Carboxyl Methylation of Non-Histone Chromosomal Proteins

The in vitro methylation of non-histone chromosomal proteins (NHCP) was investigated in nuclei isolated from the brain, liver, and thymus of 6-8-day-old rats. After incubating the nuclei in the presence of 20 μM S-adenosyl-L-[methyl-3H]methionine(l Ci/mmol), the NHCP were separated from histones on hydroxylapatite, and fractionated further on SDS acrylamide slab-gel electrophoresis. After the gels were dried, autoradiography was used to detect 3H-methyl groups associated with these proteins. Four NHCP from the liver and thymus were methylated, while six methylated proteins were detected from the brain. None of the methylated proteins in these tissues corresponded with those from other organs, except for the component with a molecular weight of 66,000. It was evident that the methyl groups were esterified to the free carboxyl groups of NHCP since they are heat labile, yielding methanol. The carboxyl methylated NHCP from these organs were tightly bound to chromatin. Nucleoplasm and loosely associated NHCP were essentially devoid of methylated protein. However, nucleoplasm contained proteins with molecular weights similar to the methylated proteins. The Carboxyl methyltransferase has a pH optimum of 6.9-7.0, was present in nucleoplasm, extractable from chromatin with 0.4 M NaCl, and non-specifically methylated nucleoplasmic protein when nucleoplasm was incubated with S-adenosyl-L-[methyl-3H]methionine. The carboxyl methylation of NHCP proteins was verified, in vivo. Six-day-old rats were given L-[methyl-3H]methionine(7 mCi/mmol) by an intraperitoneal injection. The rats were killed at varying time periods and the NHCP isolated from gradient purified nuclei. Chromosomal proteins from the cerebellum, cerebrum, kidney, liver, and thymus contained significant amounts of 3H-methyl groups. Turnover of the 3H-methyl esters was extremely rapid when compared to the turnover rates of the NHCP. Interestingly, a methylesterase, was observed to be tightly associated with chromatin, and could account for the rapid turnover of methyl esters in the absence of protein synthesis. It is possible that methylation of certain NHCP resulted in their association with chromatin. Methylated NHCP were tightly bound to chromatin while nucleoplasm contain similar molecular weight proteins that were not methylated. These observations give support to the idea that certain newly synthesized NHCP, present in nucleoplasm, were methylated and as a result of this reaction, tightly bind to chromatin. It is attractive to suppose that the methylation and demethylation of specific proteins could activate or inactivate genes by the binging or release of these proteins from chromatin. The carboxyl methylated proteins appeared to be tissue specific, and the methyl esters associated with these proteins turnover at a high rate. These observations suggested that the methylation and demethylation of specific proteins could result in gene regulation

Characterization of Upstream Mixing Cavities and a Downstream Combustion Cavity in Supersonic Flow

A key area of study in air-breathing hydrocarbon-fueled scramjet combustors is the characterization of cavity-based fuel injection and flame holding. One issue concerns oscillatory disturbances caused by trapped vortices in the main flame holder cavity as a fuel-air mixing enhancement technique. Previous research demonstrates that oscillatory disturbances can be carried downstream via the shear layer and alter the oscillatory characteristics of a downstream cavity. This study investigates the mixing effectiveness of three upstream direct-fueled mixing cavities as well as the effect on the oscillatory behavior of the downstream combustion cavity by the upstream mixing cavity. The three upstream mixing cavities are characterized in Mach 2 freestream flow with injection at three locations within each cavity. Non-intrusive visual data are collected using the nitric oxide (NO) Planar Laser Induced Fluorescence (PLIF) diagnostic technique to characterize mixing and shear layer influence. High frequency response pressure transducers measure pressure fluctuations in both the upstream and downstream cavities for comparative analysis. Injection at the upstream wall of the cavity provided greater penetration height into the freestream as well as faster mixing with the freestream compared to injection at the center or aft wall of the cavity. The pressure oscillations in each cavity showed strong similarities; however, the amplitudes of the frequencies were too small to be effective in lifting mass into the freestream

