Thermospheric Nitric Oxide Response to Shock-led Storms.

We present a multi-year superposed epoch study of the Sounding of the Atmosphere using Broadband Emission Radiometry nitric oxide (NO) emission data. NO is a trace constituent in the thermosphere that acts as cooling agent via infrared (IR) emissions. The NO cooling competes with storm time thermospheric heating resulting in a thermostat effect. Our study of nearly 200 events reveals that shock-led interplanetary coronal mass ejections (ICMEs) are prone to early and excessive thermospheric NO production and IR emissions. Excess NO emissions can arrest thermospheric expansion by cooling the thermosphere during intense storms. The strongest events curtail the interval of neutral density increase and produce a phenomenon known as thermospheric 'overcooling'. We use Defense Meteorological Satellite Program particle precipitation data to show that interplanetary shocks and their ICME drivers can more than double the fluxes of precipitating particles that are known to trigger the production of thermospheric NO. Coincident increases in Joule heating likely amplify the effect. In turn, NO emissions more than double. We discuss the roles and features of shock/sheath structures that allow the thermosphere to temper the effects of extreme storm time energy input and explore the implication these structures may have on mesospheric NO. Shock-driven thermospheric NO IR cooling likely plays an important role in satellite drag forecasting challenges during extreme events.

Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice.

Myotonic dystrophy (DM) is commonly associated with CTG repeat expansions within the gene for DM-protein kinase (DMPK). The effect of altered expression levels of DMPK, which is ubiquitously expressed in all muscle cell lineages during development, was examined by disrupting the endogenous Dmpk gene and overexpressing a normal human DMPK transgene in mice. Nullizygous (-/-) mice showed only inconsistent and minor size changes in head and neck muscle fibres at older age, animals with the highest DMPK transgene expression showed hypertrophic cardiomyopathy and enhanced neonatal mortality. However, both models lack other frequent DM symptoms including the fibre-type dependent atrophy, myotonia, cataract and male-infertility. These results strengthen the contention that simple loss- or gain-of-expression of DMPK is not the only crucial requirement for development of the disease.

Soft and flexible poly(ethylene glycol) nanotubes for local drug delivery.

Nanotubes are emerging as promising materials for healthcare applications but the selection of clinically relevant starting materials for their synthesis remains largely unexplored. Here we present, for the first time, the synthesis of poly(ethylene glycol) (PEG) based nanotubes via the photopolymerization of poly(ethylene glycol) diacrylate and other diacrylate derivatives within the pores of anodized aluminum oxide templates. Template-assisted synthesis allowed the manufacture of a diverse set of polymeric nanotubes with tunable physical characteristics including diameter (∼200-400 nm) and stiffness (405-902 kPa). PEG nanotubes were subjected to cytotoxicty assessment in cell lines and primary stem cells and showed excellent cytocompatability (IC50 > 120 µg ml-1). Nanotubes were readily drug loaded but released the majority of the drug over 5 days. Direct administration of drug loaded nanotubes to human orthotopic breast tumors substantially reduced tumor growth and metastasis and outperformed i.v. administration at the equivalent dose. Overall, this nanotube templating platform is emerging as a facile route for the manufacture of poly(ethylene glycol) nanotubes.

Effects of low-frequency stimulation on soluble and structure-bound activities of hexokinase and phosphofructokinase in rat fast-twitch muscle.

Several glycolytic enzymes exist in muscle as free and structure-bound forms. A fraction of hexokinase (HK) is associated with the outer mitochondrial membrane. Phosphofructokinase (PFK) and aldolase (ALD) bind to F-actin, and AMP deaminase (AMPase) interacts with myosin. Using low-frequency stimulation (10 Hz, 24 h/d), we studied in rat fast-twitch muscle effects of contractile activity on soluble and structure-bound forms of these enzymes. Phosphoglucose isomerase (PGI), a soluble enzyme, was also examined. Fractional extraction was applied to study the intracellular distribution of soluble and bound enzyme activities 5 min, 1 h, 3 h, 1 d, and 7 d after the onset of stimulation. Confirming previous findings, total HK activity increased 7-fold in 7-d-stimulated muscles, whereas PFK, ALD, and PGI were reduced, ranging between 55% and 80% of their normal activities. AMPase activity was unaltered. At the time points studied, no changes were found in the extraction behavior of PGI and AMPase. The fraction of bound ALD increased slightly (12%). However, the distribution of HK and PFK was markedly altered. Bound PFK increased from 50% in the control to 85% in 7-d-stimulated muscles. Bound HK rose from 52% to 83% during the same time period. The increase in PFK binding was steep and occurred mainly within the first minutes and hours. The increase in HK binding occurred with some delay, but was significant in muscles stimulated for more than 1 h. In view of the altered kinetic properties of F-actin-bound PFK (alleviated allosteric inhibition by ATP) and bound HK (elevated catalytic activity), these changes are interpreted as early responses to match the metabolic demands during maximal contractile activity imposed on a muscle not programmed for sustained activity: Enhanced binding of PFK serves to accelerate glycolytic flux immediately after the onset of stimulation, whereas mitochondrial binding of HK facilitates the phosphorylation of exogenous glucose when glycogen stores have been depleted.

