Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization

Although three-dimensional electron microscopy (3D-EM) permits structural characterization of macromolecular assemblies in distinct functional states, the inability to classify projections from structurally heterogeneous samples has severely limited its application. We present a maximum likelihood-based classification method that does not depend on prior knowledge about the structural variability, and demonstrate its effectiveness for two macromolecular assemblies with different types of conformational variability: the Escherichia coli ribosome and Simian virus 40 (SV40) large T-antigen.     

The human cytochrome c oxidase assembly factors SCO1 and SCO2 have regulatory roles in the maintenance of cellular copper homeostasis

Human SCO1 and SCO2 are metallochaperones that are essential for the assembly of the catalytic core of cytochrome c oxidase (COX). Here we show that they have additional, unexpected roles in cellular copper homeostasis. Mutations in either SCO result in a cellular copper deficiency that is both tissue and allele specific. This phenotype can be dissociated from the defects in COX assembly and is suppressed by overexpression of SCO2, but not SCO1. Overexpression of a SCO1 mutant in control cells in which wild-type SCO1 levels were reduced by shRNA recapitulates the copper-deficiency phenotype in SCO1 patient cells. The copper-deficiency phenotype reflects not a change in high-affinity copper uptake but rather a proportional increase in copper efflux. These results suggest a mitochondrial pathway for the regulation of cellular copper content that involves signaling through SCO1 and SCO2, perhaps by their thiol redox or metal-binding state.     

Effects of myeloid sirtuin 1 deficiency on hypothalamic neurogranin in mice fed a high-fat diet

Hypothalamic inflammation has been known as a contributor to high-fat diet (HFD)-induced insulin resistance and obesity. Myeloid-specific sirtuin 1 (SIRT1) deletion aggravates insulin resistance and hypothalamic inflammation in HFD-fed mice. Neurogranin, a calmodulin-binding protein, is expressed in the hypothalamus. However, the effects of myeloid SIRT1 deletion on hypothalamic neurogranin has not been fully clarified. To investigate the effect of myeloid SIRT1 deletion on food intake and hypothalamic neurogranin expression, mice were fed a HFD for 20 weeks. Myeloid SIRT1 knockout (KO) mice exhibited higher food intake, weight gain, and lower expression of anorexigenic proopiomelanocortin in the arcuate nucleus than WT mice. In particular, KO mice had lower ventromedial hypothalamus (VMH)-specific neurogranin expression. However, SIRT1 deletion reduced HFD-induced hypothalamic neurogranin. Furthermore, hypothalamic phosphorylated AMPK and parvalbumin protein levels were also lower in HFD-fed KO mice than in HFD-fed WT mice. Thus, these findings suggest that myeloid SIRT1 deletion affects food intake through VMH-specific neurogranin-mediated AMPK signaling and hypothalamic inflammation in mice fed a HFD.     

Invasion by extremes: population spread with variation in dispersal and reproduction

For populations having dispersal described by fat-tailed kernels (kernels with tails that are not exponentially bounded), asymptotic population spread rates cannot be estimated by traditional models because these models predict continually accelerating (asymptotically infinite) invasion. The impossible predictions come from the fact that the fat-tailed kernels fitted to dispersal data have a quality (nondiscrete individuals and, thus, no moment-generating function) that never applies to data. Real organisms produce finite (and random) numbers of offspring; thus, an empirical moment-generating function can always be determined. Using an alternative method to estimate spread rates in terms of extreme dispersal events, we show that finite estimates can be derived for fat-tailed kernels, and we demonstrate how variable reproduction modifies these rates. Whereas the traditional models define spread rate as the speed of an advancing front describing the expected density of individuals, our alternative definition for spread rate is the expected velocity for the location of the furthest-forward individual in the population. The asymptotic wave speed for a constant net reproductive rate R0 is approximated as (1/T)(piuR)/2)(1/2) m yr(-1), where T is generation time, and u is a distance parameter (m2) of Clark et al.'s 2Dt model having shape parameter p = 1. From fitted dispersal kernels with fat tails and infinite variance, we derive finite rates of spread and a simple method for numerical estimation. Fitted kernels, with infinite variance, yield distributions of rates of spread that are asymptotically normal and, thus, have finite moments. Variable reproduction can profoundly affect rates of spread. By incorporating the variance in reproduction that results from variable life span, we estimate much lower rates than predicted by the standard approach, which assumes a constant net reproductive rate. Using basic life-history data for trees, we show these estimated rates to be lower than expected from previous analytical models and as interpreted from paleorecords of forest spread at the end of the Pleistocene. Our results suggest reexamination of past rates of spread and the potential for future response to climate change.     

