Changes in lipid and protein synthesis during spermatozoan development and thoracic tissue maturation inDrosophila hydei

Summary The synthesis of protein and lipid was studied in the testis and thoracic tissues ofDrosophila hydei by radioisotope incorporation. The formation and accumulation of late stage gametes in the testes during maturation was accompanied by more than 4-fold increases in both lipid and protein synthesis rates. Though there was an 8-fold increase in the rate of protein synthesis during thoracic tissue maturation, the rate of lipid synthesis little more than doubled. By comparison the rates of protein synthesis were high in mature testis and thoracic tissues, whereas the rate of lipid synthesis was high in the mature testis but low in both immature and mature thoracic tissues.Label incorporation from pyruvate and L-alanine into protein in thoracic tissue appeared to be highly dependent on Krebs cycle activity, whereas pyruvate and L-alanine label incorporation into protein in the testis was primarily from L-alanine and its derivatives. Both pyruvate and L-alanine were decarboxylated prior to label incorporation into lipid in the testis and thoracic tissue.This work was supported by National Science Foundation Grant GB-13393.     

Contractile Force Is Enhanced in Aortas from Pendrin Null Mice Due to Stimulation of Angiotensin II-Dependent Signaling

Pendrin is a Cl⁻/HCO₃ ⁻ exchanger expressed in the apical regions of renal intercalated cells. Following pendrin gene ablation, blood pressure falls, in part, from reduced renal NaCl absorption. We asked if pendrin is expressed in vascular tissue and if the lower blood pressure observed in pendrin null mice is accompanied by reduced vascular reactivity. Thus, the contractile responses to KCl and phenylephrine (PE) were examined in isometrically mounted thoracic aortas from wild-type and pendrin null mice. Although pendrin expression was not detected in the aorta, pendrin gene ablation changed contractile protein abundance and increased the maximal contractile response to PE when normalized to cross sectional area (CSA). However, the contractile sensitivity to this agent was unchanged. The increase in contractile force/cross sectional area observed in pendrin null mice was due to reduced cross sectional area of the aorta and not from increased contractile force per vessel. The pendrin-dependent increase in maximal contractile response was endothelium- and nitric oxide-independent and did not occur from changes in Ca²⁺ sensitivity or chronic changes in catecholamine production. However, application of 100 nM angiotensin II increased force/CSA more in aortas from pendrin null than from wild type mice. Moreover, angiotensin type 1 receptor inhibitor (candesartan) treatment in vivo eliminated the pendrin-dependent changes contractile protein abundance and changes in the contractile force/cross sectional area in response to PE. In conclusion, pendrin gene ablation increases aorta contractile force per cross sectional area in response to angiotensin II and PE due to stimulation of angiotensin type 1 receptor-dependent signaling. The angiotensin type 1 receptor-dependent increase in vascular reactivity may mitigate the fall in blood pressure observed with pendrin gene ablation.     

Coordinate accumulation of contractile protein mRNAs during myoblast differentiation.

The synthesis of contractile protein mRNAs has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Eight contractile protein mRNAs were identified by translation of total cellular RNA isolated from differentiated myofibers in wheat germ and reticulocyte lysates. Products of the translation systems were fractionated by two-dimensional gel electrophoresis, and incorporation of [³⁵S]methionine into individual contractile proteins was quantitated by computerized densitometry of autoradiograms. These translation assay systems were used to quantitate levels of contractile protein mRNAs in cultures of myoblasts undergoing highly synchronous differentiation. Our results show that dividing myoblasts contain very little, if any, translatable contractile protein mRNA. The mRNAs coding for myosin heavy chain, the musclespecific actin, three myosin light chains, two tropomyosin subunits, and one troponin subunit begin to coordinately accumulate at fusion, when contractile protein synthesis is activated. Their levels increase 50- to 200-fold during the next 30 hr, paralleling increases in the rates of contractile protein synthesis. These results indicate that the contractile protein mRNAs accumulate coordinately during myoblast differentiation and that contractile protein synthesis is regulated by changes in the levels of these mRNAs.     