Prostaglandins and cyclic AMP stimulate creatine kinase synthesis but not fusion in cultured embryonic chick muscle cells.

Cyclic AMP levels have been measured in cultures derived from 12-day-old chick embryonic muscle. A rise in concentration was found after the onset of myoblast fusion. Cells cultured at a medium Ca2+ concentration of 0.1 microM did not fuse and exhibited only a small rise in cyclic AMP concentration during culture. Addition of 1.4 mM Ca2+ to these cells after 50 h in culture caused rapid, synchronous fusion with a concomitant rise in cyclic AMP levels. Indomethacin, an inhibitor of prostaglandin synthesis, did not inhibit fusion, but inhibited the rise in cyclic AMP concentration. Indomethacin-treated cultures exhibited lower creatine kinase levels, though no change in the ratio of the three isoenzymes was observed. Addition of prostaglandins E1 and E2 to indomethacin-treated cultures overcame this inhibition. We propose that prostaglandin synthesis is a consequence of the stimulation of myoblast fusion and that via cyclic AMP it stimulates protein synthesis.

Comparative analysis of the isoform expression pattern of Ca(2+)-regulatory membrane proteins in fast-twitch, slow-twitch, cardiac, neonatal and chronic low-frequency stimulated muscle fibers.

Although all muscle cells generate contractile forces by means of organized filament systems, isoform expression patterns of contractile and regulatory proteins in heart are not identical compared to developing, conditioned or mature skeletal muscles. In order to determine biochemical parameters that may reflect functional variations in the Ca(2+)-regulatory membrane systems of different muscle types, we performed a comparative immunoblot analysis of key membrane proteins involved in ion homeostasis. Cardiac isoforms of the alpha(1)-dihydropyridine receptor, Ca(2+)-ATPase and calsequestrin are also present in skeletal muscle and are up-regulated in chronic low-frequency stimulated fast muscle. In contrast, the cardiac RyR2 isoform of the Ca(2+)-release channel was not found in slow muscle but was detectable in neonatal skeletal muscle. Up-regulation of RyR2 in conditioned muscle was probably due to degeneration-regeneration processes. Fiber type-specific differences were also detected in the abundance of auxiliary subunits of the dihydropyridine receptor, the ryanodine receptor and the Ca(2+)-ATPase, as well as triad markers and various Ca(2+)-binding and ion-regulatory proteins. Hence, the variation in innervation of different types of muscle appears to have a profound influence on the levels and pattern of isoform expression of Ca(2+)-regulatory membrane proteins reflecting differences in the regulation of Ca(2+)-homeostasis. However, independent of the muscle cell type, key Ca(2+)-regulatory proteins exist as oligomeric complexes under native conditions.

Mammalian skeletal muscle fiber type transitions.

Mammalian skeletal muscle is an extremely heterogeneous tissue, composed of a large variety of fiber types. These fibers, however, are not fixed units but represent highly versatile entities capable of responding to altered functional demands and a variety of signals by changing their phenotypic profiles. This adaptive responsiveness is the basis of fiber type transitions. The fiber population of a given muscle is in a dynamic state, constantly adjusting to the current conditions. The full range of adaptive ability spans fast to slow characteristics. However, it is now clear that fiber type transitions do not proceed in immediate jumps from one extreme to the other, but occur in a graded and orderly sequential manner. At the molecular level, the best examples of these stepwise transitions are myofibrillar protein isoform exchanges. For the myosin heavy chain, this entails a sequence going from the fastest (MHCIIb) to the slowest (MHCI) isoform, and vice-versa. Depending on the basal protein isoform profile and hence the position within the fast-slow spectrum, the adaptive ranges of different fibers vary. A simple transition scheme has emerged from the multitude of data collected on fiber type conversions under a variety of conditions.

