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PubMed2001

Cloning and expressionanalysis of two cotton (Gossypium hirsutum L.) genes encodingcell wall proline-rich proteins.

Two cotton (Gossypium hirsutum L.) genes, ghprp1 and ghprp2, encoding cell wall proline-rich proteins (PRPs) have been cloned and characterized. The ghprpl gene has an open reading frame (ORF) that encodes a PRP of 299 amino acids (aa), whereas the ghprp2 gene contains an ORF that codes for a 310-aa PRP. The GhPRP1 has an 80% identity in aa sequence with that of GhPRP2. Like other plant cell wall PRPs, both cotton PRPs have a hydrophobic signal peptide at their N-termini, followed by repeating peptide units. Northern blot analyses showed that the ghprpl gene is predominantly expressed in the fiber during the elongation stage of fiber development. Reverse transcription (RT)-PCR analysis showed that ghprpl is expressed in both fiber and root tissues, whereas ghprp2 is in roots only. The ghprpl gene was shown to be present in the A1, A2, D1 and D5 genomes of Gossypium by PCR amplification, whereas the ghprp2 gene is only present in the A1 and A2 genomes. The ghprpl gene was over-expressed in the yeast Pichia pastoris, and the expressed GhPRP1 protein was used as an antigen to raise polyclonal antibodies (anti-GhPRP1). Western analysis using the anti-GhPRP1 probe detected a major protein band (50 kDa) in 5-31-day postanthesis (DPA) fibers. However, the 50 kDa protein was absent in other cotton tissues.

Amino Acid Sequence↗

PubMed2004

Acetylcholinesterase function is dispensable for sensory neurite growth but is critical for neuromuscular synapse stability.

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine. In addition, AChE is thought to play several 'non-classical' roles that do not require catalytic function. Most prominent among these is facilitation of neurite growth. Here, we report that the zebrafish zieharmonika (zim) locus encodes AChE. We show that one mutant zim allele is caused by a pre-mature stop codon, resulting in a truncated protein that lacks both the catalytic site and the carboxy-terminal neuritogenic domain. To explore the 'non-classical' role of AChE, we examined embryos mutant for this allele. In contrast to previous results using a catalytic-inactive allele, our analysis demonstrates that AChE is dispensable for muscle fiber development and Rohon-Beard sensory neuron growth and survival. Moreover, we show that in the absence of AChE, acetylcholine receptor clusters at neuromuscular junctions initially assemble, but that these clusters are not maintained. Taken together, our results demonstrate that AChE is dispensable for its proposed non-classical roles in muscle fiber formation and sensory neuron development, but is crucial for regulating the stability of neuromuscular synapses.

Acetylcholine↗

PubMed1998

A fiberless seed mutation in cotton is associated with lack of fiber cell initiation in ovule epidermis and alterations in sucrose synthase expression and carbon partitioning in developing seeds

Fiber cell initiation in the epidermal cells of cotton (Gossypium hirsutum L.) ovules represents a unique example of trichome development in higher plants. Little is known about the molecular and metabolic mechanisms controlling this process. Here we report a comparative analysis of a fiberless seed (fls) mutant (lacking fibers) and a normal (FLS) mutant to better understand the initial cytological events in fiber development and to analyze the metabolic changes that are associated with the loss of a major sink for sucrose during cellulose biosynthesis in the mutant seeds. On the day of anthesis (0 DAA), the mutant ovular epidermal cells lacked the typical bud-like projections that are seen in FLS ovules and are required for commitment to the fiber development pathway. Cell-specific gene expression analyses at 0 DAA showed that sucrose synthase (SuSy) RNA and protein were undetectable in fls ovules but were in abundant, steady-state levels in initiating fiber cells of the FLS ovules. Tissue-level analyses of developing seeds 15 to 35 DAA revealed an altered temporal pattern of SuSy expression in the mutant relative to the normal genotype. Whether the altered programming of SuSy expression is the cause or the result of the mutation is unknown. The developing seeds of the fls mutant have also shown several correlated changes that represent altered carbon partitioning in seed coats and cotyledons as compared with the FLS genotype.

