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Distribution of alpha- and beta-myosin heavy chains in the ventricular fibers of the postnatal developing rat.

Four monoclonal antibodies, two raised against alpha-myosin heavy chain (MHC) and two against beta-MHC, have been used to investigate in situ the fiber distribution of alpha- and beta-MHC in rat cardiac ventricles during postnatal development. Eighteen ventricles from 2-day-old to 1-year-old rats were analyzed. Three fiber populations were determined according to their immunofluorescent labeling: one with only alpha-MHC, one only beta-MHC, and one with mixed alpha- and beta-MHC. Large variations in the proportions of these three fiber populations according to age indicate that: (1) alpha-MHC are expressed in all fibers until the second month; they then disappear in a small endocardial fiber population and in a few apparently conductive fibers around the vessels. (2) beta-MHC are also first expressed in all fibers and then disappear gradually from epicardium to endocardium between the second and fourth weeks, except in the conductive fibers; they reappear during the second month sequentially from endocardium to epicardium; and they are then expressed in almost all fibers, except in a small epicardial fiber population, proportionally larger in the right ventricle than in the left. Immunological characterization of MHC isolated from a 22-day-old-rat ventricle, using anti-beta immunoaffinity chromatography, suggests that MHC of conductive fibers are probably at least partially in an alpha beta heterodimeric form.

Aging↗

Myosin-free ghosts of single fibers and an attempt to re-form myosin filaments in the ghost fibers.

With the final aim of replacing myosin in a single muscle fiber, a technique for removing myosin almost completely from single fibers was developed and an attempt to "re-form" thick filaments in the myosin-free ghosts of single fibers was made. Complete removal of myosin from single glycerol-treated rabbit psoas fibers with Hasselbach-Schneider solution was difficult. However, when skinned glycerol-treated fibers were used and 1% (v/v) Triton X-100 was added to the Hasselbach-Schneider solution, almost complete removal of myosin was possible. The myosin-free ghosts of skinned single fibers were very fragile but retained the overall structure. In the ghost fibers, Z-membranes and thin filaments remained. The ghost fibers, after irrigation with myosin, underwent contraction upon addition of Mg-ATP. In the myosin-irrigated fibers, thick filaments were re-formed in lengths from one Z-membrane to the other Z-membrane of a sarcomere, running parallel to the thin filaments. The packing of these two filaments was not good. The isometric tension developed by the irrigated fibers upon addition of mg-ATP was about 10% of the tension developed by untreated fibers. The weak tension developed by irrigated fibers is probably due to the irregular packing of the thick and thin filaments in the fibers. The ghost fibers also contracted, though only slightly, upon addition of Mg-ATP after irrigation with heavy meromyosin.

Adenosine Triphosphate↗

PRDX1 as a novel urinary biomarker for bladder cancer: Development of an integrated fiber optic sensing platform.

In this study, integrated proteomic and transcriptomic analyses identified peroxiredoxin 1 (PRDX1) as a novel urinary biomarker for bladder cancer (BC). PRDX1 was significantly upregulated in BC tissues and was associated with poorer overall survival. In vitro experiments further demonstrated that PRDX1 promotes malignant phenotypes of BC cells, including proliferation, migration, and invasion. Silencing PRDX1 in BC cells significantly reduced the invasiveness and proliferation ability.To address the clinical need for rapid and non-invasive detection, we developed an innovative optical fiber biosensor based on surface plasmon resonance (SPR) technology for the quantitative detection of urinary PRDX1. The biosensor exhibited excellent analytical performance, including high sensitivity (limit of detection: 0.06 ng/mL), a wide linear range (0-25 ng/mL), rapid response (∼14 s), as well as good stability and selectivity. In clinical validation involving 97 BC patients and 30 healthy controls, the biosensor demonstrated outstanding diagnostic performance, with an area under the receiver operating characteristic curve (AUC) of 0.91 and an overall diagnostic accuracy of 86.6%, outperforming conventional enzyme-linked immunosorbent assay (ELISA). Collectively, this study not only identifies PRDX1 as a promising biomarker for non-invasive diagnosis and prognostic evaluation of BC, but also establishes an efficient SPR-based optical fiber sensing platform, providing new insights into both clinical detection and the functional role of PRDX1 in BC progression.