Study of the protective effects of exogenous heat shock protein 70 kDa in the model of sleep deprivation in pigeons Columba livia

Electroencephalographic methods were used to study effects of the preparation of the exogenous heat shock protein with molecular mass 70 kDa (Hsp70i/Hsc70) on the time characteristics of sleep and waking, brain temperature, peripheral vasomotor reactions and thoracic muscle contractile activity after the 5-hour sleep deprivation in pigeons (Columba livia). The microinjections of Hsp70i/Hsc70 were performed into the third brain ventricle after the end of sleep deprivation. It was shown that Hsp70i/Hsc70 eliminated the disturbances of sleep-wake cycle and evoked a decrease in the thoracic muscle contractile and brain temperature during the first hour of postdeprivation period. During the following hours Hsp70i/Hsc70 evoked an increase in the total time of deep sleep and a decrease in the total time of rapid-eye-movement sleep. We suppose that the protective effects of Hsp70i/Hsc70 could be associated with its capacity to weaken the activity of the hypothalamo-hypophyseal-adrenal axis and to enhance the stress-limiting function of non-rapid-eye-movement sleep.     

Study of protective effects of exogenous heat shock protein 70 kDa in model of sleep deprivation in pigeon <Emphasis Type="Italic">Columba livia</Emphasis>

Electroencephalographic methods were used to study effects of preparation of the exogenous heat shock protein with molecular mass of 70 kDa (Hsp70i/Hsc70) on time characteristics of sleep and wakefulness, brain temperature, peripheral vasomotor reactions, and thoracic muscle contractile activity after the 5-hour forceful sleep deprivation in the pigeon Columba livia. Administration of Hsp70i/Hsc70 into the third brain ventricle at once after the end of sleep deprivation eliminated disturbances in the sleep-wakefulness cycle organization and decreased the thoracic muscle contractile activity and the brain temperature as early as for the first hour of postdeprivation period. For the subsequent hours, the Hsp70i/Hsc70 action was characterized by an increase of the total time of deep sleep and a decrease of the total time of the rapid eye movement sleep. We suggest that the protective effects of the exogenous Hsp70i/Hsc70 preparation are associated with its ability to decrease activity of the hypothalamo-pituitary-adrenal axis and to enhance the stress-limiting function of the slow eye movement sleep.     

Protein phosphorylation and oocyte maturation. I. Induction of starfish oocyte maturation by intracellular microinjection of a phosphatase inhibitor, alpha-naphthylphosphate

Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. alpha-Naphthylphosphate (alpha-NP), a widely used phosphatase inhibitor/substrate, was found to induce oocyte maturation when microinjected intracellularly (50% maturation of 3.5 mM; 100% above 6mM, final intracellular concentration) into oocytes of Marthasterias and Asterias but not of Astropecten. As 1-MeAde, alpha-NP triggers a complete maturation, i.e. germinal vesicle breakdown, extrusion of the two polar bodies and formation of the female pronucleus. The kinetics of alpha-NP-induced maturation (35-45 min) is, however, longer than the kinetics of 1-MeAde-induced maturation (18-20 min). The addition of alpha-NP externally to oocytes does not trigger maturation. Among several reported phosphatase inhibitors, including two natural protein phosphatase inhibitors and several products structurally related to alpha-NP, only alpha-NP was found capable of inducing maturation when microinjection into oocytes. alpha-NP triggers the appearance of MPF activity in the cytoplasm of oocytes into which it has been injected. Although alpha-NP-induced maturation is insensitive to inhibitors whose action is known to be restricted to the hormone-dependent period (such as the protease inhibitor leupeptin), it is blocked by inhibitors of MPF action (such as nicotinamide and lithium). Finally it was found that alpha-NP-induced maturation is inhibited by simultaneous microinjection of protein phosphatase-2A; also, alpha-NP, classically used as an inhibitor of acid and alkaline phosphatases, is able to inhibit protein phosphatases, is able to inhibit protein phosphatases 1 and 2 A. The addition of alpha-NP to oocytes increases the level of phosphorylated proteins. These results constitute direct evidence that an elevated level of phosphorylated proteins is sufficient to trigger MPF activity and to induce maturation.     

Changes in cardiac troponins with gestational age explain changes in cardiac muscle contractility in the sheep fetus