Journal Article↗

PubMed2002

Optimizing ventricular fibers: uniform strain or stress, but not ATP consumption, leads to high efficiency.

The aim of this study was to investigate the influence of fiber orientation in the left ventricular (LV) wall on the ejection fraction, efficiency, and heterogeneity of the distributions of developed fiber stress, strain and ATP consumption. A finite element model of LV mechanics was used with active properties of the cardiac muscle described by the Huxley-type cross-bridge model. The computed variances of sarcomere length (SL(var)), developed stress (DS(var)), and ATP consumption (ATP(var)) have several minima at different transmural courses of helix fiber angle. We identified only one region in the used design space with high ejection fraction, high efficiency of the LV and relatively small SL(var), DS(var), and ATP(var). This region corresponds to the physiological distribution of the helix fiber angle in the LV wall. Transmural fiber angle can be predicted by minimizing SL(var) and DS(var), but not ATP(var). If ATP(var) was minimized, then the transverse fiber angle was considerably underestimated. The results suggest that ATP consumption distribution is not regulating the fiber orientation in the heart.

Adenosine Triphosphate↗

PubMed2006

Epicardium-derived cells are important for correct development of the Purkinje fibers in the avian heart.

During embryonic development, the proepicardial organ (PEO) grows out over the heart surface to form the epicardium. Following epithelial-mesenchymal transformation, epicardium-derived cells (EPDCs) migrate into the heart and contribute to the developing coronary arteries, to the valves, and to the myocardium. The peripheral Purkinje fiber network develops from differentiating cardiomyocytes in the ventricular myocardium. Intrigued by the close spatial relationship between the final destinations of migrating EPDCs and Purkinje fiber differentiation in the avian heart, that is, surrounding the coronary arteries and at subendocardial sites, we investigated whether inhibition of epicardial outgrowth would disturb cardiomyocyte differentiation into Purkinje fibers. To this end, epicardial development was inhibited mechanically with a membrane, or genetically, by suppressing epicardial epithelial-to-mesenchymal transformation with antisense retroviral vectors affecting Ets transcription factor levels (n=4, HH39-41). In both epicardial inhibition models, we evaluated Purkinje fiber development by EAP-300 immunohistochemistry and found that restraints on EPDC development resulted in morphologically aberrant differentiation of Purkinje fibers. Purkinje fiber hypoplasia was observed both periarterially and at subendocardial positions. Furthermore, the cells were morphologically abnormal and not aligned in orderly Purkinje fibers. We conclude that EPDCs are instrumental in Purkinje fiber differentiation, and we hypothesize that they cooperate directly with endothelial and endocardial cells in the development of the peripheral conduction system.

Animals↗

PubMed1997

Identification of a cotton fiber-specific acyl carrier protein cDNA by differential display.

Transcripts from immature fibers and stripped ovules (fibers removed) of cotton (Gossypium hirsutum L.) were compared by differential display to identify cDNA fragments that represent mRNAs that are expressed primarily in cotton fibers. Eight independent fiber-specific cDNA fragments were isolated. One of these cDNAs had strong sequence similarity with acyl carrier protein (ACP). A full-length cDNA for the cotton fiber-specific ACP was isolated using a PCR cDNA library screening technique. This 713 bp cDNA has an open reading frame that encodes a 136 amino acid polypeptide. Overall nucleotide and amino acid sequence identities with other plant ACP gene sequences averaged 66% and 60% respectively. A 19 amino acid sequence surrounding the prosthetic group attachment site is nearly identical to other plant ACP genes. Northern blot analyses showed that transcripts homologous to this fiber-specific ACP cDNA were predominantly expressed during the elongation stage of fiber development. Initial genomic Southern blot analysis indicated that a single copy of the fiber-specific ACP gene may be present in both the cotton A and D genomes, since diploid Gossypium species with A or D genomes gave identical bands. We speculate that this putative fiber-specific ACP may play an important role in rapidly elongating cotton fibers by contributing to the synthesis of membrane lipids. It is also apparent that during the evolution of cotton a member of the ACP gene family has been recruited for specific expression in cotton fibers.

