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Biochemical and histochemical studies on skeletal muscle in rat during the course of development. The biochemical properties of type 2C muscle fiber.

1. Biochemical and histochemical analyses were performed on skeletal rat muscles over the course of development from neonate to adult. 2. A small amount of triglyceride and a large amount of glycogen were found to be contained in neonatal type 2C muscle fiber; based on this finding the main energy metabolism in this fiber was thought to be anaerobic glycolysis. 3. Moreover at 10 days type 2C muscle fiber was shown to contain a large amount of triglyceride. 4. These results suggest that, although histochemical characteristics remain the same, biochemical properties change during the course of muscle development.

Animals↗

Developmental potential of rat L6 myoblasts in vivo following injection into regenerating muscles.

To examine the relative importance of myoblast lineage and environmental influences on the development of muscle fiber types in vivo, the phenotype of muscle fibers formed from rat L6 myoblasts was examined following their injection into different regenerating adult muscles. Myoblasts were infected with a retroviral vector carrying a LacZ reporter gene and their fate in vivo was examined using a panel of antibodies against various myosin heavy chain (MyHC) isoforms. Since L6 myoblasts express IIX MyHC following differentiation in vitro, we wanted to determine if they would form IIX muscle fibers in vivo and whether innervation would alter this fate. Following injection, L6 cells either fused with each other to form homotypic fibers or fused with host muscle cells to form heterotypic fibers. Initially, homotypic fibers expressed embryonic MyHC-similar to L6 myotubes in vitro. However, by 4 weeks postinjection IIX MyHC had replaced embryonic MyHC as the predominant isoform. Single fiber analysis using an antibody specific for NCAM indicated that this transition was independent of innervation. Analysis of heterotypic fibers resulting from the incorporation of donor L6 myoblasts into host fast IIA and IIB fibers revealed that L6-derived nuclei express embryonic and IIX MyHCs for up to 8 weeks postinjection, often as nuclear domains surrounding L6 nuclei. These results suggest that MyHC expression in muscle fibers derived from L6 myoblasts is regulated, in part, by intrinsic factors that limit the fiber type potential of these cells in vivo.

Animals↗

Appearance of PLP mRNA in specific regions of the developing rat lumbosacral spinal cord as revealed by in situ hybridization.

The capacity of specific regions and fiber tracts of developing rat lumbosacral spinal cord to express proteolipid protein (PLP) mRNA was examined using in situ hybridization. PLP mRNA was undetectable at Day 18 postfertilization but present in the white matter by Postnatal Day 0 (P0). Initially, levels of PLP mRNA were higher in ventral than in dorsal funiculi. Hybridization signal was observed in the corticospinal tract by P10. Appearance of PLP message in gray matter lagged several days behind that in white matter. The expression of PLP mRNA in gray matter rose between P10 and P40 and the abundance of PLP transcript remained high in white matter during the same period. In the adult spinal cord some oligodendrocytes in the Lissauer tract and in the posterior commissure expressed low levels of PLP mRNA, which was undetectable in the substantia gelatinosa. Comparing the time of emergence of PLP mRNA to the histological appearance of myelination gave evidence that the message is expressed in oligodendrocytes that are not many days away from myelin formation. These results provide new information on the topographic distribution of PLP mRNA during myelination of the rat spinal cord.

Animals↗

Screening method for polycyclic aromatic hydrocarbons in soil using hollow fiber membrane solvent microextraction.

A fast, inexpensive screening method for polycyclic aromatic hydrocarbons in soil has been developed. Using hollow fiber membrane solvent microextraction, 8 microl of octane extraction solvent was placed inside a porous, polypropylene fiber. Following an 8 min analyte preconcentration step, 4 microl of extract was injected into a gas chromatograph. Separation was achieved in less than 10 min with a detection limit of 0.13 mg/kg for 2-methylnaphthalene. Results of both spiked and real soil samples are presented.

Calibration↗

[Investigations and review of literature relating to carcinogenesis. I. Communication: Cancer from asbestos, schistosomiasis, and cicatrization (author's transl)].