The development of the adult cardiac troponin complex in conjunction with changes in cardiac function and cardiomyocyte binucleation has not been systematically characterized during fetal life in a species where maturation of the cardiomyocytes occurs prenatally as it does in the human. The aim of this study was to correlate the expression of each of the major adult troponin isoforms (T, I, and C) during late gestation (term of 150 days) to changes in both Ca(2+) sensitivity and maximum Ca(2+)-activated force of the contractile apparatus and the maturation of cardiomyocytes. The percentage of mononucleated cardiomyocytes in the right ventricle decreased with gestational age to 46% by 137-142 days of gestation. The length of binucleated cardiomyocytes did not change with gestational age, but the length of binucleated cardiomyocytes relative to heart weight decreased with gestational age. There was no change in the expression of adult cardiac troponin T with increasing gestation. The contractile apparatus was significantly more sensitive to Ca(2+) at 90 days compared with either 132 or 139 days of gestation, consistent with an ～30% increase in the expression of adult cardiac troponin I between 90 and 110 days of gestation. Maximum Ca(2+)-activated force significantly increased from 90 days compared with 130 days consistent with an increase of ～40% in cardiac troponin C protein expression. These data show that increased adult cardiac troponin I and C protein expression across late gestation is consistent with reduced Ca(2+) sensitivity and increased maximum Ca(2+)-activated force. Furthermore, changes in cardiac troponin C, not I, protein expression track with the timing of cardiomyocyte binucleation.     

Entactin promotes adhesion and long-term maintenance of cultured regenerated skeletal myotubes.

The basal lamina protein, laminin, has been shown to promote migration and proliferation of cultured skeletal myoblasts, resulting in increased myotube formation. However, skeletal myotubes adhere poorly to a laminin substrate, and long-term cultures of skeletal myotubes on laminin have not been achieved. We have found that cultured satellite cells from bupivacaine-damaged rat skeletal muscle actively proliferate and differentiate on a diluted Matrigel substrate composed of laminin, type IV collagen, heparan sulfate proteoglycan, and entactin. Myotubes cultured on diluted Matrigel are contractile and have never been observed to detach from the culture dish; rather, myotubes generally atrophy after 2-3 weeks in culture. Antibodies directed against the various protein components of Matrigel were used to determine the role of each component in enhancing muscle differentiation. Anti-laminin impaired satellite cell adhesion, whereas antibodies against either type IV collagen or heparan sulfate proteoglycan had no effect. Anti-entactin did not inhibit attachment, proliferation, or fusion of cultured satellite cells; however, myotubes exposed to anti-entactin failed to adhere to the culture dish after spontaneous myotube contractions began. We conclude that entactin is responsible for long-term maintenance and maturation of contractile skeletal myotubes on a diluted Matrigel substrate. This is the first study to assign a biological function for entactin in myogenesis.     

Tellurite effect on ATPase activity of contractile membrane protein.

The effect of tellurite on ATPase activity of the contractile membrane protein in human erythrocytes was studied. Tellurite, even at a concentration of 0.01 mM, inhibited 25 per cent of the saponin-stimulated ATPase activity of the contractile membrane protein; the inhibition increased with increasing tellurite concentration, and was reversible. On the other hand, in erythrocytes preincubated with tellurite, the ATPase activity of the membrane contractile protein, non-stimulated by saponin, increased, and the increase was further enhanced by washing the erythrocytes. The behaviour is analogous to the tellurite effect on erythrocyte morphology: incubation of erythrocytes with tellurite caused morphological changes which were more pronounced after removing the tellurite by washing. The complex effect of tellurite is discussed.     

Role of Dystrophin in Airway Smooth Muscle Phenotype,Contraction and Lung Function

Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD) and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC) and calponin and reduced Ca²⁺ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3β and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh) when compared to genetic control BL10ScSnJ mice (wild-type). In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM.     

Vascular development in early ovine gestation: carotid smooth muscle function, phenotype, and biochemical markers

Vascular smooth muscle (VSM) maturation is developmentally regulated and differs between vascular beds. The maturation and contribution of VSM function to tissue blood flow and blood pressure regulation during early gestation are unknown. The carotid artery (CA) contributes to fetal cerebral blood flow regulation and well being. We studied CA VSM contractility, protein contents, and phenotype beginning in the midthird of ovine development. CAs were collected from early (88-101 day of gestation) and late (138-150 day; term = day 150) fetal (n = 14), newborn (6-8 day old; n = 7), and adult (n = 5) sheep to measure forces in endothelium-denuded rings with KCl, phenylephrine, and ANG II; changes in cellular proteins, including total and soluble protein, actin and myosin, myosin heavy chain isoforms (MHC), filamin, and proliferating cell nuclear antigen; and vascular remodeling. KCl and phenylephrine elicited age- and dose-dependent contraction responses (P < 0.001) at all ages except early fetal, which were unresponsive. In contrast, ANG II elicited dose responses only in adults, with contractility increasing greater than fivefold vs. that shown in fetal or neonatal animals (P < 0.001). Increased contractility paralleled age-dependent increases (P < 0.01) in soluble protein, actin and myosin, filamin, adult smooth muscle MHC-2 (SM2) and medial wall thickness and reciprocal decreases (P < 0.001) in nonmuscle MHC-B, proliferating cell nuclear antigen and medial cellular density. VSM nonreceptor- and receptor-mediated contractions are absent or markedly attenuated in midgestation and increase age dependently, paralleling the transition from synthetic to contractile VSM phenotype and, in the case of ANG II, paralleling the switch to the AT(1) receptor. The mechanisms regulating VSM maturation and thus blood pressure and tissue perfusion in early development remain to be determined.     