Humans↗

Development of acetylcholine sensor using carbon fiber (amperometric determination).

An enzyme sensor is developed using carbon fiber to measure acetylcholine concentration. The mechanism is based on the detection of H2O2 which is a product of the sequential enzyme reactions of acetylcholinesterase and choline oxidase. The fabrication of the electrode is described. The sensor is polarized at 1.2 V. Enzymes are co-immobilized in polyvinyl alcohol containing styryl pyrydinium (photo-crosslinkable polymer). A fast response time of 0.8 min is obtained. A linear correlation is observed between 0.2 and 1.0 mM. Other optimal operational conditions with respect to pH, temperature and stability are discussed. The use of carbon fiber containing co-immobilized enzymes could offer several novel advantages especially in neuroscience research. In conclusion, the aims of the present work are centered on carbon fiber electrode fabrication, immobilization and electrochemical measurements.

Acetylcholine↗

Disruption of interfascicular fiber differentiation in an Arabidopsis mutant.

Arabidopsis develops interfascicular fibers in stems for needed support of shoots. To study the molecular mechanisms controlling fiber differentiation, we isolated an interfascicular fiber mutant (ifl1) by screening ethyl methanesulfonate-mutagenized Arabidopsis populations. This mutant lacks normal interfascicular fibers in stems. Interestingly, some interfascicular cells were sclerified in the upper parts but not in the basal parts of the ifl1 stems. These sclerified cells were differentiated at a position different from that of interfascicular fibers in the wild type. Lack of interfascicular fibers correlated with a dramatic change of stem strength. Stems of the mutant could not stand erect and were easily broken by bending. Quantitative measurement showed that it took approximately six times less force to break basal stems of the mutant than of the wild type. In addition, noticeable morphological changes were associated with the mutant, including long stems, dark green leaves with delayed senescence, and reduced numbers of cauline leaves and branches. Genetic analysis showed that the ifl1 mutation was monogenic and recessive. The ifl1 locus was mapped to a region between the 17C2 and 7H9L markers on chromosome 5. Isolation of the ifl1 mutant provides a novel means to study the genetic control of fiber differentiation.

Arabidopsis↗

Segmental cytochrome c-oxidase deficiency in CPEO: teased muscle fiber analysis.

In an attempt to elucidate the pathogenesis of focal cytochrome c-oxidase (COX) deficiency in skeletal muscle from patients with chronic progressive external ophthalmoplegia (CPEO), we examined the longitudinal distribution of COX activity in single muscle fibers from 6 CPEO patients with muscle mitochondrial DNA (mtDNA) deletions. A new method for teasing single muscle fibers, recently developed in our laboratory, revealed fibers with COX-positive and -negative segments in all 6 patients. The borders between the enzyme-positive and -negative segments in these fibers were sharply delineated, so that the length of each COX-negative segments could be accurately measured. The proportion of the sum of the lengths of the enzyme-negative segments to the total length of the muscle fibers correlated well with the proportion of deleted mtDNA, suggesting that abnormal mitochondria harboring mutant mtDNA may be responsible for the focal loss of COX activity.

Adolescent↗

Temporal expression of three mouse lens fiber cell membrane protein genes during early development.

PURPOSE: Three different lens fiber cell intrinsic membrane proteins, MIP (Major Intrinsic Protein), MP19, and connexin50 (Cx50), have separately been implicated as causative candidates for congenital cataracts. The aim of this study was to determine gene transcript expression of these three proteins during successive stages of mouse embryonic development. METHODS: Total RNA was prepared from mouse embryos taken at days 9-10 (E9-E10) of gestation, heads of day 11-13 (E11-E13) embryos, and lenses of adult mice. Coupled reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine gene transcript expression of MIP, Cx50, and MP19 during embryonic development. The products of RT-PCR were further cloned into the TOPO(TM) TA vector, and further analyzed by double strand nucleotide sequencing. RESULTS: Cx50 gene expression was observed throughout the developmental period observed (E9-E13). MIP transcripts were first observed at mouse embryonic day 11.5 (E11.5) and synthesis continued throughout the developmental period observed. The gene for MP19 (Lim2) begins to express at mouse embryo day 12 (E12) and synthesis continued throughout the developmental period observed. mRNA levels for all three proteins appear to remain steady from these early embryonic stages through adulthood. CONCLUSIONS: The identified early expression of Cx50, MIP, and Lim2 transcripts in mouse embryonic stages suggests that all three proteins play very important, probably quite different, roles in lens fiber cell differentiation. Variation in the temporal expression of these three genes during the course of development suggests a critical gene-coordinated regulation throughout lens fiber cell development. These three genes clearly play important roles in early normal lens development since it is known that mutations in the sequence of each membrane protein results in cataractogenesis.