In Vitro Studies on the Methylation of DNA in Rat Brain and Liver Chromatin

The properties of DNA methyltransferase in various age rats were investigated to better characterize the enzyme. When purified chromatin from 5-day-old rat brain or liver nuclei was incubated with S-adenosyl-L-[methyl-3H]-methionine, 5-[methyl-3H]methyl-cytosine was the only product of methylation. The optimum pH of the DNA methyltransferase was approximately 8.0 with little or no change in activity over a pH range of 6.9 to 8.3. The Km value of the chromosomal bound DNA methyltransferase for S-adenosyl-L-methionine (AdoMet) was 3.5 ± 0.15 μm and 4.2 ± 0.13 μm, in the liver and brain, respectively. The Vmax values for the enzyme from liver and brain of 5-day-old rats were 0.17 ± 0.04 and 0.23 ± 0.09 pmol of 3H-methyl groups incorporated/mg DNA/min, respectively. S-adenosyl-L-homocysteine was found to inhibit competitively the DNA methyltransferase in the brain with a Ki value of 3.5 ± 1.6 μm. The enzyme was firmly bound to chromatin. After ten extractions with water or 20 mM potassium phosphate buffer, pH 7.0, only trace amounts of enzyme could be eluted from the chromatin. Measurable amounts of the enzyme could be extracted from chromatin with 0.4 M NaCl. However, measurable amounts of the DNA methyltransferase were still associated with the NaCl extracted chromatin. The rate of the reaction catalyzed by the chromosomal-bound DNA methyltransferase was dependent on the age of the rat and the type of cell from which the chromatin was isolated. In the liver, the rate declined from 0.28 pmol of methyl-3H incorporated/mg DNA/min at birth to 0.13 pmol of methyl-3H incorporated/mg DNA/min at 30 days. Enzyme activity remained at this level throughout life. In the brain, the rate decreased rapidly from 0.48 pmol of methyl-3H incorporated/mg DNA/min at birth to 0.05 pmol of methyl-3H incorporated/mg DNA/min at 30 days. Little or no activity could be detected after 30 days. Chromosomal DNA methyltransferase activity was found to be dependent on the availability of methyl acceptors and enzyme concentration. The number of methylation sites available for in vitro methylation in liver decreased from 3.5 pmol of 5-[methyl-3H]methylcytosine/mg DNA at birth to 1.5 pmol of 5-[methyl-3H]methylcytosine/mg DNA at 14 days, remaining constant thereafter. The number of methyl acceptors in the brain decreased from 8.5 pmol of 5-[methyl-3H]methylcytosine/mg DNA at birth to 2.5 pmol of 5-[methyl-3H]methylcytosine/mg DNA at 40 days, remaining constant thereafter. The level of extractable enzyme from the cerebellum increased from 0.13 pmol of methyl-3H incorporated/mg protein/min at birth to 0.29 pmol of methyl-3H incorporated/mg protein/min at 10 days. The level of the enzyme decreased thereafter to 0.07 pmol of methyl-3H incorporated/mg protein/min in 30-day-old rats, remaining at this level throughout life. The amount of extractable enzyme from the cerebrum decreased from 0.13 pmol of methyl-3H incorporated/mg protein/min at birth to 0.07 methyl-3H incorporated/mg DNA/min in 30-day-old rats, remaining at this level of activity thereafter. The extractable enzyme from the liver increased from 0.45 pmol of methyl-3H incorporated/mg protein/min at birth to 0.60 pmol of methyl-3H incorporated/mg protein/min at 16 days. In 30-day-old rat liver the enzyme decreased to 0.10 pmol of methyl-3H incorporated/mg protein/min, remaining at this level until 100 days. At this time the extractable enzyme increased to 0.25 pmol of methyl-3H incorporated/mg protein/min at 300 days, remaining at approximately this level, thereafter. The level of DNA methyltransferase and the number of in vitro methylation sites available on DNA correlated well with the rate of DNA synthesis in these organs. The synthesis of DNA methyltransferase appears to be coupled to DNA synthesis

Phase fluctuations and Non-Fermi Liquid Properties of 2D Fermi-system with attraction

The effect of static fluctuations in the phase of the order parameter on the normal and superconducting properties of a 2D system with attractive four-fermion interaction has been studied. Analytic expressions for the fermion Green function, its spectral density and the density of states are derived. The resultant single-particle Green function clearly demonstrates non-Fermi liquid behavior. The results show that as the temperature increases through the 2D critical temperature the width of the quasiparticle peaks broadens significantly. At the same time one retains the gap in quasiparticle spectrum. The spectral density for the dynamical fluctuations can also be obtained. Clearly the dynamical fluctuations fill the gap giving the observed pseudogap behaviour.Comment: 4 pages, LaTeX; invited paper presented at New^3SC-2, Las Vegas, USA, 199

