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Ag+ produced two different types of transient tension development in single frog toe muscle fibers in which the Ca2+ channel had been blocked by pretreatment with 2mM Co2+. These contractions were never observed in detubulated fibers, indicating that the Ag+ -induced contraction is produced through Ca2+ channels located on the T-tubular membrane.
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Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens. Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre- and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth-associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.
We have estimated from electron micrographs the numbers of axons in the optic nerves of a series of albino rats at 8 different ages ranging from embryonic day (E)18 through postnatal day 28. The number of axons was found to reach a maximum of about 325,000 (324,790 +/- 38,589) on E20, and to decline to about 275,000 (273,744 +/- 20,973) by the day of birth. By the middle of the second postnatal week, the number was further reduced to a stable figure of just over 100,000 (105,809 +/- 7,610). This represents a loss of two-thirds of the axons from the peak value at E20, and suggests that there is a comparable degree of cell death among the retinal ganglion cells. The reduction in the number of optic nerve fibers is not affected significantly by the removal of the opposite eye at birth.
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Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.
Cervix cancer is a curable disease when diagnosed at an early stage. Screening of cervical lesions by cytology and colposcopy with in situ staining has allowed for substantial progress in early diagnosis and consequently the cure of cervix cancer. Nevertheless, because of its low specificity, this approach generally implies repetitive tissue sampling and, thus a relative long time before the treatment of the lesions. Furthermore, the cost of preparation and analysis of biopsy samples is sufficiently high to represent a burden for industrialized countries and a virtual impossibility for the developing world. To overcome these problems, various biophotonic methods using optical fibers have been developed to allow for detection of cervical epithelial anomalies in a specific, fast and non-invasive way. This process, known as "optical biopsy", is based on the measurement of light-tissue interactions, which are analysed by various mathematical and data processing methods, to provide information on the metabolism and morphology of epithelial tissue. Currently investigated methods can be distinguished according to the type of signal used to probe the tissue (fluorescence, reflectance), the depth of analysed tissue (surface analysis, confocal imaging, tomography), the analysis modalities (spectral measurements or imaging), and the use of additive molecules (contrasting or photosensitizing agents, inorganic fluorophores). While most of the methods remain experimental, constant progress in the understanding of the mechanisms of light behavior in biological environments as well as advances in optical fibers technology, will make a number of these methods soon available for clinical practice to contribute efficiently to the reduction of biopsy number and cost of cervical screening.
The embryonic precursors of the avian slow (type I and III) and fast (type II) fibers can be distinguished from each other early in muscle formation (stage 28, V. Hamburger and H. L. Hamilton, J. Morphol, 88, 49-92, 1951) on the basis of the differential sensitivity of their myosin ATPases. To test the neural dependence of fiber type differentiation, the source of motor innervation was eliminated by excision of the brachial neural tube at stages 16-18 before muscles are innervated. Removal of the brachial neural tube did not affect the number of primary myotubes in a sample muscle of the forelimb (ulnimetacarpalis dorsalis, UMD) up until stage 36. Myosin ATPase staining at a variety of pHs revealed the typical patterns of fiber types in muscles of neural-tube free embryos in stages 35-37. These muscles included the anterior latissimus dorsi, brachialis, and UMD which showed presumptive type III staining (type IIIEMB), the pronator superficialis and flexor carpi ulnaris which showed embryonic type II staining (type IIEMB), and the triceps brachii muscles which showed characteristic arrangements of both type IEMB and type IIEMB fibers. The normal patterns of type IEMB and type IIEMB myotubes were also seen in muscles containing a heterogeneous mixture of fiber types such as the biceps brachii, extensor metacarpi radialis, and adductor indicis muscles, although the intensity of acid-stable ATPase staining of the type IEMB myotubes in these muscles was lower than in innervated muscles. It is concluded that the earliest differentiation of muscle fiber types is independent of the nervous system.
Osmotic-response element-binding protein (OREBP), also known as TonEBP or NFAT5, is thought to be responsible for the induction of osmolyte-accumulating genes when cells are under hypertonic stress. Recent studies suggest that OREBP also plays a role in water reabsorption in the kidney, T-cell proliferation, and embryonic development. We developed transgenic mice that express the dominant-negative OREBP (OREBPdn) specifically in the lens because our earlier studies showed that it is particularly sensitive to osmotic stress. The transgenic mice developed nuclear cataract soon after birth, suggesting defects in lens development. The developing transgenic lenses showed incomplete elongation of fiber cells and formation of vacuoles. This is accompanied by evidence of DNA strand breaks, activation of p53, and induction of checkpoint kinase, suggesting that the developing fiber cells lacking OREBP are in a similar physiological state as cells experiencing hypertonic stress. These results indicate that OREBP-mediated accumulation of osmolytes is essential during elongation of the lens fiber cells.