Animals↗

Role of gravitational loading in the development of rat soleus muscle fibers.

Effects of hindlimb unloading during the first 3 months after birth on the development of soleus muscle fibers were studied in rats. The mean absolute weigh and cross-sectional area of whole soleus muscle in the unloaded rats were -1/3 and 1/4 of those in the controls, respectively. But the unloading did not affect the lengths of muscle, at 90 degrees of ankle joint angle, and of muscle fibers sampled from tendon to tendon, and the total sarcomere number. Since the total number of fibers in soleus was not affected either, the inhibited increase of muscle mass following unloading was mainly due to the smaller CSA of individual fibers. Numbers of both myonuclei and satellite cells were significantly less in unloaded than control rats. The % distribution of fibers expressing pure type I myosin heavy chain was significantly less in unloaded than controls (-23 %). Further, muscle fibers with multiple innervation were noted in the unloaded rats. It is suggested that the development and/or differentiation of soleus muscle fibers are closely associated with gravitational loading and that the growth-associated increase in fiber number may be genetically programmed.

Journal Article↗

Tgfbeta receptor expression in lens: implications for differentiation and cataractogenesis.

TGFbeta induces changes characteristic of some forms of cataract. However, the responsiveness of lens epithelial cells to TGFbeta is age-dependent; weanling and adult, but not neonatal, lens epithelial cells respond. This study investigated TGFbeta receptor (TbetaRI and TbetaRII) expression during rat lens development and the effects of FGF-2 on TGFbeta responsiveness and TbetaR expression. Immunofluorescence, immunoblotting, RT-PCR and in situ hybridization were used to examine the spatio-temporal expression patterns of TbetaR. Lens explants were used to investigate the effects of FGF-2 on TGFbeta responsiveness and TbetaR expression. In the lens epithelium, little or no immunoreactivity was detected at P3 but at P21 there was distinct reactivity for TbetaRI and TbetaRII. Reactivity for both receptors was also found in the differentiating fibers in the transitional zone and cortex at both ages. Western blotting of lens membrane extracts identified multiple molecular weight forms of TbetaRI (30, 50, 90 kDa) and TbetaRII (70-120 kDa). In situ hybridization with a rat probe for Alk5 (TbetaRI) showed that the lens expresses Alk5 mRNA in epithelium and fibers throughout development. A rat TbetaRII probe revealed distinct expression of a TbetaRII mRNA in lens fibers throughout development and in the lens epithelium at P21 but not at P3. In vitro studies showed that lens epithelial explants from P9 rats did not undergo cataractous changes in response to TGFbeta but P13 explants did. Addition of FGF-2 to P9 explants induced increased TbetaR immunoreactivity and enhanced the competency of lens epithelial cells to respond to TGFbeta. These data indicate that the overall increased expression of TGFbeta receptors in lens epithelium during postnatal development (P3-P21) underlies an age-related change in TGFbeta responsiveness. The results also suggest that lens cells may express multiple forms of TbetaR. Expression of TbetaR in lens fibers throughout lens development and the induction of enhanced TbetaR expression by FGF suggest a role for TGFbeta signaling during FGF-induced responses and fiber differentiation.

Aging↗

Fiber outgrowth and pathfinding in the developing auditory brainstem.

