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Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers.

In animals, the small GTP-binding proteins, Rac and Rho, of the ras superfamily participate in the signal transduction pathway that regulates the organization of the actin cytoskeleton. We report here on the characterization of two distinct cDNA clones isolated from a cotton fiber cDNA library that code for homologs of animal Rac proteins. Using gene-specific probes, we have determined that amphidiploid cotton contains two genes that code for each of the two Rac proteins, designated Rac13 and Rac9, respectively. The gene for Rac13 shows highly enhanced expression in developing cotton fibers, with maximal expression occurring at the time of transition between primary and secondary wall synthesis. This is also the time at which reorganization of the cytoskeleton occurs, and thus the pattern of expression of Rac13 is consistent with its possible role, analogous to animal Rac, in the signal transduction pathway that controls cytoskeletal organization.

Amino Acid Sequence

Spatial association of renin-containing cells and nerve fibers in developing rat kidney.

The development of renin-containing cells and nerve fibers was studied in Sprague-Dawley rat kidneys during the last third of gestation and the first 15 days of postnatal life. Kidney tissue sections were stained for nerve fibers or double stained employing an anti-rat renin polyclonal antibody and a monoclonal antibody (TUJ1) directed against a neuron-specific class III beta-tubulin isotype. Renin-containing cells and nerve fibers were detected at 17 days of gestation, in close spatial relationship along the main branches of the renal artery. During fetal life, renin-containing cells and nerve fibers were spatially associated along arcuate and interlobular arteries, renin-containing cells being also present throughout the entire length of afferent arterioles supplying juxtamedullary glomeruli. During postnatal life the distribution of renin-containing cells progressively shifted to a restricted juxtaglomerular position in afferent arterioles. Simultaneously, density and organization of nerve fibers increased with age along the arterial vascular tree. Our results suggest that innervation of renin-containing cells is present in fetal life and follows the centrifugal pattern of renin distribution and nephrovascular development.

Animals

Isometric tension differences in fibers of red and white muscles.

Measurements of maximum isometric tension and rate of onset of tension in fibers of glycerinated rat muscle indicate that the red soleus fibers develop significantly higher tensions than do white medial gastrocnemius fibers. Gastrocnemius fibers develop tension at a significantly faster rate than soleus fibers, however. These differences seem to be related to formation of actinmyosin cross-links.

Animals

Uninjured C-fiber nociceptors develop spontaneous activity and alpha-adrenergic sensitivity following L6 spinal nerve ligation in monkey.

We investigated whether uninjured cutaneous C-fiber nociceptors in primates develop abnormal responses after partial denervation of the skin. Partial denervation was induced by tightly ligating spinal nerve L6 that innervates the dorsum of the foot. Using an in vitro skin-nerve preparation, we recorded from uninjured single afferent nerve fibers in the superficial peroneal nerve. Recordings were made from 32 C-fiber nociceptors 2-3 wk after ligation and from 29 C-fiber nociceptors in control animals. Phenylephrine, a selective alpha1-adrenergic agonist, and UK14304 (UK), a selective alpha2-adrenergic agonist, were applied to the receptive field for 5 min in increasing concentrations from 0.1 to 100 microM. Nociceptors from in vitro control experiments were not significantly different from nociceptors recorded by us previously in in vivo experiments. In comparison to in vitro control animals, the afferents found in lesioned animals had 1) a significantly higher incidence of spontaneous activity, 2) a significantly higher incidence of response to phenylephrine, and 3) a higher incidence of response to UK. In lesioned animals, the peak response to phenylephrine was significantly greater than to UK, and the mechanical threshold of phenylephrine-sensitive afferents was significantly lower than for phenylephrine-insensitive afferents. Staining with protein gene product 9.5 revealed an approximately 55% reduction in the number of unmyelinated terminals in the epidermis of the lesioned limb compared with the contralateral limb. Thus uninjured cutaneous C-fiber nociceptors that innervate skin partially denervated by ligation of a spinal nerve acquire two abnormal properties: spontaneous activity and alpha-adrenergic sensitivity. These abnormalities in nociceptor function may contribute to neuropathic pain.

Adrenergic alpha-Agonists

Functional significance of myosin transitions in single fibers of developing soleus muscle.