Type I protein is a slow isoform of troponin T.

Type I protein, a myofibrillar protein thought to be specific to slow-twitch skeletal muscle fibers, was purified. Two-dimensional electrophoresis indicated its identity with the purified slow troponin-T1s isoform. Immunochemical analyses using antibodies raised against type I protein and slow Tn-T1s, further substantiated the identity of the two proteins.

Three fast myosin heavy chains in adult rat skeletal muscle.

A new fast myosin heavy chain isoform was electrophoretically detected in adult rat skeletal muscles. It was present at high levels in diaphragm and, therefore, designated as MHCIId. Appreciable amounts of MHCIId were detected in tongue musculature, the extraocular muscles, and in the deep red portions of various fast muscles. Its concentration in fast-twitch muscle was greatly increased by chronic stimulation.

PCR-based assignment of two myosin heavy chain cDNA clones to biochemically and histochemically defined single type IIB and IID fibers of rabbit muscle.

The present study assigns two as yet unidentified fast myosin cDNA clones to specific myosin heavy chain (MHC) isoforms and their mRNAs in different fiber types of rabbit skeletal muscle. Specific oligonucleotide primers were used for reverse transcription and polymerase chain reaction (PCR) to yield products of defined lengths. The method was sensitive enough to detect specific mRNA sequences in total RNA extracts from microdissected, freeze-dried, single-fiber fragments down to 16 ng dry weight. The fibers were typed histochemically and biochemically by their electrophoretically assessed MHC complement. The following results were obtained: clone pMHC20-40 was assigned to type IIB fibers and clone pMHC24-79 to type IID fibers.

Reverse transcriptase-polymerase chain reaction detects induction of cardiac-like alpha myosin heavy chain mRNA in low frequency stimulated rabbit fast-twitch muscle.

Using reverse transcriptase-polymerase chain reaction we quantified in rabbit skeletal muscles expression levels of the highly homologous cardiac alpha and beta myosin heavy chain (alpha MHC, beta MHC) mRNA isoforms. Masseter muscle displayed highest levels of a cardiac-like alpha MHC mRNA. This isoform was present at 20-fold lower amounts in slow soleus and at 200-fold lower levels in several fast-twitch muscles. Low-frequency stimulation periods exceeding 20 days drastically induced the alpha MHC mRNA in fast tibialis anterior. The alpha MHC mRNA was 140-fold elevated after 60 days when beta MHC mRNA had increased 50-fold. Our results demonstrate the wide distribution of a cardiac-like alpha MHC mRNA in skeletal muscle and its marked induction during fast-to-slow transition as induced by low-frequency stimulation.

Non-radioactive reverse transcriptase/polymerase chain reaction for quantification of myosin heavy chain mRNA isoforms in various rabbit muscles.

A method was established for measuring molecule numbers of three different myosin heavy chain (MHC) mRNA isoforms in total RNA preparations. The quantification was based on a combination of primer-directed reverse transcriptase and polymerase chain reactions with 5'-digoxigenin-labeled oligonucleotides, using external standards. The sensitivity of the method allowed the quantitation of mRNA amounts down to the range of 1,000 molecules (detection limit 50 molecules). The numbers determined for eight different rabbit muscles are in the range of 10(3)-10(9)/micrograms total RNA. In soleus muscle, the value of 1.11 x 10(9) MHCI mRNA molecules corresponds to approximately 8% of the total mRNA. With reference to myonuclei, this amount corresponds to 1-2 x 10(4) molecules/nucleus. A quantitative comparison of the two fast MHC mRNA isoforms with the distribution of different MHC isoforms at the protein level indicates that one of these two fast sequences is specific to MHCIIb and the other to MHCIId. However, our data point to the existence of additional MHCIId mRNA subtypes.

Slow-to-fast transitions in myosin expression of rat soleus muscle by phasic high-frequency stimulation.

Denervated soleus muscles of euthyroid and hyperthyroid rats were exposed to phasic high-frequency stimulation for periods of up to 40 days and analysed for their myosin heavy chain (MHC) composition. Denervation alone induced appreciable amounts of the fast MHCIId/x and minute amounts of MHCIIb. However, the effects of phasic high-frequency stimulation exceeded by far those of denervation, leading to marked increases of these two isoforms, as well as to pronounced decreases in slow MHCI. In addition, the present study suggested a greater impact of neural activity on myosin expression than thyroid hormone.