In order to study the initial outgrowth of cochlear nucleus fibers, the lipophilic carbocyanine dye DiI was used to label these neurites in rats at E13 and E15. At E13, prior to leaving the ventricular zone, cochlear neurons have already extended axons along the marginal edge of the hindbrain and a few fibers have reached the midline. By E15, many more fibers have entered the midline region and some appear to contact glial cells at the midline. Based on axon trajectory and growth cone morphology it does not appear likely that there are significant intermediate decision points for trapezoid body fibers at these early stages of development. Contact between growth cones and glial cells at the midline is consistent with these cells playing a role in providing guidance cues for developing auditory fibers.

Animals↗

Muscle organizers in Drosophila: the role of persistent larval fibers in adult flight muscle development.

In many organisms muscle formation depends on specialized cells that prefigure the pattern of the musculature and serve as templates for myoblast organization and fusion. These include muscle pioneers in insects and muscle organizing cells in leech. In Drosophila, muscle founder cells have been proposed to play a similar role in organizing larval muscle development during embryogenesis. During metamorphosis in Drosophila, following histolysis of most of the larval musculature, there is a second round of myogenesis that gives rise to the adult muscles. It is not known whether muscle founder cells organize the development of these muscles. However, in the thorax specific larval muscle fibers do not histolyze at the onset of metamorphosis, but instead serve as templates for the formation of a subset of adult muscles, the dorsal longitudinal flight muscles (DLMs). Because these persistent larval muscle fibers appear to be functioning in many respects like muscle founder cells, we investigated whether they were necessary for DLM development by using a microbeam laser to ablate them singly and in combination. We found that, in the absence of the larval muscle fibers, DLMs nonetheless develop. Our results show that the persistent larval muscle fibers are not required to initiate myoblast fusion, to determine DLM identity, to locate the DLMs in the thorax, or to specify the total DLM fiber volume. However, they are required to regulate the number of DLM fibers generated. Thus, while the persistent larval muscle fibers are not obligatory for DLM fiber formation and differentiation, they are necessary to ensure the development of the correct number of fibers.

Actins↗

Anatomical distribution of glycoprotein 93 (gp93) on nerve fibers during rat brain development.

Recent studies have implicated glycoconjugates on the membrane of growth cones as the necessary markers and intermediaries for axonal recognition, axonal motility, and pathway development. One such glycoconjugate, glycoprotein 93 (gp93), has been characterized, but the relative distribution of gp93 has yet to be described for the embryonic brain. In this study, the anatomical distribution of gp93 has been analyzed at embryonic day 15 (E15) and E18, and on postnatal day 3 in the rat by using a polyclonal gp93 antibody. Furthermore, fetal brain tissue transplanted into the adult rat eye has been tested for gp93 immunoreactivity, since central noradrenergic neurons in brainstem transplants are known to provide a continuous source of growing axons, even in adult tissue. In general, a greater abundance of gp93 immunoreactivity is apparent in the earlier embryonic stages (E15 and E18), whereas less is seen in the postnatal brain. The regions showing unique dispersal patterns of gp93 are the neuroepithelium, cerebral cortex, septo-hippocampal pathways, brainstem, and midbrain. This study has therefore focused on these areas and found implications for gp93 distribution appearing in the early development of specific neuronal pathways. Moreover, axons stain densely for gp93 within brain tissue transplants. The presence of gp93 in areas of extensive axonal outgrowth in the normal brain and in transplants suggests that this antibody is used as an early marker for axonal growth. Furthermore, gp93 might be used to map normal development in order to improve our understanding of diseases arising from developmental abnormalities.

Animals↗

Pro-opiomelanocortin neuropeptide receptors on developing and dystrophic muscle fibers.

Autoradiography was used to investigate the presence of corticotropin and beta-endorphin receptors on soleus, extensor digitorum longus, and diaphragm muscles of normal and dystrophic adult mice and developing mice. In both adult and developing mice, specific binding sites for both [125I]ACTH and [125I]beta-endorphin were present in some fibers in all of the muscles examined. The specific binding sites appeared to be distributed over the length of the surface membrane in the fibers that expressed them. There were significantly higher proportions of fibers exhibiting the specific beta-endorphin and the specific ACTH binding sites in the three muscle types in mice of 5 d of age compared to the muscles of the normal adult. There was also a higher proportion of fibers with the putative ACTH and beta-endorphin receptors in the three muscle types in dystrophic mice.