Acyl Carrier Protein↗

PubMed1998

Simian virus 40 and human cancer.

Deoxyribonucleic acid (DNA) oncoviruses can induce neoplastic transformation by interfering with proliferative proteins. Simian virus 40 (SV40) has been shown to induce brain tumors, osteosarcoma, lymphoid tumors and malignant mesothelioma in hamsters and SV40-like DNA sequences corresponding to the Rb-pocket binding domain of SV40 T-antigen (Tag) have been detected in the same human tumors. Since only a small percentage of people exposed to asbestos fibers develop a malignant mesothelioma, SV40 has been suspected to co-operate with the fibers in the neoplastic transformation or even to itself induce the onset of malignant mesothelioma in patients without expositive history. The mechanism that seems to be involved in the SV40-induced carcinogenesis process is mediated by interaction of Tag, both with p53 and Rb proteins, leading to their functional inactivation that is responsible for the removal of their inhibitory cell cycle effect which determines the increase of the number of cells entering the G1-S phase. Up to now the source of SV40 human infections has not yet been completely identified even though administration from 1957-1965 of SV40 contaminated polio vaccines is highly suspected. Horizontal infection by sexual transmission has been also hypothesized. Due to the important public health implications further investigations are required in order to establish both the source and the carcinogenetic role of simian virus 40 in humans.

Animals↗

PubMed2000

Formation of sarcomeres in developing myotubes: role of mechanical stretch and contractile activation.

In a series of experiments, cultured myotubes were exposed to passive stretch or pharmacological agents that block contractile activation. Under these experimental conditions, the formation of Z lines and A bands (morphological structures, resulting from the specific structural alignment of sarcomeric proteins, necessary for contraction) was assessed by immunofluorescence. The addition of an antagonist of the voltage-gated Na(+) channels [tetrodotoxin (TTX)] for 2 days in developing rat myotube cultures led to a nearly total absence of Z lines and A bands. When contractile activation was allowed to resume for 2 days, the Z lines and A bands reappeared in a significant way. The appearance of Z lines or A bands could not be inhibited nor facilitated by the application of a uniaxial passive stretch. Electrical stimulation of the cultures increased sarcomere assembly significantly. Antagonists of L-type Ca(2+) channels (verapamil, nifedipine) combined with electrical stimulation led to the absence of Z lines and A bands to the same degree as the TTX treatment. Western blot analysis did not show a major change in the amount of sarcomeric alpha-actinin nor a shift in myosin heavy chain phenotype as a result of a 2-day passive stretch or TTX treatment. Results of experiments suggest that temporal Ca(2+) transients play an important factor in the assembly and maintenance of sarcomeric structures during muscle fiber development.

Actinin↗

PubMed1982

Population of muscle fibers during postnatal development in the rat hindlimb.

Changes in the numbers of muscle fibers during the postnatal development (1, 7, 14 and 21 days old) of rat EDL and SOL muscles were studied. Quantitative analysis of fibers was performed using cryosectioned specimens. The 1 micrometer-thick sections were taken from epon-embedded blocks of these muscle, and light micrographs were obtained. Thereafter, the same 1 micrometer-thick specimens were embedded again for EM studies. Many small cells, which could not be confirmed as myofibers at the light microscopic level, were identified as skeletal muscle fibers with certainty by electron microscopy. The population of muscle fibers apparently increased by ca. 45% in the 7-day-old rat and by ca. 67% in the 21-day-old rat, when compared with 1-day-old rat EDL and SOL muscles. This number increased gradually during postnatal development over several weeks. It was suggested that a small cell (myofiber) adjacent to a large myofiber with adherent junctions and with a common basement membrane develops into a real muscle fiber.

Aging↗

PubMed1996

Heat-shock protein 90 and ubiquitin: developmental regulation during myogenesis.

Heat-shock/stress proteins are constitutive and stress-inducible proteins, regulated by a number of factors including developmental processes. The 90-kD heat-shock protein (hsp90) and ubiquitin are up-regulated in regenerating fibers and diseased fibers of Duchenne muscular dystrophy. The aim of the present study was to investigate whether the heat-shock response in regenerating fibers is developmentally regulated or disease-associated. Immunohistochemistry and immunoblot analysis were employed to compare the expression of hsp90 and ubiquitin in normal immature muscle from infants and regenerating fibers in polymyositis and dermatomyositis with the basal expression in normal mature muscle from adults. A significant up-regulation of hsp90 and ubiquitin in regenerating fibers and developing infantile fibers suggests that hsp90 and ubiquitin, during myogenesis, are largely regulated by the activation of developmental mechanisms rather than being primarily disease-related. Modulation of the stress response may promote myogenesis and provide a new therapeutic approach in myopathies.

Adult↗

PubMed1975

The development of sympathetic innervation and the functional state of the cardiovascular system in newborn dogs.

The present study in dogs indicates that the peripheral sympathetic fibers develop mostly after birth and reach a full maturity at about 2 months of life. The norepinephrine content of the heart, spleen, intestine, salivary glands, and adrenal glands increased from birth to 56 days of age. In contrast, the content of the stellate ganglia decreased during this period. In most of the organs studied, the uptake of [3H] norepinephrine developed in parallel with the norepinephrine content, except in the right atrium and salivary glands where it was fully developed soon after birth. During development, the systemic blood pressure increased from 40 to 100 mm Hg. Bilateral adrenal vessel clamping failed to induce a fall in blood pressure in growing dogs which indicates that the adrenal medulla or the baroreceptors did not fully compensate for the lack of peripheral sympathetic fibers and for the lower blood pressure in newborn animals. Although cardiac norepinephrine content was still very low in 10-day-old animals, cardiovascular responses to direct and reflex sympathetic stimulation were similar to those observed in 56-day-old animals. These results indicate that the sympathetic nervous system becomes functional before the fibers reach their full maturity.

Adrenal Glands↗

PubMed1987

Early uncrossed component of the developing optic nerve with a short extracerebral course: a light and electron microscopic study of fetal ferrets.

During the study of the developing optic nerve described in the preceding paper (Guillery and Walsh, '87), small bundles of nerve fibers were seen passing between the optic nerve and the ipsilateral hypothalamus of 24-to 27-day-old prenatal ferrets. The bundles appear before any other fiber groups of the retinofugal pathway and are identifiable while the main portions of the retinofugal system are growing into the optic tracts. The bundles, made up of 50 or more axons, leave the optic nerve, emerge through the otherwise continuous layer of subpial glia and through the basal lamina of the nerve, run a short, naked, extracerebral course among collagen fibers and presumed fibroblasts, and then re-enter the central nervous system, passing rostrally and dorsally to the superficial parts of the ipsilateral hypothalamus away from the region of the chiasm. These fibers represent the earliest link between the optic nerve and the brain, but their course is not followed by the majority of retinofugal fibers developing later, which pass toward one or the other optic tract.

Animals↗

PubMed1986

Prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemical study.

Prenatal development of the GABAergic system in the rat brain has been studied using an antiserum to GABA-glutaraldehyde-hemocyanin conjugates, specific for GABAergic neurons. The gamma-aminobutyric acid (GABA) system has been found to differentiate very early relative to other transmitter-identified neurons, such that by embryonic day 13 a well developed fiber network exists in the brainstem, mesencephalon and diencephalon, including a large projection in the posterior commissure and adjacent areas on the surface of the mesencephalon and tectum. Although no cell bodies are visible at this time, it appears that these fibers originate from the caudal brainstem and spinal cord. GABAergic cell bodies begin to appear on embryonic day 14 in the lateral cortical anlage. By embryonic day 16, they are also visible in the basal forebrain and in all regions of cortex where they are located in three zones: in layer I, below the cortical plate, and in the intermediate zone. Also contained in the outer part of layer I is a dense fiber plexus which stains intensely for GABA. These fibers may be part of the first contingent of cortical afferents to invade the telencephalic vesicle, an event which is thought to be a stimulus for the beginning of neuronal differentiation in this region. By E18, two bands of immunoreactivity are visible in layer I, which probably contain both cell bodies and fibers. The trajectories taken by growing GABAergic fibers in the brainstem, mesencephalon and diencephalon at embryonic day 13 and at subsequent stages of development are coincident with regions of both monoaminergic and peptidergic differentiation and appear to correspond to recently reported patterns of benzodiazepine receptors which appear slightly later. The early differentiation of the GABAergic system could indicate a trophic role for GABA in early brain development, possibly involving receptors for this neurotransmitter or related substances.