Pseudogap phase formation in the crossover from Bose-Einstein condensation to BCS superconductivity in low dimensional systems

A phase diagram for a 2D metal with variable carrier density has been studied using the modulus-phase representation for the order parameter in a fully microscopic treatment. This amounts to splitting the degrees of freedom into neutral fermion and charged boson degrees of freedom. Although true long range order is forbidden in two dimensions, long range order for the neutral fermions is possible since this does not violate any continuous symmetry. The phase fluctuations associated with the charged degrees of freedom destroy long range order in the full system as expected. The presence of the neutral order parameter gives rise to new features in the superconducting condensate formation in low dimensional systems. The resulting phase diagram contains a new phase which lies above the superconducting (here Berezinskii-Kosterlitz-Thouless) phase and below the normal (Fermi-liquid) phase. We identify this phase with the pseudogap phase observed in underdoped high-$T_{c}$ superconducting compounds above their critical temperature. We also find that the phase diagram persists even in the presence of weak 3-dimensionalisation.Comment: 4 pages, LaTeX; invited paper presented at New^3SC-1, Baton Rouge, USA, 1998. To be published in Int.J.Mod.Phys.

Generating intrafusal skeletal muscle fibres in vitro: Current state of the art and future challenges

Intrafusal fibres are a specialised cell population in skeletal muscle, found within the muscle spindle. These fibres have a mechano-sensory capacity, forming part of the monosynaptic stretch-reflex arc, a key component responsible for proprioceptive function. Impairment of proprioception and associated dysfunction of the muscle spindle is linked with many neuromuscular diseases. Research to-date has largely been undertaken in vivo or using ex vivo preparations. These studies have provided a foundation for our understanding of muscle spindle physiology, however, the cellular and molecular mechanisms which underpin physiological changes are yet to be fully elucidated. Therefrom, the use of in vitro models has been proposed, whereby intrafusal fibres can be generated de novo. Although there has been progress, it is predominantly a developing and evolving area of research. This narrative review presents the current state of art in this area and proposes the direction of future work, with the aim of providing novel pre-clinical and clinical applications

Neuregulin 1 Drives Morphological and Phenotypical Changes in C2C12 Myotubes: Towards De Novo Formation of Intrafusal Fibres In Vitro

Muscle spindles are sensory organs that detect and mediate both static and dynamic muscle stretch and monitor muscle position, through a specialised cell population, termed intrafusal fibres. It is these fibres that provide a key contribution to proprioception and muscle spindle dysfunction is associated with multiple neuromuscular diseases, aging and nerve injuries. To date, there are few publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro. To this end, current models of skeletal muscle focus on extrafusal fibres and lack an appreciation for the afferent functions of the muscle spindle. The goal of this study was to produce and define intrafusal bag and chain myotubes from differentiated C2C12 myoblasts, utilising the addition of the developmentally associated protein, Neuregulin 1 (Nrg-1). Intrafusal bag myotubes have a fusiform shape and were assigned using statistical morphological parameters. The model was further validated using immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. The addition of Nrg-1 treatment resulted in a 5-fold increase in intrafusal bag myotubes (as assessed by morphology) and increased protein and gene expression of the intrafusal specific transcription factor, Egr3. Surprisingly, Nrg-1 treated myotubes had significantly reduced gene and protein expression of many intrafusal specific markers and showed no specificity towards intrafusal bag morphology. Another novel finding highlights a proliferative effect for Nrg-1 during the serum starvation-initiated differentiation phase, leading to increased nuclei counts, paired with less myotube area per myonuclei. Therefore, despite no clear collective evidence for specific intrafusal development, Nrg-1 treated myotubes share two inherent characteristics of intrafusal fibres, which contain increased satellite cell numbers and smaller myonuclear domains compared with their extrafusal neighbours. This research represents a minimalistic, monocellular C2C12 model for progression towards de novo intrafusal skeletal muscle generation, with the most extensive characterisation to date. Integration of intrafusal myotubes, characteristic of native, in vivo intrafusal skeletal muscle into future biomimetic tissue engineered models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications