Lysosomal chat maintains the balance

The original idea that each protein follows a particular proteolytic pathway for its degradation is no longer supported. Instead, different proteolytic systems can simultaneously contribute to the degradation of a particular protein, or they can alternate in this task depending, for the most part, on the cellular conditions. It is thus reasonable to expect that some level of communication exists among different proteolytic systems to orchestrate these coordinated activities. Direct cross-talk between two forms of autophagy, macroautophagy and chaperone-mediated autophagy (CMA) has been recently demonstrated. Cells respond to blockage of CMA by upregulating macroautophagy. Although macroautophagy cannot completely substitute for the lack of CMA, the partial redundancy between both pathways allows some level of compensation, enough to maintain protein degradation and preserve cell homeostasis. Understanding the cross-talk among different autophagic pathways and with other proteolytic systems is important to predict the type of compensatory mechanisms that could be elicited in response to failure of one of these systems, and to understand the consequences that manipulating one of these pathways for therapeutic purposes could have on the activity of the other pathways.     

In-cell NMR for protein-protein interactions (STINT-NMR)

We describe an in-cell NMR-based method for mapping the structural interactions (STINT-NMR) that underlie protein-protein complex formation. This method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring their interactions using in-cell NMR spectroscopy. The resulting NMR data provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution. Unlike the case where interacting proteins are simultaneously overexpressed in the labeled medium, in STINT-NMR the spectral complexity is minimized because only the target protein is labeled with NMR-active nuclei, which leaves the interactor protein(s) cryptic. This method can be combined with genetic and molecular screens to provide a structural foundation for proteomic studies. The protocol takes 4 d from the initial transformation of the bacterial cells to the acquisition of the NMR spectra.     

Echolocating bats use a nearly time-optimal strategy to intercept prey

Acquisition of food in many animal species depends on the pursuit and capture of moving prey. Among modern humans, the pursuit and interception of moving targets plays a central role in a variety of sports, such as tennis, football, Frisbee, and baseball. Studies of target pursuit in animals, ranging from dragonflies to fish and dogs to humans, have suggested that they all use a constant bearing (CB) strategy to pursue prey or other moving targets. CB is best known as the interception strategy employed by baseball outfielders to catch ballistic fly balls. CB is a time-optimal solution to catch targets moving along a straight line, or in a predictable fashion--such as a ballistic baseball, or a piece of food sinking in water. Many animals, however, have to capture prey that may make evasive and unpredictable maneuvers. Is CB an optimum solution to pursuing erratically moving targets? Do animals faced with such erratic prey also use CB? In this paper, we address these questions by studying prey capture in an insectivorous echolocating bat. Echolocating bats rely on sonar to pursue and capture flying insects. The bat's prey may emerge from foliage for a brief time, fly in erratic three-dimensional paths before returning to cover. Bats typically take less than one second to detect, localize and capture such insects. We used high speed stereo infra-red videography to study the three dimensional flight paths of the big brown bat, Eptesicus fuscus, as it chased erratically moving insects in a dark laboratory flight room. We quantified the bat's complex pursuit trajectories using a simple delay differential equation. Our analysis of the pursuit trajectories suggests that bats use a constant absolute target direction strategy during pursuit. We show mathematically that, unlike CB, this approach minimizes the time it takes for a pursuer to intercept an unpredictably moving target. Interestingly, the bat's behavior is similar to the interception strategy implemented in some guided missiles. We suggest that the time-optimal strategy adopted by the bat is in response to the evolutionary pressures of having to capture erratic and fast moving insects.     

Interaction between glutamate and GABA systems in the integration of sympathetic outflow by the paraventricular nucleus of the hypothalamus

The paraventricular nucleus (PVN) of the hypothalamus is a central site known to modulate sympathetic outflow. Excitatory and inhibitory neurotransmitters within the PVN dictate final outflow. The goal of the present study was to examine the role of the interaction between the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter GABA in the regulation of sympathetic activity. In alpha-chloralose- and urethane-anesthetized rats, microinjection of glutamate and N-methyl-D-aspartate (NMDA; 50, 100, and 200 pmol) into the PVN produced dose-dependent increases in renal sympathetic nerve activity, blood pressure, and heart rate. These responses were blocked by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP-5). Microinjection of bicuculline, a GABA(A) receptor antagonist, into the PVN (50, 100, and 200 pmol) also produced significant, dose-dependent increases in renal sympathetic nerve activity, blood pressure, and heart rate; AP-5 also blocked these responses. Using microdialysis and HPLC/electrochemical detection techniques, we observed that bicuculline infusion into the PVN increased glutamate release. Using an in vitro hypothalamic slice preparation, we found that bicuculline increased the frequency of glutamate-mediated excitatory postsynaptic currents in PVN-rostral ventrolateral medullary projecting neurons, supporting a GABA(A)-mediated tonic inhibition of this excitatory input into these neurons. Together, these data indicate that 1) glutamate, via NMDA receptors, excites the presympathetic neurons within the PVN and increases sympathetic outflow and 2) this glutamate excitatory input is tonically inhibited by a GABA(A)-mediated mechanism.     