Animals↗

PubMed2003

Evolution and expression of MYB genes in diploid and polyploid cotton.

R2R3-MYB transcription factors have been implicated in a diversity of plant-specific processes. Among the functions attributed to myb factors is the determination of cell shape, including regulation of trichome length and density. Because myb transcription factors are likely to play a role in cotton fiber development, the molecular evolutionary properties of six MYB genes previously shown to be expressed in cotton fiber initiation were examined. In accordance with their presumed central role, each of the genes display conservative substitution patterns and limited sequence divergence in diploid members of the genus Gossypium, and this pattern is conserved in allotetraploid cottons. In contrast to highly reiterated rDNA repeats, GhMYB homologues (duplicated gene pairs) exhibit no evidence of concerted evolution, but instead appear to evolve independently in the allopolyploid nucleus. Expression patterns for the MYB genes were examined in several organs to determine if there have been changes in expression patterns between the diploids (G. raimondii and G. arboreum) and the tetraploid (G. hirsutum) or between the duplicated copies in the tetraploid. Spatial and temporal expression patterns appear to have been evolutionarily conserved, both during divergence of the diploid parents of allopolyploid cotton and following polyploid formation. However, the duplicated copies of MYB1 in the tetraploid are not expressed at equal levels or equivalently in all organs, suggesting possible functional differentiation.

Blotting, Southern↗

PubMed1993

Differential effects of experimentally induced chronic pancreatitis on neuropeptide immunoreactivities in the feline pancreas.

The distribution and concentration of calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and gastrin-releasing peptide (GRP) immunoreactivities in the pancreas of cats with experimentally induced chronic pancreatitis and of age- and sex-matched controls were investigated. By narrowing the main pancreatic duct between the head and the body to approximately 25% of its normal diameter, we induced within 5 weeks chronic pancreatitis restricted to the body and tail. In control animals, peptide immunoreactive nerves were distributed to the islets, acini, and ducts; the latter were predominantly innervated by fibers immunoreactive for NPY, VIP, or CGRP. The vasculature received an abundant supply of NPY-, CGRP-, and, to a lesser extent, SP-containing axons. Within intrapancreatic ganglia, peptide immunoreactivities were identified in fibers and ganglion cells, with the exception of CGRP and SP immunostaining, which could be visualized only in fibers. In animals with chronic pancreatitis, the innervation pattern of each peptidergic system was comparable to that described in controls. However, there was a remarkable increase in the density and staining intensity of VIP and NPY immunoreactive fibers in the exocrine parenchyma and fibrous septa of the body and tail, where chronic pancreatitis developed. Fibers immunoreactive for CGRP and SP also were moderately denser than in controls, whereas those containing GRP immunoreactivity did not show any detectable changes. In addition, a marked increase of the immunostaining for VIP and, to a much lesser extent, for NPY and GRP, was observed in neurites supplying the head of the pancreas, which appeared devoid of histologically detectable pathological alterations. Radioimmunoassay analysis confirmed the immunohistochemical observations. The increased density of distinct peptidergic nerves in the pancreas with induced chronic pancreatitis might be the result of compensatory phenomena in response to the inflammatory process.

Animals↗

PubMed1988

Innervation of developing intrafusal muscle fibers in the rat.

