A central role for hepatocyte growth factor in adipose tissue angiogenesis

Hepatocyte growth factor (HGF) is a potent mitogenic and angiogenic factor produced in human adipose tissue. In this study, we use 3T3-F442A preadipocytes to study the contribution of HGF to angiogenesis in an in vivo fat pad development model. As observed for human adipocytes, HGF is synthesized and secreted by 3T3-F442A preadipocytes and mature adipocytes. HGF knockdown with small-interfering RNA reduced HGF mRNA expression 82.3 +/- 4.2% and protein secretion 82.9 +/- 1.4% from 3T3-F442A preadipocytes. Silencing of HGF resulted in a 70.5 +/- 19.0% reduction in endothelial progenitor cell migration to 3T3-F442A-conditioned medium in vitro. 3T3-F442A preadipocytes injected under the skin of mice form a fat pad containing mature, lipid-filled adipocytes and a functional vasculature. At 72 h postinjection, expression of the endothelial cell genes TIE-1 and platelet endothelial cell adhesion molecule (PECAM)-1 was decreased 94.4 +/- 2.2 and 91.5 +/- 2.5%, respectively, in 3T3-F442A fat pads with HGF silencing. Knockdown of HGF had no effect on differentiation of 3T3-F442A preadipocytes to mature adipocytes in vitro or in vivo. In developing fat pads under the skin of HGF overexpressing transgenic mice, TIE-1 and PECAM-1 mRNA was increased 16.5- and 21.4-fold, respectively, at 72 h postinjection. The increase in gene expression correlated with immunohistochemical evidence of endothelial cell migration in the developing fat pad. These data suggest that HGF has a central role in regulating angiogenesis in adipose tissue.     

Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers

The assembly of AMPA-type glutamate receptors (AMPARs) into distinct ion channel tetramers ultimately governs the nature of information transfer at excitatory synapses. How cells regulate the formation of diverse homo- and heteromeric AMPARs is unknown. Using a sensitive biophysical approach, we show that the extracellular, membrane-distal AMPAR N-terminal domains (NTDs) orchestrate selective routes of heteromeric assembly via a surprisingly wide spectrum of subunit-specific association affinities. Heteromerization is dominant, occurs at the level of the dimer, and results in a preferential incorporation of the functionally critical GluA2 subunit. Using a combination of structure-guided mutagenesis and electrophysiology, we further map evolutionarily variable hotspots in the NTD dimer interface, which modulate heteromerization capacity. This 'flexibility' of the NTD not only explains why heteromers predominate but also how GluA2-lacking, Ca(2+)-permeable homomers could form, which are induced under specific physiological and pathological conditions. Our findings reveal that distinct NTD properties set the stage for the biogenesis of functionally diverse pools of homo- and heteromeric AMPAR tetramers.     

Non-stressful death of 23S rRNA mutant G2061C defective in puromycin reaction

Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown. Here we report a study of the lethal G2061C mutant of Escherichia coli 23S ribosomal RNA (rRNA). The G2061C mutation completely impaired the puromycin reaction and abolished formation of the active firefly luciferase in an in vitro translation system, while poly(U)- and short synthetic mRNA-directed peptidyl transferase reaction with aminoacylated tRNAs in vitro was seemingly unaffected. Study of the cellular proteome upon expression of the 23S rRNA gene carrying the G2061C mutation compared to cells expressing wild-type 23S rRNA gene revealed substantial differences. Most of the observed effects in the mutant were associated with reduced expression of stress response proteins and particularly proteins associated with the ppGpp-mediated stringent response.     

Ca is involved in phytochrome A-dependent regulation of the succinate dehydrogenase gene sdh1-2 in Arabidopsis

The mechanism of transduction of the phytochrome signal regulating the expression of succinate dehydrogenase in Arabidopsis has been investigated. Using the phytochrome mutants of Arabidopsis, it is demonstrated that the inhibition of succinate dehydrogenase in the light may result from the phytochrome A-dependent modulation of Ca²⁺ amount in the nuclear fraction of leaves. This leads to the activation of expression of the gene pif3 encoding the phytochrome-interacting factor PIF3, which binds to the promoter of the gene sdh1-2 encoding the SDHA subunit of succinate dehydrogenase and suppresses its expression. It is concluded that Ca²⁺ ions are involved in the phytochrome A-mediated inhibition of succinate dehydrogenase activity in the light.     