The maximal velocity of shortening and myosin heavy chain (MHC) composition of single, chemically skinned fibers from neonatal and adult rat soleus muscles were examined to determine the relationship between these parameters during slow muscle development in the rat. In addition, the MHC composition of bundles of fibers from soleus muscles at the same ages was studied. The MHC compositions were examined using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The results from the bundles of fibers indicate that from 3 days to 5 mo postnatal, the rat soleus contains predominantly MHCs that migrate in the vicinity of the MHC from adult slow muscle. From 14 days to 2 mo postnatal, there are also significant amounts of additional MHCs that comigrate on SDS gels with those characteristic of adult rat fast muscle. All the fibers studied at 3 and 7 days postnatal and at 5 mo and the majority of fibers from 14 days to 2 mo postnatal had relatively low shortening velocities. A few fibers from the latter group had significantly higher velocities. The faster fibers at each age had greater amounts of the MHCs that comigrate with the adult fast-type MHC on SDS gels. Thus the velocity of shortening of single fibers from the rat soleus muscle appears to be related to MHC composition during postnatal development.

Animals

Gonadotropin-releasing hormone containing neurons and olfactory fibers during development: from lamprey to mammals.

Gonadotropin releasing-hormone (GnRH) regulates the hypothalamo-pituitary-gonadal axis in all vertebrates. The vast majority of GnRH neurons are thought to be derived from progenitor cells in medial olfactory placodes. Several antibodies and lectins that recognize cell surface carbohydrates have been useful for delineating the migratory pathway from the olfactory placodes and vomeronasal organ, through the nasal compartment, and across the cribriform plate into the brain. In rats, alpha-galactosyl-linked glycoconjugates (immunoreactive with the CC2 monoclonal antibody) are expressed on fibers along the GnRH migration pathway and approximately 10% of the GnRH neuronal population. In lamprey, the alpha-galactosyl binding lectin, Grifonia simplicifolia-I (GS-1), identifies cells and fibers of the developing olfactory system. In contrast to the CC2 immunoreactive GnRH neurons in rats, the GS-1 does not label a subpopulation of presumptive GnRH neurons in lamprey. Results from these and other experiments suggest that GnRH neurons in developing lamprey do not originate within the olfactory placode, but rather within proliferative zones of the diencephalon. However, the overlap of olfactory- and GnRH-containing fibers from prolarval stages to metamorphosis, suggest that olfactory stimuli may play a major role in the regulation of GnRH secretion in lamprey throughout life. By contrast, olfactory fibers are directly relevant to the migration of GnRH neurons from the olfactory placodes in mammalian species. Primary interactions between olfactory fibers and GnRH neurons are likely transient in mammals, and so in later life olfactory modulation of GnRH secretion is likely to be indirect.

Aging

Effects of clofilium on ischemic subendocardial Purkinje fibers 1 day postinfarction.

One day after ligation of the canine anterior descending coronary artery, clofilium, a long-acting class III antiarrhythmic agent, was studied for its effects on normal and ischemic Purkinje fibers. In normal Tyrode's solution (4 mM potassium, 2.7 mM calcium) clofilium (10(-7) to 10(-5) M) increased action potential duration. Although only 2 of 10 normal Purkinje fibers developed early after depolarizations and early afterdepolarization-initiated triggered activity, 10 of 11 ischemic Purkinje fibers developed these features. Consequently, action potentials in ischemic fibers were prolonged to durations greater than 10 s. The triggered activity in the ischemic Purkinje fibers produced repetitive activity in adjacent normal ventricular muscle. In vivo, 3 days after ligation, the administration of 3 to 10 mg/kg clofilium induced grouped beating. Action potentials recorded subsequently from these same hearts in vitro showed early afterdepolarizations, triggered activity and a similar grouping of responses. Therefore, clofilium differentially produced early afterdepolarizations in ischemic Purkinje fibers. This is a mechanism by which clofilium could be arrhythmogenic in an ischemic heart.

Action Potentials

Development of the retinotectal system in normal quail embryos: cytoarchitectonic development and optic fiber innervation.

