microscopy of the zonular fibers in the rat eye

Summary The three-dimensional arrangement of the zonular fibers of Zinn and their ultrastructure was studied with the aid of scanning and transmission electron microscopy.Most of the thicker zonular fibers are arranged in straight bundles between the ciliary body and the lens, while the thinner fibers form a complex three-dimensional network interconnecting all the zonular fibers. These do originate from the limiting membrane covering the ciliary body. The zonular fibers are subdivided close to lens and form a complicated network on the surface of the lens capsule, i. e. the zonular lamella. The latter consists of a dense network of fibers and is from a structural point of view closely related to the zonular fibers and not to the lens capsule.The zonular fibers are continuous with those in the vitreous body close to the ciliary body but never in the lenticular two thirds of the zonular fibers or in the retrolental area.The ground substance is possible to demonstrate in freeze-dried specimens by scanning electron microscopy. It appeared granular or amorphous and coated the zonular fibers. It does not form membranes or fill all available space in contrast to its properties in the vitreous body. The many structural similarities between the zonular fibers and the vitreous body indicate perhaps a common origin.Supported by grants from Magnus Bergwalls Stiftelse and the Swedish Medical Research Council (B70-12 X -2543-02, B71-12 X -2543-03).