Adrenocorticotropic Hormone↗

The development and structure of principal fibers and cellular cementum in rat molars.

The principal fibers and cellular cementum were examined in various developmental stages in rat molars by light and electron microscopy, and their development and structure were studied. The principal fibers increased in thickness with root development. At any given stage, the principal fibers consisted of small fiber bundles on the cementum surface and showed a branching structure. Sharpey's fibers also showed a similar branching structure and increased in thickness, as traced superficially in the cementum. It was suggested that the principal fibers increased in thickness by means of the aggregation of fiber bundles. The development of cellular cementum was divided into two (early and late) phases on the basis of the fiber arrangement. The early cementum was believed to contain a few Sharpey's fibers and many randomly arranged intrinsic fibers. The late cementum contained thicker Sharpey's fibers with branches and the intrinsic fibers which appeared to encircle Sharpey's fibers and/or meander among them.

Animals↗

Ciliary neurotrophic factor increases muscle fiber number in the developing levator ani muscle of female rats.

In addition to its well characterized neurotrophic properties, ciliary neurotrophic factor (CNTF) also has myotrophic effects in several experimental paradigms. We have previously observed that the volume of the levator ani (LA) muscle is increased in female rats treated with CNTF during the perinatal period. In order to determine the cellular basis for the effect of CNTF on LA muscle volume, female rat pups were given daily perineal injections of CNTF or a control solution from postnatal day 1 through 6. Mean cross-sectional area of LA muscle fibers and LA fiber number were assessed on postnatal day 7. CNTF treatment increased LA muscle fiber number more than 300% while having no effect on LA fiber size. We conclude that CNTF prevents muscle fiber degeneration and/or increases myogenesis in the developing LA muscle.

Animals↗

Targeted expression of shibire ts and semaphorin 1a reveals critical periods for synapse formation in the giant fiber of Drosophila.

In order to determine the timing of events during the assembly of a neural circuit in Drosophila we targeted expression of the temperature-sensitive shibire gene to the giant fiber system and then disrupted endocytosis at various times during development. The giant fiber retracted its axon or incipient synapses when endocytosis was blocked at critical times, and we perceived four phases to giant fiber development: an early pathfinding phase, an intermediate phase of synaptogenesis, a late stabilization process and, finally, a mature synapse. By co-expressing shibire(ts) and semaphorin 1a we provided evidence that Semaphorin 1a was one of the proteins being regulated by endocytosis and its removal was a necessary part of the program for synaptogenesis. Temporal control of targeted expression of the semaphorin 1a gene showed that acute excess Semaphorin 1a had a permanent disruptive effect on synapse formation.

Animals↗

Pentobarbital prevents the development of C-fiber-induced hyperalgesia in the rat.

Noxious stimuli applied to the skin can produce long-lasting, C-fiber-dependent, secondary hyperalgesia that is mediated by central mechanisms. NMDA receptor antagonists and low doses of morphine can preferentially block the development of hyperalgesia without significantly altering unpotentiated responses to nociceptive stimuli. The aim of our study was to determine if low doses of pentobarbital can also preferentially alter either hyperalgesic or unpotentiated responses to nociceptive heat stimuli in spinalized and intact rats. Our results demonstrate the following. (1) Mustard oil applied above the ankle joint or electrical stimulation of the sciatic nerve at C-fiber intensity in spinalized, unanesthetized rats decreased the latency to withdrawal of the foot from water maintained at 47-49 degrees C. This secondary hyperalgesia to thermal stimulation persisted for at least 1 h and was most likely mediated by central mechanisms. (2) Pentobarbital in both spinalized and spinal cord-intact rats prevented the development of the late component (42-120 min) but only partially decreased the early (2-6 min) component of hyperalgesia. In contrast, pentobarbital had relatively minimal effects on unpotentiated withdrawal responses. Thus, pentobarbital is similar to morphine in its ability to prevent hyperalgesia, but may differ from the anesthetic isoflurane, which does not interfere with the development of hyperalgesia.

Anesthesia↗