The Use of Phosphite‐Type Ligands in the Ir‐Catalyzed Asymmetric Hydrogenation of Heterocyclic Compounds

A series of chiral phosphite‐type ligands was tested in asymmetric Ir‐catalyzed hydrogenation of quinolines and 2,4,5,6‐tetrahydro‐1H‐pyrazino(3,2,1‐j,k)carbazole. Hydrogenation of quinaldine hydrochloride provided superior enantioselectivity up to 65% ee compared to quinaldine free base. The ligands were tested for the first time in the asymmetric Ir‐Ircatalyzed hydrogenation of 2,4,5,6‐tetrahydro‐1H‐pyrazino(3,2,1‐j,k)carbazole yielding the antidepressant drug, pirlindole. Chirality 26:56–60, 2013. © 2013 Wiley Periodicals, Inc.     

Effect of subdomain interactions on methyl group dynamics in the hydrophobic core of villin headpiece protein

Thermostable villin headpiece protein (HP67) consists of the N‐terminal subdomain (residues 10–41) and the autonomously folding C‐terminal subdomain (residues 42–76) which pack against each other to form a structure with a unified hydrophobic core. The X‐ray structures of the isolated C‐terminal subdomain (HP36) and its counterpart in HP67 are very similar for the hydrophobic core residues. However, fine rearrangements of the free energy landscape are expected to occur because of the interactions between the two subdomains. We detect and characterize these changes by comparing the µs‐ms time scale dynamics of the methyl‐bearing side chains in isolated HP36 and in HP67. Specifically, we probe three hydrophobic side chains at the interface of the two subdomains (L42, V50, and L75) as well as at two residues far from the interface (L61 and L69). Solid‐state deuteron NMR techniques are combined with computational modeling for the detailed characterization of motional modes in terms of their kinetic and thermodynamic parameters. The effect of interdomain interactions on side chain dynamics is seen for all residues but L75. Thus, changes in dynamics because of subdomain interactions are not confined to the site of perturbation. One of the main results is a two‐ to threefold increase in the value of the activation energies for the rotameric mode of motions in HP67 compared with HP36. Detailed analysis of configurational entropies and heat capacities complement the kinetic view of the degree of the disorder in the folded state.     

Comparison of the Wild-Type Obligate Methylotrophic Bacterium Methylophilus quaylei and its Isogenic Streptomycin-Resistant Mutant via Metal Nanoparticle Generation

Biological Trace Element Research - Metal nanoparticles synthesized by green methods with the use of microorganisms are currently one of the most closely studied types of nanomaterials. It has...     

High-resolution atomic force microscopy visualization of metalloproteins and their complexes

Background

Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level.

Orexinergic Neurotransmission in Temperature Responses to Methamphetamine and Stress: Mathematical Modeling as a Data Assimilation Approach

Experimental Data

Orexinergic neurotransmission is involved in mediating temperature responses to methamphetamine (Meth). In experiments in rats, SB-334867 (SB), an antagonist of orexin receptors (OX1R), at a dose of 10 mg/kg decreases late temperature responses (t>60 min) to an intermediate dose of Meth (5 mg/kg). A higher dose of SB (30 mg/kg) attenuates temperature responses to low dose (1 mg/kg) of Meth and to stress. In contrast, it significantly exaggerates early responses (t<60 min) to intermediate and high doses (5 and 10 mg/kg) of Meth. As pretreatment with SB also inhibits temperature response to the stress of injection, traditional statistical analysis of temperature responses is difficult.

Mathematical Modeling

We have developed a mathematical model that explains the complexity of temperature responses to Meth as the interplay between excitatory and inhibitory nodes. We have extended the developed model to include the stress of manipulations and the effects of SB. Stress is synergistic with Meth on the action on excitatory node. Orexin receptors mediate an activation of on both excitatory and inhibitory nodes by low doses of Meth, but not on the node activated by high doses (HD). Exaggeration of early responses to high doses of Meth involves disinhibition: low dose of SB decreases tonic inhibition of HD and lowers the activation threshold, while the higher dose suppresses the inhibitory component. Using a modeling approach to data assimilation appears efficient in separating individual components of complex response with statistical analysis unachievable by traditional data processing methods.