Biochemical studies on bovine adenovirus type 3. II. Incomplete virus.

Incomplete virus of oncogenic bovine adenovirus type 3 (BAV3) was highly purified and its biological activity was studied. The production of incomplete virus was found to increase with a high multiplicity of infection and with a large amount of arginine in the growth medium. On infection of contact-inhibited mouse cells, incomplete virus induced cellular DNA synthesis and focus formation. Moreover, this virus was oncogenic to newborn hamsters. On infection of calf kidney cells, a permissive cell line, viral early and late RNA, viral DNA, and almost all the viral late proteins were produced, but no mature progeny virus was detected. It is, therefore, suggested that incomplete virus of BAV3 may be unable to synthesize a protein(s) (perhaps a kind of maturation protein[s]) essential for assembly of viral macromolecules for maturation.     

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β(1). The failure by TGF-β(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.     

Modulation of temperature-induced tone by vasoconstrictor agents.

One of the primary cardiovascular adjustments to hyperthermia is a sympathetically mediated increase in vascular resistance in the viscera. Nonneural factors such as a change in vascular tone or reactivity may also contribute to this response. Therefore, the aim of this study was to determine whether vascular smooth muscle tone is altered during heating to physiologically relevant temperatures >37 degrees C. Gradually increasing bath temperature from 37 degrees C (normothermia) to 43 degrees C (severe hyperthermia) produced graded contractions in vascular ring segments from rat mesenteric arteries and thoracic aortae. In untreated rings these contractions were relatively small, whereas hyperthermia elicited near-maximal increases in tension when rings were constricted with phenylephrine or KCl before heating. In phenylephrine-treated mesenteric arterial rings, the contractile responses to heating were markedly attenuated by the Ca2+ channel antagonists nifedipine and diltiazem. Diltiazem also blocked the contractile responses to heating in thoracic aortic rings. These results demonstrate that hyperthermia has a limited effect on tension generation in rat vascular smooth muscle in the absence of vascular tone. However, in the presence of agonist-induced tone, tension generation during heating is markedly enhanced and dependent on extracellular Ca2+. In conclusion, these data suggest that local regulation of vascular tone can contribute to the hemodynamic adjustments to hyperthermia.     

Cyclic AMP and the positive inotropic effect of norepinephrine and phenylephrine.

Time-response studies of the effects of norepinephrine and phenylephrine revealed that both agonists caused an increase in cyclic AMP levels before increases in contractile force in either the electrically stimulated left atria or spontaneously beating right atria of the rat. Norepinephrine caused a nearly sixfold increase in cyclic AMP, whereas phenylephrine produced only a 50% increase in the nucleotide. Pretreatment with reserpine did not affect the norepinephrine cyclic AMP response; however, the phenylephrine cyclic AMP response was abolished. Reserpine pretreatment did not significantly affect the contractile responses of either amine. In the presence of propranolol, norepinephrine was found to have the ability to produce an increace in contractile force in which cyclic AMP was apparently not involved. The time course of the contractile response induced by adrenergic amines was found to be remarkably influenced by the chronotropic response in spontaneously beating preparations while the cyclic AMP response was not greatly affected. This difference in the contractile response may be due to the ability of the chronotropic response to influence the flux of calcium through the cell membrane.     

The flight performance of tsetse flies.

The four species of tsetse used are shown to be aerodynamically rather similar. The wingbeat frequency, aerodynamic lift, flight duration, and number of wingbeats per flight are seen to undergo large increases during adult maturation. Tsetse can produce unusually large aerodynamic lifts, although the flight performance declines very quickly during individual flights. The large lifts appear to be associated with the high percentage of muscle in the fly, and the rapid decline with the inability of the fly to replenish rapidly its proline reserves. Wing mutilation has been shown to increase the wingbeat frequency. The potentiation of the flight performance during maturation appears to be causally linked to increases in the thoracic muscle, cuticle, and proline levels. The results presented here are at variance with those of an earlier study on the physics of flight in tsetse.