The development of the optic tectum and the establishment of retinotectal projections were investigated in the quail embryo from day E2 to hatching day (E16) with Cresyl violet-thionine, silver staining and anterograde axonal tracing methods. Both tectal cytodifferentiation and retinotectal innervation occur according to a rostroventral-caudodorsal gradient. Radial migration of postmitotic neurons starts on day E4. At E14, the tectum is fully laminated. Optic fibers reach the tectum on day E5 and cover its surface on day E10. 'Golgi-like' staining of optic fibers with HRP injected in vitro on the surface of the tectum reveals that: growing fronts are formed exclusively by axons extending over the tectal surface; fibers penetrating the outer tectal layers are always observed behind the growing fronts; the penetrating fibers are either the tip of the optic axons or collateral branches; as they penetrate the tectum, optic fibers give off branches which may extend for long distances within their terminal domains; the optic fiber terminal arbors acquire their mature morphology by day E14. The temporal sequence of retinotectal development in the quail was compared to that already established for the chick, thus providing a basis for further investigation of the development of the retinotectal system in chimeric avian embryos obtained after xenoplastic transplantation of quail tectal primordia into the chick neural tube.

Age Factors

Metabolic specialization in fast and slow muscle fibers of the developing rat.

Individual fibers of prospective fast (extensor digitorum longus; EDL) and slow (soleus) muscles of rats have been analyzed to determine the profiles of key energy-generating enzymes at successive stages of postnatal development. Mean activities of lactate dehydrogenase (LDH) and adenylokinase (AK), 2 enzymes associated with contractile function, are significantly different in the 2 fiber populations at birth; furthermore, wide variations in enzyme activities exist among the individual fibers. There is a progressive refinement of enzyme levels in the soleus into a more uniform fiber population, while the fibers in the EDL progressively diverge into 2 distinct phenotypes. Changes in EDL and soleus are punctuated by periods of rapid change, with the period between 10 and 21 d being most eventful. Generally, the maturation profiles of LDH and AK coincide with the transition from neonatal to adult fast myosins and closely reflect the timing of energy demands imposed by contractile activity patterns. In contrast, activities of the oxidative enzymes malate dehydrogenase and beta-hydroxyacyl CoA dehydrogenase are similar in both muscles at birth and steadily increase during the first 3 weeks, suggesting a progressive adaptation to the aerobic extrauterine environment. After 30 d, there are differential changes in the oxidative profiles of enzymes for fatty acid and glucose metabolism. The profiles follow dietary changes associated with weaning, which suggests a phenotypic dependence of neonatal muscle on the particular available energy substrate. All enzymes are low in all fibers of EDL and soleus at birth, indicating their modest metabolic capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxyacyl CoA Dehydrogenases

An experimental study in dogs of transseptal fiber arrangement between teeth which have emerged in rotated or non-rotated positions.

Transseptal fibers of the periodontal ligament are thought to play a role in relapse after orthodontic derotation. The aim of this study was to compare the location of the attachment site and the arrangement of the transseptal fibers in rotated emerged and non-rotated emerged teeth. In five dogs, unilaterally, the first upper premolars were replaced into a rotated position, before emergence. Histological evaluation showed that transseptal fibers developed in the experimental as well as in the control specimens. In all cases, transseptal fibers bridged the shortest distance between two adjacent teeth. It was concluded that the attachment site of the transseptal fibers was not determined by the tooth anatomy itself, but by the tooth position and its orientation in the dental arch during transseptal fiber development.

Animals

The influence of maternal nutrition on muscle fiber number development in the porcine fetus and on subsequent postnatal growth.

In the pig, undernutrition in utero causes low birth weight, a decrease in muscle fiber number, and a reduction in postnatal growth rate. The effect on fiber number is mediated via a reduced secondary fiber population. Within a litter of pigs, lighter-weight pigs have probably suffered some deficit in muscle fiber number. In an attempt to improve the number of fibers in the lighter-weight pig fetuses, four maternal feeding regimens were used, one serving as the control. Maternal feed intake was doubled for one of three time periods during pregnancy: 1) d 25 to 50 (HE) immediately before fiber hyperplasia; 2) d 50 to 80 (HL) during fiber hyperplasia; or 3) d 25 to 80 (HT) covering both developmental events. Controls were fed at levels routinely used for pregnant sows on the farm. Sows farrowed normally and pig birth weights were recorded. Estimates were made of total myofiber number, total primary fiber number, and mean secondary:primary fiber number ratio (S:P) for the semitendinosus of each pig at 5 wk postnatal or 80 kg (HT and two control litters only). The progeny of all supplemented sows had a significantly greater mean S:P ratio (P < .05), and the HE pigs tended to have a greater number of muscle fibers than control pigs (403,840 +/- 8,197 vs 370,970 +/- 12,720). Postnatal growth rate to 80 kg was also investigated for the HT group of pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance

Distribution of histamine-, 5-hydroxytryptamine-, and tyrosine hydroxylase-immunoreactive neurons and nerve fibers in developing rat brain.