This review covers the following aspects: Cancer associated with asbestos and other fibers: Epidemiology. - Cancer types and location (mesothelioma; bronchogenic carcinoma; cancer of the upper respiratory tract; abdominal cancer). - Experimental asbestos cancer. - Other kinds of fibers and cancer (wool and cotton; glass; talc; others). - Cancer determining or influencing factors (individual predisposition; species susceptibility; type of material; shape and size of fibers; smoking). - Preneoplastic signs in man (fibrosis; ferruginous bodies; pleural plaques; milky spots). - Preneoplastic development in animals. - Fiber effects on cell cultures (macrophages; fibroblasts). Cancer associated with schistosomiasis: Epidemiology. - Patient age and cancer latency. - Pathology. - Foreign body reaction and preneoplastic development. Scar cancer. Foreign body cancer: In man. - Experimental (species differences in susceptibility; individual genetic differences in tumor incidence and latency; influence of sex, age, nutrition; tumor histopathology and ultrastructure; tumor growth, invasiveness, metastases, transplantability, immunology; search for tumor viruses). - Properties of foreign body materials in relation to tumorigenicity (chemical and physical properties; size and shape; surface properties; porosity). - Investigations and findings concerning the origin of foreign body sarcomas (the foreign body reaction; search for foci of tumor origin; an analytical method; monoclonal tumor origin; heterogenicity of carcinogenic events; surface dependency; identification of originator cells; time and location of the emergence of tumor originator cells; the carcinogenic initiation event; surface-independent and dependent preneoplastic maturation; the carcinogenic role of the foreign body). - Earlier hypotheses and theories in the light of new experimental findings. The results of experimental foreign body tumorigenesis in relation to foreign body-, asbestos-, schistosomiasis-, and scar-cancer in man. (Common factors of promotion; differences regarding induction mechanisms, cells of origin, latencies, frequencies; immune defense). Consequences for prevention: Asbestos cancer. - Fiber cancer. - Schistosomiasis cancer. - Foreign body cancer (assessing the peril in man; testing of materials for carcinogenicity; recommendations).

Animals↗

Masticatory function and properties of masseter muscle fibers in microphthalmic (mi/mi) mice during postnatal development.

It is well known that teeth do not erupt in microphthalmic (mi/mi) mice, a type of osteopetrotic mice, due to bone resorption failure. Therefore, we surmise that the masticatory function of these mice is different from that of normal mice. In this study, the differences to the properties of masseter muscle fibers were clarified morphologically and immunohistochemically in the mi/mi and normal mice. Morphological observations revealed that the muscle fibers in the mi/mi mice were smaller than those in normal mice at 9 weeks of age. However, no marked differences between mi/mi and normal mice at 2 and 4 weeks of age. Immunohistochemical observations showed myosin heavy chain (MHC) slow type fibers, which were usually seen at only early stages of development, in 4-week old mi/mi mice. There were also differences in isoform compositions between the mi/mi and normal mice. These results suggest that differences in masticatory function affect the properties of its muscle fibers.

Aging↗

Hyperplastic development and hypertrophic growth of muscle fibers in the white seabass (Atractoscion nobilis)

Because skeletal muscle is the main contributor to body weight in most fish, it is probable that the size of a fish is limited by the growth of this tissue. Several aspects of both somatic size and skeletal muscle growth were investigated in white seabass (Atractoscion nobilis). One hundred and four subjects ranging in size from 1.3-91.8 cm standard length were examined to discern growth patterns throughout the entire life span of this species. Relationships of somatic growth were evaluated by weight, length, and age comparisons while muscle growth was assessed from cross-sectional area measurements of white muscle fibers. The average cross-sectional fiber area increased from approximately 150 &mgr;m(2) to 4300 &mgr;m(2) as the fish increased in size, indicating that hypertrophy plays an important role in growth. Hyperplastic fiber recruitment accounted for approximately 93% of muscle growth in recently hatched fish and gradually decreased with fish length. Although the contribution of hyperplasia declined to less than 1% in the largest subject, the persistence of hyperplasia at standard lengths equal to or greater than 91.8 cm as observed in the white seabass has yet to be documented in other fish species. This sustained fiber recruitment may be responsible for the impressive ultimate standard length of 133 cm (41 kg) that white seabass are capable of attaining. The white seabass currently represents the largest marine fish species for which the dynamics of muscle fiber growth have been described. J. Exp. Zool. 284:299-308, 1999. Copyright 1999 Wiley-Liss, Inc.

Journal Article↗

The topography of long nuclear chain intrafusal fibers in the cat muscle spindle.

Muscle spindles were studied histochemically in serial transverse sections of specimens of the cat tenuissimus muscle. The nuclear chain intrafusal muscle fibers were separated into three subtypes, called long, intermediate and typical. The long chain and intermediate chain fibers tended to assume a particular position within the axial bundle of intrafusal fibers. The fibers were usually located in that layer of chain fibers that was positioned farthest away from the bag2 fiber. Furthermore, they were usually situated adjacent to the bag1 fiber throughout much of the extent of the spindle pole. Some long chain and intermediate chain fibers had several fiber nuclei abreast at the equator rather than a single row of central nuclei, as in most nuclear chain fibers. The relative position of intrafusal fibers within the cat spindle may reflect their order of formation during development, with the fibers retaining, to a variable degree, their association with the bag2 fiber which acted as template. Thus, the axial position of long chain and intermediate chain fibers suggests that they are among the first nuclear chain fibers to form. This may play a role in the known preferential innervation of these chain fibers by skeleto-fusimotor axons.

Adenosine Triphosphatases↗

Effects of halothane on Ca2+-activated tension development in mechanically disrupted rabbit myocardial fibers.

The effect of halothane on maximal and submaximal Ca2+-activated tension in mechanically disrupted right ventricular papillary muscle from rabbits was studied. Steady-state isometric tension generation was measured in the muscle bundle. The relaxing solution contained (in mM) [Mg2+] = 1, [K+] = 70, [MgATP2-] = 2, [creatine phosphate2-] = 15, [EGTA total] = 7 and imidazole proprionate. The contracting solution contained in addition Ca2+ in various concentrations. In all solutions ionic strength was maintained at 0.15 and pH at 7.00 +/- 0.02 at 20 degrees C. Each fiber bundle was immersed in control solutions equilibrated with 100% N2 and test solutions equilibrated with various concentrations of halothane-N2 mixture. Increasing doses of halothane (1--4%) significantly shifted the relationship between Ca2+ and tension towards higher [Ca2+] and depressed the maximum Ca2+-activated tension. The maximum tension generated at pCa = 3.8 was depressed 5% per 1% increase in halothane concentration. The percentage of maximum tension at submaximum Ca2+ concentrations (pCa = 5.6--5.0) was not significantly decreased until halothane concentration was greater than 2%. It is concluded that halothane slightly but significantly depressed the interactions of contractile proteins and to a lesser degree Ca2+-activation of the regulatory proteins. The halothane-induced depression was completely reversible.

Adenosine Triphosphatases↗

Histologic architecture of submucosal connective tissues in human eustachian tube with supplemental reference to the effects of aging.

Serial sections of 74 human eustachian tubes from 37 autopsy cases were examined microscopically to determine the histologic architecture of submucosal connective tissues. Abundant elastic fibers ran just beneath the mucosal epithelium of the eustachian tube. Two kinds of layers of collagenous fiber were observed an inner circular layer and an outer longitudinal layer. An "elastic fiber band" perfectly encircled the tubal lumen at the pharyngeal part of the eustachian tube. This band became less sharply defined at the osseous part. The two layers of collagenous fiber were contiguous, especially in the upper part of the tube, and ran from the pharyngeal orifice to the isthmus. Submucosal connective tissues were found to be undeveloped in fetal eustachian tubes. In children as well, elastic fibers were not fully developed, and circular banding of collagenous fibers was less marked. With advancing age, elastic fibers increased in number and became large and brittle. Both layers of collagenous fibers became thicker. The present data indicate that the submucosal connective tissues may play an important role in the elasticity of the eustachian tube itself and in the maintenance of the tubal framework. Gerontologic changes in the eustachian tube stroma have been considered pathophysiologically.

Adolescent↗

Development of a fluorinated polyimide hollow fiber for medical devices.

We fabricated an asymmetric polyimide hollow fiber for medical devices. A dry/wet phase inversion process was applied to a spinning process to prepare the hollow fiber. The outer diameter was 330 microm with a wall thickness of 70 microm. Transfer rates of O2 and CO2 in the asymmetric polyimide fiber were 6.9 x 10(-3) and 5.5 x 10(-3) cm3 (STP)/(cm2 s cmHg), respectively, which are approximately 10 times higher than those measured in the Menox and Si-polypropylene fibers of the presently available membrane oxygenators. The blood compatibility of the polyimide hollow fiber was evaluated in vivo, indicating that polyimide had excellent blood compatibility when compared with silicone-coated fiber. Additionally, we fabricated a novel porous membrane with three-dimensional fine structure from cylindrical microscale pores and examined possibility of a porous membrane for use in hemodialysis.

Animals↗

The influence of adenovirus fiber structure and function on vector development for gene therapy.

The collective attributes of adenoviruses (Ads), including ease of accomplishing replication deficiency, readily achievable high titers, encoding of large expression cassettes, efficiency of gene delivery to most cell types, and well-characterized biology, have made Ads, particularly Ad serotype 5 (Ad 5), some of the most utilized vectors for gene delivery. In recent years, however, it has become apparent that additional aspects of basic Ad virology must be uncovered for this vector system to succeed in the clinic. While local gene delivery is generally efficient, the broad tropism of Ad 5 and its tendency to home to the liver after systemic administration have proved to be limitations for other therapeutic applications, such as the treatment of disseminated cancers and cardiovascular disease. This has refocused research into the biology of Ad capsid components, particularly the main tropism determinant, the fiber/penton base complex, and their influence on transduction of selected cell types in vivo.

Adenoviridae↗

The tail movement of bull spermatozoa. Observations and model calculations.

Detailed observations of the tail movement of non-rotating and rotating bull spermatozoa have been carried out. For rotating sperm a helical tail wave was found with a ratio of the amplitudes of the two perpendicular components of approximately 3 to 1. For both types of cells the variation of the amplitude and the phase shift of the wave as it travels from the proximal to the distal part are reported. Model calculations indicate that the stiffness of the tail originates in the fibrous sheath, which has a Young's modulus of 3 x 10(7) dynes/cm(2). Active contractile elements distributed continuously along the tail are found necessary to maintain the amplitude of the tail wave against damping by the fluid drag. If the longitudinal fibers are identified with the contractile elements the maximum tension to be developed by these fibers is 4 x 10(6) dynes/cm(2). The energy dissipated by the "active" part of the tail wave is at least approximately 70 percent of the total dissipation.

Animals↗

The effect of ATP analogs on posthydrolytic and force development steps in skinned skeletal muscle fibers.

ATP, 2-deoxy ATP (dATP), CTP, and UTP support isometric force and unloaded shortening velocity (Vu) to various extents (Regnier et al., Biophys. J. 74:3044-3058). Vu correlated with the rate of cross-bridge dissociation after the power stroke and the steady-state hydrolysis rate in solution, whereas force was modulated by NTP binding and cleavage. Here we studied the influence of posthydrolytic cross-bridge steps on force and fiber shortening by measuring isometric force and stiffness, the rate of tension decline (kPi) after Pi photogeneration from caged Pi, and the rate of tension redevelopment (ktr) after a sudden release and restretch of fibers. The slope of the force versus [Pi] relationship was the same for ATP, dATP, and CTP, but for UTP it was threefold less. ktr and kPi increased with increasing [Pi] with a similar slope for ATP, dATP, and CTP, but had an increasing magnitude of the relationship ATP < dATP < CTP. UTP reduced ktr but increased kPi. The results suggest that the rate constant for the force-generating isomerization increases with the order ATP < dATP < CTP < UTP. Simulations using a six-state model suggest that increasing the force-generating rate accounts for the faster kPi in dATP, CTP, and UTP. In contrast, ktr appears to be strongly affected by the rates of NTP binding and cleavage and the rate of the force-generating isomerization.

