Cytosolic Ca2+ regulates the energization of isolated brain mitochondria by formation of pyruvate through the malate-aspartate shuttle

The glutamate-dependent respiration of isolated BM (brain mitochondria) is regulated by Ca2+(cyt) (cytosolic Ca2+) (S0.5=225±22 nM) through its effects on aralar. We now also demonstrate that the α-glycerophosphate-dependent respiration is controlled by Ca2+(cyt) (S0.5=60±10 nM). At higher Ca2+(cyt) (>600 nM), BM accumulate Ca2+ which enhances the rate of intramitochondrial dehydrogenases. The Ca2+-induced increments of state 3 respiration decrease with substrate in the order glutamate>α-oxoglutarate>isocitrate>α-glycerophosphate>pyruvate. Whereas the oxidation of pyruvate is only slightly influenced by Ca2+(cyt), we show that the formation of pyruvate is tightly controlled by Ca2+(cyt). Through its common substrate couple NADH/NAD+, the formation of pyruvate by LDH (lactate dehydrogenase) is linked to the MAS (malate-aspartate shuttle) with aralar as a central component. A rise in Ca2+(cyt) in a reconstituted system consisting of BM, cytosolic enzymes of MAS and LDH causes an up to 5-fold enhancement of OXPHOS (oxidative phosphorylation) rates that is due to an increased substrate supply, acting in a manner similar to a 'gas pedal'. In contrast, Ca2+(mit) (intramitochondrial Ca2+) regulates the oxidation rates of substrates which are present within the mitochondrial matrix. We postulate that Ca2+(cyt) is a key factor in adjusting the mitochondrial energization to the requirements of intact neurons.     

Disruption of muscle renin-angiotensin system in AT1a-/- mice enhances muscle function despite reducing muscle mass but compromises repair after injury

The role of the renin-angiotensin system (RAS) in vasoregulation is well established, but a localized RAS exists in multiple tissues and exerts diverse functions including autonomic control and thermogenesis. The role of the RAS in the maintenance and function of skeletal muscle is not well understood, especially the role of angiotensin peptides, which appear to contribute to muscle atrophy. We tested the hypothesis that mice lacking the angiotensin type 1A receptor (AT(1A)(-/-)) would exhibit enhanced whole body and skeletal muscle function and improved regeneration after severe injury. Despite 18- to 20-wk-old AT(1A)(-/-) mice exhibiting reduced muscle mass compared with controls (P < 0.05), the tibialis anterior (TA) muscles produced a 25% higher maximum specific (normalized) force (P < 0.05). Average fiber cross-sectional area (CSA) and fiber oxidative capacity was not different between groups, but TA muscles from AT(1A)(-/-) mice had a reduced number of muscle fibers as well as a higher proportion of type IIx/b fibers and a lower proportion of type IIa fibers (P < 0.05). Measures of whole body function (grip strength, rotarod performance, locomotor activity) were all improved in AT(1A)(-/-) mice (P < 0.05). Surprisingly, the recovery of muscle mass and fiber CSA following myotoxic injury was impaired in AT(1A)(-/-) mice, in part by impaired myoblast fusion, prolonged collagen infiltration and inflammation, and delayed expression of myogenic regulatory factors. The findings support the therapeutic potential of RAS inhibition for enhancing whole body and skeletal muscle function, but they also reveal the importance of RAS signaling in the maintenance of muscle mass and for normal fiber repair after injury.     

Angle-resolved Photoemission Spectroscopy At Ultra-low Temperatures

The physical properties of a material are defined by its electronic structure. Electrons in solids are characterized by energy (ω) and momentum (k) and the probability to find them in a particular state with given ω and k is described by the spectral function A(k, ω). This function can be directly measured in an experiment based on the well-known photoelectric effect, for the explanation of which Albert Einstein received the Nobel Prize back in 1921. In the photoelectric effect the light shone on a surface ejects electrons from the material. According to Einstein, energy conservation allows one to determine the energy of an electron inside the sample, provided the energy of the light photon and kinetic energy of the outgoing photoelectron are known. Momentum conservation makes it also possible to estimate k relating it to the momentum of the photoelectron by measuring the angle at which the photoelectron left the surface. The modern version of this technique is called Angle-Resolved Photoemission Spectroscopy (ARPES) and exploits both conservation laws in order to determine the electronic structure, i.e. energy and momentum of electrons inside the solid. In order to resolve the details crucial for understanding the topical problems of condensed matter physics, three quantities need to be minimized: uncertainty* in photon energy, uncertainty in kinetic energy of photoelectrons and temperature of the sample.In our approach we combine three recent achievements in the field of synchrotron radiation, surface science and cryogenics. We use synchrotron radiation with tunable photon energy contributing an uncertainty of the order of 1 meV, an electron energy analyzer which detects the kinetic energies with a precision of the order of 1 meV and a He³ cryostat which allows us to keep the temperature of the sample below 1 K. We discuss the exemplary results obtained on single crystals of Sr₂RuO₄ and some other materials. The electronic structure of this material can be determined with an unprecedented clarity.     

CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species

Insulators can block an enhancer of one gene from activating a promoter on another nearby gene. Almost all described vertebrate insulators require binding of the regulatory protein CTCF for their activity. We show that CTCF copurifies with the nucleolar protein nucleophosmin and both are present at insulator sites in vivo. Furthermore, exogenous insulator sequences are tethered to the nucleolus in a CTCF-dependent manner. These interactions, quite different from those of the gypsy insulator element in Drosophila, may generate similar loop structures, suggesting a common theme and model for enhancer-blocking insulator action.     