PPARgamma activation, by reducing oxidative stress, increases NO bioavailability in coronary arterioles of mice with Type 2 diabetes

We tested the hypothesis that short-term treatment of mice with Type 2 diabetes mellitus (DM) with rosiglitazone (ROSI), an agonist of peroxisome proliferator-activated receptor-gamma, ameliorates the impaired coronary arteriolar dilation by reducing oxidative stress via a mechanism unrelated to its effect on hyperglycemia and hyperinsulinemia. Control and Type 2 DM (db/db) mice were treated with ROSI (3 mg x kg(-1) x day(-1)) for 7 days, which did not significantly affect their serum concentration of glucose and insulin. Compared with controls, in db/db mice serum levels of 8-isoprostane and dihydroethydine-detectable superoxide production in carotid arteries were significantly elevated and were reduced by ROSI treatment. In coronary arterioles (diameter, approximately 80 microm) isolated from db/db mice, the reduced dilations to ACh, the nitric oxide (NO) donor NONOate, and increases in flow were significantly augmented either by in vitro administration of apocynin, an inhibitor of NAD(P)H-oxidase, or by in vivo ROSI treatment, responses that were then significantly reduced by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. In aortas of db/db mice, activity of SOD and catalase was reduced, whereas NAD(P)H oxidase activity was enhanced. ROSI treatment enhanced catalase and reduced NAD(P)H oxidase activity but did not affect the activity of SOD. These findings suggest that ROSI treatment enhances NO mediation of coronary arteriolar dilations due to the reduction of vascular NAD(P)H oxidase-derived superoxide production and enhancement of catalase activity. Thus, in addition to the previously revealed beneficial metabolic effects, the antioxidant action of rosiglitazone may protect coronary arteriolar function in Type 2 DM.     

Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats

We hypothesized that aging is characterized by a reduced release of nitric oxide (NO) in response to shear stress in resistance vessels. Mesenteric arterioles and arteries of young (6 mo) and aged (24 mo) male Fischer 344 rats were isolated and cannulated. Shear stress (15 dyn/cm(2))-induced dilation was significantly reduced and shear stress (1, 5, 10, and 15 dyn/cm(2))-induced increases in perfusate nitrite were significantly smaller at all shear stress levels in vessels of aged rats. Inhibition of NO synthesis abolished shear stress-induced release of nitrite. Furthermore, shear stress (15 dyn/cm(2))-induced release of nitrate was significantly higher and total nitrite (nitrite plus nitrate) was significantly lower in vessels of aged rats. Tiron or SOD significantly increased nitrite released from vessels of aged rats, but this was still significantly less than that in young rats. Superoxide production was increased and the activity of SOD was decreased in vessels of aged rats. There were no differences in endothelial NO synthase (eNOS) protein and basal activity or in Cu/Zn-SOD and Mn-SOD proteins in vessels of the two groups, but extracellular SOD was significantly reduced in vessels of aged rats. Maximal release of NO induced by shear stress plus ACh (10(-5) M) was comparable in the two groups, but phospho-eNOS in response to shear stress (15 dyn/cm(2)) was significantly reduced in vessels of aged rats. These data suggest that an increased production of superoxide, a reduced activity of SOD, and an impaired shear stress-induced activation of eNOS are the causes of the decreased shear stress-induced release of NO in vessels of aged rats.     

