Behavioral and anatomical consequences of early versus late symbol training in macaques

Distinct brain regions, reproducible from one person to the next, are specialized for processing different kinds of human expertise, such as face recognition?and reading. Here, we explore the relationship between age of learning, learning ability, and specialized brain structures. Specifically, we ask whether the existence of reproducible cortical domains necessarily means that certain abilities are innate, or innately easily learned, or whether reproducible domains can be formed, or refined, by interactions between genetic programs and common early experience. Functional MRI showed that intensive early, but not late, experience caused the formation of category-selective regions in macaque temporal lobe for stimuli never naturally encountered by monkeys. And behaviorally, early training produced more fluent processing of these stimuli than the same training in adults. One explanation for these results is that in higher cortical areas, as in early sensory areas, experience drives functional clustering and functional clustering determines how that information is processed.     

Insights into the regulation of the ryanodine receptor: differential effects of Mg2+ and Ca2+ on ATP binding

Calcium ions are frequently used second messengers in most living organisms. Members of the family of ryanodine sensitive calcium channels (ryanodine receptors, RyRs) are responsible for many important Ca(2+) signaling events in both excitable and nonexcitable cells. The biological activity of these membrane proteins is modulated and regulated by a great variety of different cellular and extracellular effectors, proteins, and small molecules. However, very little is still understood about how the modulators work on a molecular level. The very large size of more than 2 million Da per functional tetrameric RyR unit and its membrane association have made more detailed biochemical and structural analysis extremely challenging.     

Rhodopsin photointermediates in two-dimensional crystals at physiological temperatures

Bovine rhodopsin photointermediates formed in two-dimensional (2D) rhodopsin crystal suspensions were studied by measuring the time-dependent absorbance changes produced after excitation with 7 ns laser pulses at 15, 25, and 35 degrees C. The crystalline environment favored the Meta I(480) photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspensions at 35 degrees C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the 380 nm absorbing product of Meta I(480) decay did not display the anomalous pH dependence characteristic of classical Meta II in the native disk membrane. Crystal suspensions bleached at 35 degrees C and quenched to 19 degrees C showed that a rapid equilibrium existed on the approximately 1 s time scale, which suggests that the unprotonated predecessor of Meta II in the native membrane environment (sometimes called MII(a)) forms in 2D rhodopsin crystals but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and does not result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data of the 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changes and the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in the native membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach due to the random orientation of linearly dichroic crystals relative to the linearly polarized laser was explored as a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nm product of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activation suggests that determination of its structure would be of interest.     

Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis is a fatal childhood neurological disorder caused by a deficiency in the lysosomal protease tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian member of the S53 family of serine proteases, a group characterized by a subtilisin-like fold, a Ser-Glu-Asp catalytic triad, and an acidic pH optimum. TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is proteolytically processed into the active enzyme after exposure to low pH in vitro or targeting to the lysosome in vivo. In this study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing four Asn-linked N-acetylglucosamines that is indistinguishable from fully glycosylated TPP1 in terms of autocatalytic processing of the proform and enzymatic properties of the mature protease. The crystal structure of deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large 151-residue C-shaped prodomain makes extensive contacts as it wraps around the surface of the catalytic domain with the two domains connected by a 24-residue flexible linker that passes through the substrate-binding groove. The proenzyme structure reveals suboptimal catalytic triad geometry with its propiece linker partially blocking the substrate-binding site, which together serve to prevent premature activation of the protease. Finally, we have identified numerous processing intermediates and propose a structural model that explains the pathway for TPP1 activation in vitro. These data provide new insights into TPP1 function and represent a valuable resource for constructing improved TPP1 variants for treatment of late infantile neuronal ceroid lipofuscinosis.Late infantile neuronal ceroid lipofuscinosis (LINCL)³ (OMIM number 204500) is a neurodegenerative lysosomal storage disease of childhood that presents typically between the ages of 2 and 4 years with the onset of seizures. Disease progression is reflected by blindness, dementia, mental retardation, and an increase in the severity of seizures. LINCL is always fatal, and the life span of patients is typically 6-15 years. LINCL is caused by mutations in TPP1 (previously named CLN2, for ceroid lipofuscinosis neuronal type 2 gene) (1), which normally encodes a lysosomal protease, tripeptidyl-peptidase 1 (TPP1, EC 3.4.14.9) (2, 3).There is currently no treatment of demonstrated efficacy for LINCL, but promising progress is being made in some directions. Proof-of-principle for virus-mediated gene therapy has been established in a mouse model of LINCL, with a significant improvement in disease phenotype achieved with the use of adeno-associated virus vectors expressing TPP1 (4-7). Affected children have also been treated with adeno-associated virus vectors, although it is too soon to determine whether significant clinical benefits have been achieved in these early trials (8). Enzyme replacement therapy, an approach that has proven successful in a number of other lysosomal storage diseases, has also been investigated in LINCL. Purified recombinant human TPP1 that contains the mannose 6-phosphate lysosomal targeting modification can be taken up by LINCL fibroblasts where it degrades storage material (9), and the protein has been introduced into the cerebrospinal fluid of the LINCL mouse model via intraventricular injection, resulting in significant uptake into the brain and some correction of neuropathology (10).For therapeutic approaches that rely upon replacing a mutant gene product with a functional protein via recombinant methods, e.g. gene and enzyme replacement therapy, a thorough understanding of the biological and biophysical properties of the protein in question are essential for success. Thus, for LINCL, considerable effort has been directed toward the investigation of TPP1, and as a result, this is a well characterized enzyme at the functional and molecular levels (reviewed in Refs. 11, 12). TPP1 encodes a 563-residue preproprotein with a cleavable N-terminal 19-residue signal sequence. The proenzyme (residues 20-563) is a soluble monomer that undergoes proteolytic cleavage in the lysosome, converting the zymogen to an active, mature protease (residues 196-563) (1). Studies on purified pro-TPP1 demonstrate that maturation is autocatalytic in vitro (13, 14) but may involve other proteases in vivo (15). TPP1 is glycosylated, and its N-linked oligosaccharides have been implicated in maturation, activity, targeting, and stability of the processed enzyme (16, 17).TPP1 is a serine protease (14) that possesses two catalytic functions as follows: a primary tripeptidyl exopeptidase activity with a pH optimum of ∼5.0 that catalyzes the sequential release of tripeptides from the unsubstituted N termini of substrates (18), and a much weaker endoproteolytic activity with a pH optimum of ∼3.0 (19). TPP1 exhibits broad substrate specificity (20) and is the only mammalian member of the S53 sedolisin family (reviewed in Ref. 21), which includes a number of unusual bacterial serine peptidases (22). High resolution crystal structures of both free and inhibitor-bound complexes have been determined for three bacterial members of this family (sedolisin (23-26), kumamolisin (27, 28), and kumamolisin-As (29, 30)), and for one (kumamolisin), the structure of a mutant, inactive precursor form has also been obtained (28). These proteins share a common subtilisin-like fold, an octahedrally coordinated calcium-binding site, and an active site that contains an unusual Ser-Glu-Asp (SED) catalytic triad, rather than the Ser-His-Asp (SHD) triad of subtilisin (31, 32). Chemical modification studies of TPP1 have revealed that Ser⁴⁷⁵ is the active site nucleophile (14). Modeling studies suggest that Glu²⁷² and Asp²⁷⁶ complete the catalytic triad and that Asp³⁶⁰ is homologous to the conserved Asn in the subtilisin family in its role in stabilization of the oxyanion of the tetrahedral intermediate during catalysis (33). Site-directed mutagenesis studies are consistent with these conclusions (14, 34).A detailed understanding of the tertiary structure of TPP1 may have implications for developing or improving therapeutic strategies. First, a high resolution model would provide the basis for targeted protein engineering efforts to design TPP1 derivatives with increased half-life prior to and/or upon delivery to the lysosome. Successful creation of a longer lived TPP1 molecule could significantly enhance gene or enzyme replacement approaches to LINCL. Second, a structural model for TPP1 could be valuable in designing derivatives tagged with protein transduction domains to facilitate crossing of the blood-brain barrier for delivery to the central nervous system from the bloodstream. In this study, we present the crystal structure of the proform of human TPP1 at 1.85 Å resolution. This model provides novel insights into the structural basis for the pH-induced auto-activation of the proform of TPP1. A structure of glycosylated pro-TPP1 has been independently determined, displaying features similar to those of deglycosylated TPP1.⁴     

NO-sensitive guanylyl cyclase β1 subunit is peripherally associated to chromosomes during mitosis. Novel role in chromatin condensation and cell cycle progression