A high-throughput screen for single gene activities: isolation of apoptosis inducers

We describe a novel genetic screen that is performed by transfecting every individual clone of an expression library into a separate population of cells in a high-throughput mode. The screen allows one to achieve a hitherto unattained sensitivity in expression cloning which was exploited in a first read-out to clone apoptosis-inducing genes. This led to the isolation of several genes whose proteins induce distinct phenotypes of apoptosis in 293T cells. One of the isolated genes is the tumor suppressor cytochrome b(L) (cybL), a component of the respiratory chain complex II, that diminishes the activity of this complex for apoptosis induction. This gene is more efficient and specific for causing cell death than a drug with the same activity. These results suggest further applications, both of the isolated genes and the screen.     

Adenoviral infection decreases mortality from lipopolysaccharide-induced liver failure via induction of TNF-alpha tolerance

Effects of adenoviral infection on in vivo responses to LPS mediated by TNF-alpha were evaluated in a murine model. Adenovirus-infected mice showed decreased mortality from fulminant hepatitis induced by administration of LPS or staphylococcal enterotoxin B in the presence of D-galactosamine. Importantly, TNF-alpha resistance genes within adenoviral E3 region were not required, because E1,E3-deleted vectors showed similar effects. Adenovirus-infected mice exhibited higher TNF-alpha levels after LPS stimulation, no difference in TNFR1 expression, and similar mortality from Fas-induced fulminant hepatitis. Decreased production of IL-6 and KC in response to exogenous TNF-alpha, in addition to protection from TNF-alpha, suggested that adenoviral infection results in TNF-alpha tolerance.     

Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery

Assembled modular complexes for targeted drug delivery can be based on strong non-covalent interactions between a cargo module containing an adapter protein and a docking tag fused to a targeting protein. We have recently constructed a completely humanized adapter/docking tag system based on interactions between 15 amino acid (Hu-tag) and 110 amino acid (HuS) fragments of human ribonuclease I (RNase I). Although recombinant HuS can be expressed and refolded into a functionally active form, the purification procedure is cumbersome and expensive, and more importantly, it yields a significant proportion of improperly folded proteins. Here we describe engineering, high-yield expression, and purification of a chimeric bovine/human RNase (BH-RNase) comprising 1-29 N-terminal amino acids of bovine ribonuclease A and 30-127 amino acids of human RNase I. Unlike RNase I, the chimeric BH-RNase can be cleaved by either subtilisin or proteinase K between A20 and S21, providing a functionally active HuS. The HuS obtained from chimeric BH-RNase differs from wild-type HuS by an N24T substitution; therefore, we have reverted this substitution by mutating N24 to T24 in BH-RNase. This BH-RNase mutant can also be cleaved by subtilisin or proteinase K yielding wild-type HuS. The affinity of HuS obtained from BH-RNase to Hu-tag is approximately five times higher than that for recombinant HuS, reflecting a higher percentage of properly folded proteins.     

Ligand-dependent localization and intracellular stability of sigma-1 receptors in CHO-K1 cells

Background

Sigma-1 receptors are involved in regulation of neuronal activities presumably through regulation of the activity of ion channels. Sigma-1 receptors also play a role in growth and metastasis of cancerous cells. Intracellular distribution of sigma-1 receptors have been linked to sphingolipid-enriched domains.

Results

We report that in CHO-K1 cells sigma-1 receptors target to focal adhesion contacts (FAC) where they colocalize with Talin and Kv1.4 potassium channels. The levels of sigma-1 receptors in the FAC were significantly increased by application of sigma-1 receptor ligands and by filamentous actin (F-actin) polymerization with phalloidin. The total length of FAC (measured by the focal adhesion marker, talin) was concomitantly increased in the presence of sigma-1 receptors upon phalloidin treatment. Only sigma-1 receptor ligands, however, resulted in an increase of sigma-1 receptors in the FAC, independent of talin. Additionally, a novel approach was utilized to allow an assessment of the half life of endogenous sigma-1 receptors in CHO-K1 cells, which was measured to be at least 72 hours.

Conclusion

Ligand activated sigma-1 receptors translocate into FAC from a pool of receptors stored in ER lipid rafts presumably for inhibition of Kv1.4 channels. Stabilization of actin filaments is likely to be important for targeting sigma-1 receptors to Focal Adhesion Contacts in CHO-K1 cells.     

Estimation of stature in Turkish adults using knee height

Body height is an important clinical indicator to derive body mass index (BMI), which is a useful screening tool for both excess adiposity and malnutrition. Height measurement in the elderly may impose some difficulties and the reliability is doubtful. Stature estimation from knee height is one of the commonly used methods; nevertheless no study has been carried out so far on the Turkish population. A cross sectional anthropometric study was conducted to develop body height estimation equations by using knee height measurement for Turkish people. Measurements of height and knee height were taken according to the International Biological Programme procedures from 1422 adults (610 males, 812 females) aged 18-90 years from Ankara, the capital city of Turkey. Samples were randomly split into two sub-samples, training and validation (control group) sub-samples. Height estimation equations were developed from the knee height measurements by linear regression analysis according to age groups and sexes. Males were significantly taller and have higher knee height values than females in all age groups. Height and the knee height variables showed a gradual decrease (P 50) with aging in females and males. Evaluated knee height equations for stature estimation were tested through the validation sample and the results showed high accuracy. The study presents sex and age specific regression equations for height estimation by using the knee height measurement for Turkish adults and suggests facilitating the accurate usage of knee height.