A new technique for treating skinned cardiac muscle fibers has been developed in which troponin I is extracted, giving rise to unregulated fibers. The effect of the 12-residue troponin I peptide on these fibers indicates that this region of troponin I is solely responsible for muscle relaxation (inhibition of force). Furthermore, troponin I peptide-troponin C reconstituted fibers are stable through several contraction-relaxation cycles indicating the peptide can switch binding sites between actin and troponin C. The troponin I peptide can substitute for the native protein as part of the calcium-sensitive molecular switch that controls muscle regulation.
The content and distribution of vasopressin and oxytocin were determined during fetal development in the rat brain and pituitary by means of radioimmunoassay and immunocytochemistry. The vasopressin content in the fetal brain showed a gradual rise from day 16 of pregnancy onwards, while pituitary vasopressin rapidly increased from fetal day 19 until birth. The oxytocin content in the fetal brain was considerably lower than the vasopressin content. A decrease in oxytocin content was seen between day 16 and day 18 while from day 18 of pregnancy onwards a slight increase was found. The pituitary oxytocin content starts to rise between day 17 and 18 of pregnancy, but at term the pituitary oxytocin content was only 1/20 of the vasopressin value. Immunocytochemistry revealed that vasopressin levels in the fetal rat brain were not only due to the presence of the classical hypothalamoneurohypophyseal system, but also to the early development of exohypothalamic fibers. Vasopressin containing cells were seen from fetal day 16 in the supraoptic nucleus, and from fetal day 18 in the paraventricular nucleus. The fiber outgrowth of these cells towards the pituitary and extrahypothalamic brain sites seems to be well synchronized, as on day 17 vasopressin containing fibers could be demonstrated in the olfactory bulb as well as in the median eminence. No positive staining for oxytocin could be obtained in the fetal rat, while during the entire fetal period no positive staining was found in cell bodies in the region of the suprachiasmatic nucleus. The early peptidergic innervation of the brain, which enabled the tracing of the source of some exohypothalamic fibers, might be related to several central processes among which brain development itself is included.
In the vertebrate embryo, skeletal muscle originates from somites and is formed in discrete steps by different classes of progenitor cells. After myotome formation, embryonic myoblasts give rise to primary fibers in the embryo, while fetal myoblasts give rise to secondary fibers, initially smaller and surrounding primary fibers. Satellite cells appear underneath the newly formed basal lamina that develops around each fiber, and contribute to post-natal growth and regeneration of muscle fibers. Recently, different types of non somitic stem-progenitor cells have been shown to contribute to muscle regeneration. The origin of these different cell types and their possible lineage relationships with other myogenic cells as well as their possible role in muscle regeneration will be discussed. Finally, possible use of different myogenic cells in experimental protocols of cell therapy will be briefly outlined.
An examination has been made of the five dorsal muscles of the cat neck which insert into the lamboidal crest. The three larger muscles, splenius, biventer cervicis, and complexus, are characterized by the presence of tendinous inscriptions which serve as the insertion points of shorter muscle fibers which do not run the length of the muscle. Longer fibers are bound by the inscriptions and, thus can transmit tension developed by shorter fibers. These three muscles are multiply innervater by nerves emerging from upper cervical spinal roots. Multiple innervation is not associated with multiple end plates but with arrangement so that lower roots innervate a high proportion of short fibers and higher roots, a high proportion of long fibers. Occipitoscapularis and rectus capitis major are, by comparison, simple muscles, each with a single motor nerve. Of the muscles examined, occipitoscapularis alone does not have its origin on the vertebral column but on the scapula, and is unlikely to have any major function in head movement.
We have theoretically and experimentally investigated an optical fiber with circular polarization modes on one end and linear polarization modes on the other end. We call this fiber a polarization-transforming fiber because the local modes, or polarization states they represent, are converted from linear to circular, and visa versa, in the fiber. We have developed and implemented a postdraw process for making polarization-transforming fiber samples 30 mm long with losses less than 1 dB and a polarization-mode conversion from circular to linear greater than 20 dB. Also, we have modeled and measured the dependence on wavelength and temperature of polarization-transforming fiber samples. The measured normalized wavelength dependence of a sample fiber 30 mm long was approximately 1.4 x 10(-4) nm(-1), and the measured normalized temperature dependence was approximately 6 x 10(-4) degrees C(-1). These values are better in some cases than values for conventional high-birefringent fiber quarter-wave plates.
We developed a new variant of coral-derived red fluorescent protein, DsRed S197Y, which is brighter and essentially free from secondary fluorescence peak. This makes it an ideal reporter for double labeling with green fluorescent protein (GFP). Though purified protein shows only 20% stronger fluorescence emission, culture cells that express DsRed S197Y exhibit a 3-3.5 times higher level of fluorescence than the cells that express wild-type DsRed. The much slower fluorescence maturation of DsRed than that of GFP is a beneficial feature for a fluorescent developmental timer application. When GFP and DsRed S197Y are expressed simultaneously, emissions start at different latency. This provides information about the time after the onset of expression. It reflects the order of cell differentiation if the expression is activated upon differentiation of certain types of cells. We applied this system to the developing brain of Drosophila and visualized, for the first time, the formation order of neural fibers within a large bundle. Our results showed that newly extending fibers of the mushroom body neurons mainly run into the core rather than to the periphery of the existing bundle. DsRed-based timer thus presents an indispensable tool for developmental biology and genetics of model organisms.
All mammalian muscle fibers are formed by cell fusion, but fiber formation occurs in two successive waves. Initially a small number of large fibers (primaries) are produced and are used as a scaffold for the formation of a larger number of smaller later forming fibers (secondaries). It has been reported that different populations of myoblasts are present at different ages, but it is unclear whether separate populations of cells contribute to primary and secondary fibers during the period of fetal development. We have tested this by injecting two replication-deficient retroviruses, carrying a marker gene (lacZ), into the hind limbs of Embryonic Day (E) 15 and E17 mouse fetuses. All fetuses were killed at E19. Clusters of marked fibers (assumed to correspond to single clones) found after injection at both ages contained both primary and secondary fibers. This indicates that at these ages, a single population of cells contributes to both primary and secondary fibers.
The relative contributions of innervation and androgen to three muscle fiber properties--twitch type, size, and number--were examined in the sexually dimorphic, androgen-sensitive laryngeal muscle of Xenopus laevis. In adults, the muscle contains all fast-twitch fibers in males and fast- and slow-twitch fibers in females; laryngeal muscle fibers are larger and more numerous in males than in females. Juvenile larynges are female-like in both sexes; male laryngeal muscle is subsequently masculinized by androgen secretion during postmetamorphic development. Because both laryngeal motor neurons and muscle fibers are androgen sensitive during masculinization, we examined the role of the nerve in androgen-regulated muscle fiber development. Laryngeal muscle of male and female juvenile frogs was unilaterally denervated, and effects on muscle fiber type, size, and number were examined 4 weeks later. Half of the frogs received a dihydrotestosterone pellet at the time of denervation. Androgen treatment converts laryngeal muscle from mixed slow and fast to all fast twitch in both innervated and denervated muscle. Thus, the nerve is not required for androgen-regulated fiber type expression in either sex. Denervation produces muscle fiber atrophy and androgen treatment induces muscle fiber hypertrophy in male and female larynx. Nerve and hormone effects are independent and additive; fiber size in androgen-treated denervated muscle is greater than in untreated innervated muscle, and fiber size in androgen-treated denervated muscle is smaller than in androgen-treated innervated muscle. There is no sex difference in the effects of innervation or androgen on fiber size. Denervation causes laryngeal muscle fiber loss in males but not in females. Androgen treatment protects male laryngeal muscle from denervation-induced fiber loss and causes fiber addition in innervated female laryngeal muscle. We conclude that there is a sexually dimorphic interaction between innervation and androgen in control of laryngeal muscle fiber number.
In animal experiments, the probability that pleural tumors will develop after mineral fiber implantation is dependent upon the fiber's size. This was originally analyzed in relation to the number of injected fibers with specific dimensional characteristics and, more recently, in relation to average aspect ratio (average length/diameter ratio). New experimental data on various mineral fibers suggest that the carcinogenic potency of a solid particle depends on two parameters: the nature of the mineral and therefore the nature of active surface sites, and the aspect ratio of the particle. These two parameters can intervene in a mechanism of model reaction involving the basic properties of lateral faces of fibers.