Microvascular dysfunction after transient high glucose is caused by superoxide-dependent reduction in the bioavailability of NO and BH(4)

We hypothesized that transient high-glucose concentration interferes with mediation by nitric oxide (NO) of flow-induced dilation (FID) of arterioles due to enhanced production of superoxide. In isolated, pressurized (80 mmHg) rat gracilis muscle arterioles ( approximately 130 microm) after transient high-glucose treatment (tHG; incubation with 30 mM glucose for 1 h), FID was reduced (maximum: control, 38 +/- 4%; after tHG, 17 +/- 3%), which was not further diminished by the NO synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME; 18 +/- 2%). Correspondingly, an enhanced polyethylene-glycol-SOD (PEG-SOD)-sensitive superoxide production was detected after tHG in carotid arteries by dihydroethydine (DHE) staining. Presence of PEG-SOD during tHG prevented the reduction of FID (41 +/- 3%), which could be inhibited by l-NAME (20 +/- 4%). Administration of PEG-SOD after tHG did not prevent the reduction of FID (22 +/- 3%). Sepiapterin, a precursor of the NO synthase cofactor tetrahydrobiopterin (BH(4)), administered during tHG did not prevent the reduction of FID (maximum, 15 +/- 5%); however, it restored FID when administered after tHG (32 +/- 4%). Furthermore, inhibition of either glycolysis by 2-deoxyglucose or mitochondrial complex II by 2-thenoyltrifluoroacetone reduced the tHG-induced DHE-detectable enhanced superoxide production in carotid arteries and prevented FID reduction in arterioles (39 +/- 5 and 35 +/- 2%). Collectively, these findings suggest that in skeletal muscle arterioles, a transient elevation of glucose via its increased metabolism, elicits enhanced production of superoxide, which decreases the bioavailability of NO and the level of the NOS cofactor BH(4), resulting in a reduction of FID mediated by NO.     

Interactions between glutamate transporters and metabotropic glutamate receptors at excitatory synapses in the cerebellar cortex

Five glutamate transporter genes have been identified; two of these (EAAT3 and EAAT4) are expressed in neurons and are predominantly confined to the membranes of cell bodies and dendrites. At an ultrastructural level, glutamate transporters have been shown to surround excitatory synapses in hippocampus and cerebellum [J. Neurosci. 18 (1998) 3606; J. Comp. Neurol. 418 (2000) 255]. This pattern of localization overlaps the well-described perisynaptic distribution of Group I metabotropic glutamate receptors or mGluRs [Neuron 11 (1993) 771; J. Chem. Neuroanat. 13 (1997) 77]. Both of the principal excitatory synaptic inputs to cerebellar Purkinje neurons, the parallel fiber (PF) and climbing fiber (CF) synapses, express mGluR-dependent forms of synaptic plasticity [Nat. Neurosci. 4 (2001) 467]. Prompted by the colocalization of postsynaptic glutamate transporters and mGluRs, we have examined whether glutamate uptake limits mGluR-mediated signals and mGluR-dependent forms of plasticity at PF and CF synapses in cerebellar slices. We find that, at PF and, surprisingly also at CF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release. At both PF and CF synapses, mGluR responses are strongly limited by glutamate transporters under resting conditions and are facilitated by short trains of stimuli. Nearly every Purkinje neuron expresses an mGluR-mediated synaptic current upon inhibition of glutamate transport. Global applications of glutamate achieved by photolysis of chemically caged glutamate yield similar results and argue that the colocalized transporters can effectively limit glutamate access to the mGluRs even in the face of such a large amount of transmitter. We hypothesize that neuronal glutamate transporters and Group I mGluRs located in the perisynaptic space interact to sense and then regulate the amount of glutamate escaping excitatory synapses. This hypothesis is currently being tested using electrophysiological methods and the introduction of optically tagged glutamate transporter proteins. In the brain, synaptic signals are terminated mainly by neurotransmitter transporters. Families of genes encoding transporters for the major neurotransmitters (dopamine, GABA, glutamate, glycine, norepinephrine and 5-HT) have been identified. Although transporters serve as targets for important classes of therapeutic drugs (e.g. selective serotonin reuptake inhibitors) and drugs of abuse (amphetamine, cocaine), little is known about how they operate at a molecular level or contribute to synaptic transmission.