The chronology of development of spindle neural elements was examined by electron microscopy in fetal and neonatal rats. The three types of intrafusal muscle fiber of spindles from the soleus muscle acquired sensory and motor innervation in the same sequence as they formed--bag2, bag1, and chain. Both the primary and secondary afferents contacted developing spindles before day 20 of gestation. Sensory endings were present on myoblasts, myotubes, and myofibers in all intrafusal bundles regardless of age. The basic features of the sensory innervation--first-order branching of the parent axon, separation of the primary and secondary sensory regions, and location of both primary and secondary endings beneath the basal lamina of the intrafusal fibers--were all established by the fourth postnatal day. Cross-terminals, sensory terminals shared by more than one intrafusal fiber, were more numerous at all developmental stages than in mature spindles. No afferents to immature spindles were supernumerary, and no sensory axons appeared to retract from terminations on intrafusal fibers. The earliest motor axons contacted spindles on the 20th day of gestation or shortly afterward. More motor axons supplied the immature spindles, and a greater number of axon terminals were visible at immature intrafusal motor endings than in adult spindles; hence, retraction of supernumerary motor axons accompanies maturation of the fusimotor system analogous to that observed during the maturation of the skeletomotor system. Motor endings were observed only on the relatively mature myofibers; intrafusal myoblasts and myotubes lacked motor innervation in all age groups. This independence of the early stages of intrafusal fiber assembly from motor innervation may reflect a special inherent myogenic potential of intrafusal myotubes or may stem from the innervation of spindles by sensory axons.

Animals↗

PubMed2003

Transscleral thermotherapy: short- and long-term effects of transscleral conductive heating in rabbit eyes.

OBJECTIVE: To determine the highest safe treatment temperature, at 30- and 60-second exposure durations, for transscleral thermotherapy (TSTT) of choroidal melanoma. METHODS AND DESIGN: Transscleral conductive heating was performed in 15 rabbits at 50 degrees C to 70 degrees C for 30 or 60 seconds. The thermal lesions in the ocular fundus were monitored for 4 months with ophthalmoscopic, photographic, and fluorescein angiographic examination. Histologic examination included polarized light microscopy. RESULTS: The effect of TSTT was similar for both exposure durations. Vascular occlusion in the retina and choroid developed at temperatures of 55 degrees C and higher. After heating at 60 degrees C, scleral collagen fibers developed a minimal undulation; at 65 degrees C, they became clearly undulated. The undulation resolved in the 3 to 4 months after heating. Heating at 70 degrees C caused persistent severe damage to the sclera. Retinal tears developed after heating at 65 degrees C and 70 degrees C. CONCLUSIONS: A temperature of 65 degrees C was found to be the highest temperature that did not cause permanent damage to the sclera at both exposure durations. A temperature of 60 degrees C may be the optimal temperature for TSTT of choroidal melanoma because retinal tears may develop at 65 degrees C. CLINICAL RELEVANCE: In TSTT, the temperature levels reached are cytotoxic for choroidal melanoma as well as intrascleral tumor cells. Occlusion of choroidal vessels induced by TSTT may contribute to tumor necrosis because these vessels serve as feeder vessels for the tumor.

Animals↗

PubMed2007

[Pathophysiology of pain].

Clinically relevant pain states are usually characterized as either inflammatory or neuropathic. While inflammatory pain results from tissue injury or damage, neuropathic pain results from damage or disease of nerve fibers. In either pain state, both the peripheral and the central nociceptive system contribute significantly to the generation of pain. During inflammation peripheral nociceptors ("pain fibers") are sensitized (peripheral sensitization), and upon nerve injury or nerve disease peripheral nerve fibers develop ectopic discharges originating from the site of the nerve lesion or the cell body of damaged fibers. As a consequence a complex neuronal response is evoked in the spinal cord where neurons become hyperexcitable (central sensitization).Central sensitization is a neuronal process that amplifies the activity from the periphery. Numerous molecular mechanisms are involved in peripheral and central nociceptive processes including rapid functional changes of signaling (increase of excitability) and long-term regulatory changes such as upregulation of mediator/receptor systems. The conscious pain is generated by thalamocortical networks that produce both sensory discriminative and affective components of the pain response.

Animals↗