Although the general patterns of the developing histaminergic system in the rat brain are known, no comparative studies between the development of the brain histaminergic system and the development of other neuroactive substances have yet been published. Interestingly, separate immunohistochemical studies on the development of the 5-HT system and on the catecholaminergic system in the rat imply common features in the different aminergic systems. Therefore, the spatial distribution of histamine-immunoreactive (HA-ir) neurons and nerve fibers was compared to the distribution of 5-hydroxytryptamine (5-HT)-, and tyrosine hydroxylase-immunoreactive (TH-ir) ones in the developing rat brain between embryonic days 12 (E12) and 20 (E20) by using a double-immunostaining method. The high-pressure liquid chromatography (HPLC) fluorometric method was used for determination of histamine concentration in different brain regions during the same period of development and synthetic oligonucleotide probes complementary to the rat histidine decarboxylase (HDC) to determine the origin of HA in the brain during the development with in situ hybridization. The immunohistochemical results revealed co-localization of HA and 5-HT within a subgroup of cells in the developing raphe nuclei between E14 and E18. From E18 onwards HA immunoreactivity started to gradually disappear from the rhombencephalon, and was totally abolished by E20, while 5-HT-ir cells continued to establish their adult positions. No significant colocalization of HA and TH immunoreactivities was detected. The biochemical results were in agreement with the immunohistochemical ones and confirmed that histamine detected in the early developing brain is authentic. A positive in situ hybridization signal for HDC was detected in a small area in the ventrolateral pons in the same areas as HA- and HDC-ir cell bodies at E16, suggesting that at least some HA may be synthesized locally. These results confirm that HA is one of the first neurotransmitters to appear in the developing brain. In addition, the transient co-localization of HA and 5-HT immunoreactivities and the transient HDC expression at E16 within the developing pontine raphe nuclei may imply an interesting and a more general role for HA in modification of brain development.

Animals

Elastic fiber during development and aging.

Elastin molecules aggregate in the extracellular space where they are crosslinked by stable desmosine bridges. The resulting polymer is structurally organized as branched fibers and lamellae, which, in skin, are wider (a few microns) in the deep dermis and become progressively thinner (fraction of a micron) towards the papillary dermis. Several general and local factors seem to regulate elastin gene expression, deposition and degradation. In skin, the volume density of the elastin network increases from birth up to maturity, when it accounts for about 3-4% of the tissue. However, its amount and distribution depend on dermis areas, which are different among subjects and change with age. Several matrix molecules (glycosaminoglycans, decorin, biglycan, osteopontin) have been found to be associated with elastin into the normal fiber, and several others have been recognized within pathologic elastic fiber (osteonectin, vitronectin, alkaline phosphatase in PXE). With age, and in some pathologic conditions, skin elastin may undergo irreversible structural and compositional changes, which seem to progress from localized deposition of osmiophilic materials to the substitution of the great majority of the amorphous elastin with interwoven filaments negative for elastin specific antibodies.

Adult

Histochemistry and acetylcholine receptor distribution in normal and denervated monkey extraocular muscles.

In monkey extraocular muscles (EOM), a battery of histochemical reactions delineates three muscle fiber types, coarse, fine, and granular. Normal EOM are compared with EOM denervated by intracranial oculomotor nerve section. The experimentally denervated EOM fibers did not show the constellation of histologic responses typical of denervated limb muscle, making a diagnosis of a denervation process in EOM muscle very difficult. Although the denervated fine and granular fibers (but not the coarse fibers) develop diffuse extrajunctional acetylcholine receptors (AChR) following experimental denervation, this is not a reliable criterion of denervation because not all of those fibers developed it and they did not show it beyond a 12-week period following nerve section; moreover, myopathic mechanisms have previously been shown capable of provoking diffuse extrajunctional AChR in limb-muscle fibers.