Adenosine Triphosphate↗

Effects of neonatal injury of the inferior alveolar nerve on the development and regeneration of periodontal nerve fibers in the rat incisor.

Our previous study showed that the migration of terminal Schwann cells occurred in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve (IAN) in the adult animals [Y. Atsumi, K. Matsumoto, M. Sakuda, T. Maeda, K. Kurisu, S. Wakisaka, Altered distribution of Schwann cells in the periodontal ligament of the rat incisor following resection of the inferior alveolar nerve: An immunohistochemical study on S-100 proteins, Brain Res. 849 (1999) 187-195]. The aim of the present study was to investigate the effects of neonatal transection of the IAN on the regeneration of axon elements and Schwann cells in the periodontal ligament of the rat lower incisor. Following transection of IAN at post-natal day 5 (PN 5d), when the numbers of both axon elements and the terminal Schwann cells were very small, regenerating nerve fibers appeared between post-injured days 7 (PO 7d) and PO 14d, and increased in number thereafter gradually. Although the terminal morphologies of regenerated Ruffini endings became identical to those of the adult animals by PO 54d, the number of regenerated PGP 9.5-IR nerve fibers did not recover the adult levels even by PO 56d. A small number of Schwann cells migrated into the shear zone, the border between the alveolus-related part (ARP) and the tooth-related part (TRP), but did not enter into the TRP. Following transection of the IAN at PN 14d or PN 28d, when clusters of apparent terminal Schwann cells could be recognized, axon regeneration started around PO 5d. Individual axon terminals of the regenerating Ruffini endings ramified and became identical to those of the adult animals around PO 28d, but the number of regenerated Ruffini endings was smaller than that of the adult animals. Similar to the adult animals, the migration of Schwann cells into the shear zone and TRP occurred, and disappeared prior to the completion of the axonal regeneration. The present results indicate that the migration of the Schwann cells into TRP during the regeneration of the periodontal nerve fibers following nerve injury to the IAN depends on the maturation of the terminal Schwann cells of the periodontal Ruffini endings, not on post-operative time.

Age Factors↗

Gene transfer to muscle using herpes simplex virus-based vectors.

The main goal of gene therapy for Duchenne muscular dystrophy (DMD) is to restore dystrophin into as many muscle cells as necessary to be therapeutic. Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo. The viral genome is large and can accommodate multiple or large non-viral genes including the full-length dystrophin. Here we report on the use of a replication defective HSV-1 mutant vector (DZ) deleted for the essential immediate early (IE) gene ICP4 for studies of reporter gene transfer and expression following direct inoculation of mouse skeletal muscle. Our initial experiments showed that HSV-1 can efficiently infect and express a foreign reporter gene in myoblasts and myotubes in vitro. Furthermore, the intramuscular inoculation of HSV-1 resulted in transduction of a significant number of muscle fibers in newborn mice and some muscle fibers in adult animals. We have attempted to exploit these features to develop new HSV mutant vectors for dystrophin gene delivery to DMD muscle, however two impediments to using this virus for muscle gene delivery have to be overcome: namely viral cytotoxicity and the differential transducibility with HSV-1 throughout the development of muscle fibers. To solve the first problem, virus mutants deleted for the immediate early (IE) genes (ICP4, ICP22, ICP27 and UL41) were constructed and the multiple deleted virus was greatly reduced in cytotoxicity relative to our first generation HSV vector strains. Current work is aimed at incorporating full-length dystrophin under muscle specific promoter (muscle creatine kinase MCK) into these new viral vectors. To address the second problem we have analysed by immunohistochemistry the spreading of the HSV-1 in newborn versus adult muscles to determine whether mature basal lamina which surrounds the adult muscle fibers blocks the HSV-1 entry into the mature muscle fibers.

Animals↗