Which Measure of Abdominal Adiposity Best Relates with Body Mass Index Among Older Bengalee Hindus of Kolkata, India? A Comparison of Three Measures

A cross-sectional study of 410 (210 men and 200 women) older (≥55 years) Bengalee Hindus of Kolkata, India, was undertaken to determine which measure of abdominal adiposity best relates with body mass index (BMI), an indicator of overall adiposity. Three measures of abdominal adiposity were studied: waist circumference (WC), waist–hip ratio (WHR), and conicity index (CI). Results revealed that, in both sexes, WC had the strongest partial (age controlled) correlations with BMI (men = 0.56, women = 0.80). Linear regression analyses demonstrated that BMI had the strongest significant impact on WC in both sexes. BMI alone accounted for 28.2 and 61.8% variation in WC in men and women, respectively. This strongest significant impact remained even after controlling for age. In conclusion, this study provides strong evidence that WC can be preferred over WHR and CI in studies dealing with BMI among older Bengalee Hindus. In particular, BMI and WC can be useful in studies dealing with aging and anthropometric characteristics among older Bengalees.     

Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.     

Preparation, spectral characterization, cytotoxic and thermal studies of platinum (IV) thiohydrazone complexes

Platinum (IV) complexes of type [Pt(L)₂Cl₂] [where, L=benzaldehyde-N,N-diphenyl-N-thiohydrazone (L¹), salicylaldehyde-N,N-diphenyl-N-thiohydrazone (L²), acetaphenone-N,N-diphenyl-N-thiohydrazone (L³) and cyclohexanone-N,N-diphenyl-N-thiohydrazone (L⁴)] have been synthesized. The thiohydrazones can exist as thione–thiol tautomer and coordinates as a bidentate N–S ligand. The ligands found to act in monobasic bidentate fashion. Analytical data reveals that metal to ligand stoichiometry is 1:2. The complexes have been characterized by elemental analysis, IR, mass, electronic, ¹H NMR spectroscopic studies, and TG/DTA study. Various kinetic and thermodynamic parameters like order of reaction (n), activation energy (E_a), apparent activation entropy (S^#) and heat of reaction (ΔH) have also been carried out for one complex. Cytotoxic study has also been carried out for one complex.     

Effect of normal and waxy maize starch on growth, food utilization and hepatic glucose metabolism in European sea bass (Dicentrarchus labrax) juveniles

We determined the effect of dietary starch on growth performance and feed utilization in European sea bass juveniles. Data on the dietary regulation of key hepatic enzymes of the glycolytic, gluconeogenic, lipogenic and amino acid metabolic pathways (hexokinase, HK; glucokinase, GK; pyruvate kinase, PK; fructose-1,6-bisphosphatase, FBPase; glucose-6-phosphatase, G6Pase; glucose-6-phosphate dehydrogenase, G6PD; alanine aminotransferase, ALAT; aspartate aminotransferase, ASAT and glutamate dehydrogenase, GDH) were also measured. Five isonitrogenous (48% crude protein) and isolipidic (14% crude lipids) diets were formulated to contain 10% normal starch (diet NS10), 10% waxy starch (diet WS10), 20% normal starch (diet NS20), 20% waxy starch (diet WS20) or no starch (control diet). Another diet was formulated with no carbohydrate, and contained 68% crude protein and 14% crude lipids (diet HP). Each experimental diet was fed to triplicate groups of 30 fish (initial weight: 23.3 g) on an equivalent feeding scheme for 12 weeks. The best growth performance and feed efficiency were achieved with fish fed the HP diet. Neither the level nor the nature of starch had measurable effects on growth performance of sea bass juveniles. Digestibility of starch was higher with waxy starch and decreased with increasing levels of starch in the diet. Whole-body composition and plasma metabolites, mainly glycemia, were not affected by the level and nature of the dietary starch. Data on enzyme activities suggest that dietary carbohydrates significantly improve protein utilization associated with increased glycolytic enzyme activities (GK and PK), as well as decreased gluconeogenic (FBPase) and amino acid catabolic (GDH) enzyme activities. The nature of dietary carbohydrates tested had little influence on performance criteria.     

Molecular dynamics simulations on pars intercerebralis major peptide-C (PMP-C) reveal the role of glycosylation and disulfide bonds in its enhanced structural stability and function

Fucosylation of Thr 9 in pars intercerebralis major peptide-C (PMP-C) enhances its structural stability and functional ability as a serine protease inhibitor. In order to understand the role of disulfide bonds and glycosylation on the structure and function of PMP-C, we have carried out multiple explicit solvent molecular dynamics (MD) simulations on fucosylated and non-fucosylated forms of PMP-C, both in the presence and absence of the disulfide bonds. Our simulations revealed that there were no significant structural changes in the native disulfide bonded forms of PMP-C due to fucosylation. On the other hand, the non-fucosylated form of PMP-C without disulfide bonds showed larger deviations from the starting structure than the fucosylated form. However, the structural deviations were restricted to the terminal regions while core β-sheet retained its hydrogen bonded structure even in absence of disulfide bonds as well as fucosylation. Interestingly, fucosylation of disulfide bonded native PMP-C led to a decreased thermal flexibility in the residue stretch 29-32 which is known to interact with the active site of the target proteases. Our analysis revealed that disulfide bonds covalently connect the residue stretch 29-32 to the central β-sheet of PMP-C and using a novel network of side chain interactions and disulfide bonds fucosylation at Thr 9 is altering the flexibility of the stretch 29-32 located at a distal site. Thus, our simulations explain for the first time, how presence of disulfide bonds between conserved cysteines and fucosylation enhance the function of PMP-C as a protease inhibitor.     

Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D

The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3′-phosphomonoesterase and 3′-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn²⁺• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3′-deoxynucleotide, 3′-deoxynucleotide 3′-phosphate, or 3′ ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3′-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd²⁺) and inhibitory (Zn²⁺) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn²⁺.