The ratio between intrinsic 115 kDa and 30 kDa peptides as a marker of fibre type-specific sarcoplasmic reticulum in mammalian muscles.

Sarcoplasmic reticulum displays characteristic differences in Ca2+-uptake, Ca2+-ATPase and the pattern of membrane proteins in type I, IIA and IIB fibres. The ratio between the 115 kDa Ca2+-ATPase and a 30 kDa protein is of characteristic magnitude in the sarcoplasmic reticulum of the three fibre types in rat muscles. The slow-to-fast fibre type transformation observed in rabbits during chronic nerve stimulation is accompanied by predictable changes of this ratio.

Changes in transcriptional activity of chronically stimulated fast twitch muscle.

mRNAs extracted from rabbit soleus, normal and 28-day, indirectly stimulated tibialis anterior muscles were translated in an in vitro system. Analysis for translation products by 2-dimensional electrophoresis showed fast myosin light chains in tibialis anterior, and slow myosin light chains in soleus muscle. The stoichiometry of the in vitro translated light chains varies from that seen in normal fast and slow twitch muscles. The stimulated muscle contained mRNA coding, both for fast and slow myosin light chains, although the pattern of slow myosin light chains appears not to be complete at this point of time of the transformation process.

Electrophoretic separation by an improved method of fast myosin HCIIb-, HCIId-, and HCIIa-based isomyosins with specific alkali light chain combinations.

An improved method of electrophoresis under nondenaturing conditions separated three electrophoretically distinct isomyosin triplets when applied to rat fast-twitch muscles displaying a predominance of one of the fast myosin heavy chain isoforms HCIIb, HCIId or HCIIa. The three isomyosin triplets, named FM1b-FM3b, FM1d-FM3d, FM1a-FM3a, corresponded to the three possible alkali light chain (LC) combinations (LC1f homodimer, LC1f/LC3f heterodimer, and LC3f homodimer) with each fast HC isoform. Different proportions of these various isomyosins suggested specific affinities of light chains LC1f and LC3f for the fast heavy chain isoforms.

Increased mitochondrial creatine kinase in chronically stimulated fast-twitch rabbit muscle.

Fractional extraction and isozyme electrophoresis revealed the presence of small amounts (2.5% of total cellular activity) of mitochondrial creatine kinase (CK) in rabbit fast-twitch muscle. Chronic nerve stimulation resulted in a decrease of extramitochondrial MM-CK to 60% of its normal value but induced an approx. 4-fold increase in mitochondrial CK. This increase occurred in parallel with the rise in enzyme activities of terminal substrate oxidation.

Electrophoretic analysis of myosin heavy chain isoform patterns in extraocular muscles of the rat.

Six oculorotatory muscles and the levator palpebrae muscle of the rat were analysed by SDS-PAGE for their myosin heavy chain (MHC) isoform patterns. Oculorotatory muscles display a marked predominance of fast MHC isoforms. They contain, in addition to the slow (MHCI) and fast (MHCIIb, MHCIId, MHCIIa) skeletal MHCs, the neonatal MHCneo and the extraocular MHCeom. The levator palpebrae, generally assumed to be a member of the extraocular muscle group because of its innervation by the oculomotor nerve, does not contain MHCneo and MHCeom. It resembles a fast-twitch skeletal muscle with a predominance of MHCIId.

Separation of active and inactive (nonphosphorylating) Ca(2+)-ATPase in sarcoplasmic reticulum subfractions from low-frequency-stimulated rabbit muscle.

Chronic low-frequency stimulation elicits in rabbit fast-twitch muscle a partial inactivation of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Ca(2+)-uptake activities. Inactive Ca(2+)-ATPase was enriched in a light microsomal fraction by sucrose density gradient centrifugation after calcium oxalate loading in the presence of ATP. This fraction showed a reduced specific activity and phosphoprotein formation of the Ca(2+)-transport ATPase. These results suggest that the inactivation of the Ca(2+)-ATPase as induced by increased contractile activity, is confined to a specific SR vesicle population.

Contractile activity enhances the synthesis of hexokinase II in rat skeletal muscle.

An 11-fold increase in hexokinase activity and the hexokinase II isoform was found in rat tibialis anterior muscle after 7 days of chronic, low-frequency stimulation. In vivo labeling studies showed that this increase in enzyme protein content was related to an approx. 30-fold increase in [35S] methionine incorporation.