NO-sensitive guanylyl cyclase (GCNO), the major NO target, is involved in important regulatory functions in the cardiovascular, gastrointestinal and central nervous systems. GC_NO exists as heterodimers of α(1/2) and β1 subunits. Deletion of the obligate β1 dimerizing partner abrogates NO/cGMP signaling and shortens the life span of KO mice. Localization studies in the CNS have shown that β1 is more widespread than α subunits and in some areas is the only GC_NO subunit expressed, suggesting that β1 may have GC_NO-independent functions. GC_NO is predominantly cytosolic, but association to membranes and other intracellular structures has been described. Here, we show localization of β1 in cytoplasm and nucleus of cells expressing α subunits and GC_NO activity (astrocytes, C6 cells), as well as in cells devoid of α subunits and GC_NO activity (microglia). In both cell types β1 associates peripherally to chromosomes in all phases of mitosis. Immunodepletion of β1 in C6 cells enhances chromatin condensation in an in vitro assay. Moreover, silencing β1 by siRNA induces cell cycle re-entry as determined by flow cytometry, and increases proliferation rate in a MTT-assay, whereas infection with β1-containing adenovirus has the opposite effect. These actions are independent of cGMP formation. We postulate that β1 is a multifunctional protein that regulates chromatin condensation and cell cycle progression, in addition to being an obligate monomer in functional GC_NO heterodimers.     

Validation of mega-environment universal and specific QTL associated with seed yield and agronomic traits in soybeans

The value of quantitative trait loci (QTL) is dependant on the strength of association with the traits of interest, allelic diversity at the QTL and the effect of the genetic background on the expression of the QTL. A number of recent studies have identified QTL associated with traits of interest that appear to be independent of the environment but dependant on the genetic background in which they are found. Therefore, the objective of this study was to validate universal and/or mega-environment-specific seed yield QTL that have been previously reported in an independent recombinant inbred line (RIL) population derived from the cross between an elite Chinese and Canadian parent. The population was evaluated at two field environments in China and in five environments in Canada in 2005 and 2006. Of the seven markers linked to seed yield QTL reported by our group in a previous study, four were polymorphic between the two parents. No association between seed yield and QTL was observed. The result could imply that seed yield QTL were either not stable in this particular genetic background or harboured different alleles than the ones in the original mapping population. QTL_U Satt162 was associated with several agronomic traits of which lodging was validated. Both the non-adapted and adapted parent contributed favourable alleles to the progeny. Therefore, plant introductions have been validated as a source of favourable alleles that could increase the genetic variability of the soybean germplasm pool and lead to further improvements in seed yield and other agronomic traits.     

Effects of Longevinex (modified resveratrol) on cardioprotection and its mechanisms of action

Although resveratrol has been proven to possess diverse health benefits, several recent reports have demonstrated conflicting results on some aspects of its effects, including its anti-aging properties. Considerable debate appears to exist on the dose and bioavailability of resveratrol, leading to the controversies on its effectiveness. To resolve the problem, we designed a study with a resveratrol formulation that contained resveratrol supplemented with 5% quercetin and 5% rice bran phytate (commercially known as Longevinex). These ingredients were micronized to increase the bioavailability. Sprague-Dawley rats were gavaged with either Longevinex or vehicle (5% quercetin plus 5% rice bran phytate), and rats were sacrificed after 1 or 3 months, when isolated working hearts were subjected to 30 min ischemia followed by 2 h of reperfusion. Longevinex-treated hearts, irrespective of the duration of treatments, revealed superior cardiac performance, reduced infarct size, and induction of survival signals as evidenced by increased Bcl2/Bax ratio and enhanced Akt phosphorylation. In contrast, LC3-II and Beclin were enhanced significantly after 3 months of Longevinex treatment, suggesting that autophagy occurred only after feeding Longevinex to rats for a prolonged period of time. Corroborating with the results of autophagy, Sirt1 and Sirt3 increased significantly only after 3 months of Longevinex treatment, suggesting that enhanced expression of Sirts correlated with induction of autophagy. In concert, Longevinex caused phosphorylation and nuclear translocation of FoxO1, FoxO3a, and FoxO4, indicating involvement of FoxOs with autophagy. Since Sirts and FoxOs are reliable markers of longevity, the results appear to suggest that Longevinex induces longevity after prolonged feeding via induction of autophagy, while it converts death signals into survival signals and provides cardioprotection within a relatively shorter period of time.     

Aldose reductase inhibition prevents metaplasia of airway epithelial cells

Background

Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR) regulates the mucus cell metaplasia in vitro and in vivo.

Methodology/Findings

Metaplasia in primary human small airway epithelial cells (SAEC) was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS)-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of ragweed pollen extract (RWE)-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE.

Conclusions

The results show that exposure of SAEC to IL-13 caused goblet cell metaplasia, which was significantly prevented by AR inhibition. Administration of fidarestat to mice prevented RWE-induced goblet cell metaplasia and AR null mice were largely resistant to allergen induced changes in the lung. Thus our results indicate that AR inhibitors such as fidarestat could be developed as therapeutic agents to prevent goblet cell metaplasia in asthma and related pathologies.