Acetylcholine

Formation of myoneural and myotendinous junctions in the chick embryo. Role of acetylcholinesterase-rich granules in the developing muscle fibers.

The mode of formation of the myoneural and myotendinous junctions was investigated in the thigh muscles of the chick embryo. Myotendinous junctions first appeared on day 11 of incubation, whereas myoneural junctions developed on day 12. Intracellular AChE activity in the muscles increased by the 12th day of incubation, and decreased rapidly after the formation of the myoneural junctions. Light and electron microscopically, AChE activity was demonstrated in the nuclear envelope, sarcoplasmic reticulum, Golgi complex, and in large granules which appeared to be derived from the Golgi complex. Large granules showing an intense AChE activity accumulated in the sarcoplasm at the poles of the muscle fiber before the formation of myotendinous junctions. After the translocation of this intracellular enzyme onto the sarcolemma, most likely the result of an exocytosis of the granules, the myotendinous junctions were formed. The AChE-rich granules present in the middle of myotubes developed into spindle- or comma-shaped cisternae which were located in the sarcoplasm just below the presumptive motor endplates. The present results suggest that the transport of AChE-rich granules to the sarcolamma is the first step in the formation of myoneural and myotendinous junctions.

Acetylcholinesterase

Development of elastic fibers of nuchal ligament, aorta, and lung of fetal and postnatal sheep: an ultrastructural and electron microscopic immunohistochemical study.

The morphogenesis of elastic fibers of the nuchal ligament, aorta, and lung of sheep was studied by light microscopy, transmission electron microscopy, and immunohistochemical methods for the detection of elastin. The degree of maturation of the amorphous materials of elastic fibers was assessed morphologically in preparations stained by the tannic acid and periodic acid methenamine-silver methods. With both of these methods, the amorphous components of mature fibers stained less intensely than did those of immature fibers. Elastic fibers in early stages of development consisted of many microfibrils and few, small, branching masses of immature amorphous material. Thicker fibers were formed by the coalescence of growing masses of amorphous materials. In late stages of formation of elastic fibers, the mature amorphous materials were associated with few microfibrils; and they were partially surrounded by immature amorphous materials associated with many microfibrils. Antielastin antibody reacted evenly with amorphous materials in very early stages of elastic-fiber development, but reacted only with the other zones of amorphous materials in later stages; it also reacted with the microfibrils in all stages. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin on their surfaces. This conclusion is in agreement with ultrastructural observations showing 1) that development of microfibrils precedes that of the amorphous material and 2) that the microfibrils adjacent to the immature amorphous materials are covered with small amounts of tannic acid-positive amorphous materials. These observations suggest that microfibrils serve as sites for elastin deposition, both in early elastogenesis and in subsequent growth of elastic fibers. However, the nature of the interaction between elastin and microfibrils remains unknown.

Animals

Structural characterization of genes corresponding to cotton fiber mRNA, E6: reduced E6 protein in transgenic plants by antisense gene.

Two genes, each corresponding to fiber mRNA E6, were isolated from cotton cultivars Coker 312 (Gossypium hirsutum L.) and Sea Island (G. barbadense L.). E6 is one of the predominant fiber-specific mRNAs present during early fiber development. The distinguishing feature of nucleotide-derived E6 protein is the presence of a motif where a dimer, Ser-Gly, is repeated several times. Two of the Sea Island genes contained a pentameric motif, Ser-Gly, while one of the Coker genes had one and the other had four motifs each. cDNA clones containing one or five Ser-Gly motifs were also identified. Thus, it appears that the strict conservation of this motif may not be critical to E6 protein function. Sequence characterizations of the genes and cDNAs showed that multiple members of the E6 family are transcribed in fiber and may result in proteins 238 to 246 amino acids long. The 3' ends of the genes and cDNAs showed considerable heterology among them. Transgenic plants containing antisense genes were generated to decipher E6 function. Transgenic cotton with reduced E6 protein levels in the range of 60 to 98% were identified. However, no discernible phenotypic changes in fiber development or properties were apparent. This result leads to the conclusion that E6 is not critical to the normal development or structural integrity of cotton fibers.

Amino Acid Sequence