Low-temperature effect on enzyme activities involved in sucrose–starch partitioning in salt-stressed and salt-acclimated cotyledons of quinoa (Chenopodium quinoa Willd.) seedlings

The effect of low temperature on growth, sucrose–starch partitioning and related enzymes in salt-stressed and salt-acclimated cotyledons of quinoa (Chenopodium quinoa Willd.) was studied. The growth of cotyledons and growing axes in seedlings grown at 25/20 °C (light/dark) and shifted to 5/5 °C was lower than in those only growing at 25/20 °C (unstressed). However, there were no significant differences between low-temperature control and salt-treated seedlings. The higher activities of sucrose phosphate synthase (SPS, EC 2.4.1.14) and soluble acid invertase (acid INV, EC 3.2.1.25) were observed in salt-stressed cotyledons; however, the highest acid INV activity was observed in unstressed cotyledons. ADP-glucose pyrophosphorylase (ADP-GPPase, EC 2.7.7.27) was higher in unstressed cotyledons than in stressed ones. However, between 0 and 4 days the highest value was observed in salt-stressed cotyledons. The lowest value of ADP-GPPase was observed in salt-acclimated cotyledons. Low temperature also affected sucrose synthase (SuSy, EC 2.4.1.13) activity in salt-treated cotyledons. Sucrose and glucose were higher in salt-stressed cotyledons, but fructose was essentially higher in low-temperature control. Starch was higher in low-temperature control; however, the highest content was observed at 0 day in salt-acclimated cotyledons. Results demonstrated that low temperature induces different responses on sucrose–starch partitioning in salt-stressed and salt-acclimated cotyledons. Data also suggest that in salt-treated cotyledons source–sink relations (SSR) are changed in order to supply soluble sugars and proline for the osmotic adjustment. Relationships between starch formation and SuSy activity are also discussed.     

Characterization of the smallest dimeric bile salt hydrolase from a thermophile Brevibacillus sp.

A thermophilic microorganism producing bile salt hydrolase was isolated from hot water springs, Pali, Maharashtra, India. This microorganism was identified as Brevibacillus sp. by 16S rDNA sequencing. Bile salt hydrolase (BSH) was purified to homogeneity from this thermophilic source using Q-sepharose chromatography and its enzymatic properties were characterized. The subunit molecular mass of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and, 28.2 kDa by MALDI-TOF analysis. The native molecular mass was estimated to be 56 kDa by gel filtration chromatography, indicating the protein to be a homodimer. The pH and temperature optimum for the enzyme catalysis were 9.0 and 60°C, respectively. Even though BSH from Brevibacillus sp. hydrolyzed all of the six major human bile salts, the enzyme preferred glycine conjugated substrates with apparent K _M and k _cat values of 3.08 μM and 6.32 × 10² s⁻¹, respectively, for glycodeoxycholic acid. The NH₂-terminal sequence of the purified enzyme was determined and it did not show any homology with other bacterial bile salt hydrolases. To our knowledge, this is the first report describing the purification of BSH to homogeneity from a thermophilic source. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of α-Synuclein: IMPLICATIONS FOR THE PATHOGENESIS OF PARKINSON DISEASE*S?

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln⁷⁹ and Gln¹⁰⁹ serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln⁷⁹ and Gln¹⁰⁹. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn.Parkinson disease (PD)² is a progressive movement disorder that is caused by the loss of dopaminergic neurons in the substantia nigra, the part of the brain responsible for controlling movement. Clinically, PD is manifested in symptoms that include tremors, rigidity, and difficulty in initiating movement (bradykinesia). Pathologically, PD is characterized by the presence of intraneuronal, cytoplasmic inclusions known as Lewy bodies (LB), which are composed primarily of the protein “α-synuclein” (α-syn) (1) and are seen in the post-mortem brains of PD patients with the sporadic or familial forms of the disease (2). α-Syn is a presynaptic protein of 140 residues with a “natively” unfolded structure (3). Three missense point mutations in α-syn (A30P, E46K, and A53T) are associated with the early-onset, dominant, inherited form of PD (4, 5). Moreover, duplication or triplication of the α-syn gene has been linked to the familial form of PD, suggesting that an increase in α-syn expression is sufficient to cause PD. Together, these findings suggest that α-syn plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn oligomerization and fibrillogenesis in vivo remain poorly understood. In vitro aggregation studies have shown that the mutations associated with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but only E46K and A53T α-syn show higher propensity to fibrillize than wild-type (WT) α-syn (6-8). This suggests that oligomerization, rather than fibrillization, is linked to early-onset familial PD (9). Our understanding of the molecular composition and biochemical state of α-syn in LBs has provided important clues about protein-protein interactions and post-translational modifications that may play a role in modulating oligomerization, fibrillogenesis, and LB formation of the protein. In addition to ubiquitination (10), phosphorylation (11, 12), nitration (13, 14), and C-terminal truncation (15, 16), analysis of post-mortem brain tissues from PD and Lewy bodies in dementia patients has confirmed the colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked α-syn monomers and higher molecular aggregates in LBs within dopaminergic neurons (17, 18). Tissue transglutaminase catalyzes a calcium-dependent transamidating reaction involving glutamine and lysine residues, which results in the formation of a covalent cross-link via ε-(γ-glutamyl) lysine bonds (Fig. 2F). To date, seven different isoforms of tTGs have been reported, of which only tTG2 seems to be expressed in the human brain (19), whereas tTG1 and tTG3 are more abundantly found in stratified squamous epithelia (20). Subsequent immuno-histochemical, colocalization, and immunoprecipitation studies have shown that the levels of tTG and cross-linked α-syn species are increased in the substantia nigra of PD brains (17). These findings, combined with the known role of tTG in cross-linking and stabilizing bimolecular assemblies, led to the hypothesis that tTG plays an important role in the initiation and propagation of α-syn fibril formation and that it contributes to fibril stability in LBs. This hypothesis was initially supported by in vitro studies demonstrating that tTG catalyzes the polymerization of the α-syn-derived non-amyloid component (NAC) peptide via intermolecular covalent cross-linking of residues Gln⁷⁹ and Lys⁸⁰ (21) and by other studies suggesting that tTG promotes the fibrillization of amyloidogenic proteins implicated in the pathogenesis of other neurodegenerative diseases such as Alzheimer disease, supranuclear palsy, Huntington disease, and other polyglutamine diseases (22-24). However, recent in vitro studies with full-length α-syn have shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn and inhibits, rather than promotes, its fibrillization in vitro (25, 26). The structural basis of this inhibitory effect and the exact residues involved in tTG-mediated cross-linking of α-syn, as well as structural and functional consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native (—) and cross-linked (- - -) α-syn, showing that most tTG-catalyzed cross-linking products of WT or disease-associated mutant forms of α-syn are intramolecularly linked (predominant peak with two cross-links), and up to three intramolecular cross-links can occur (left shoulder). The abbreviations M and m/cl are used to designate native and cross-linked α-synuclein, respectively. D and E, kinetic analysis of α-syn (A30P) cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic depiction of the tTG-catalyzed chemical reaction (isodipeptide formation) between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn and investigated how cross-linking these residues affects the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Using single-site mutagenesis and mass spectrometry applied to exhaustive proteolytic digests of native and cross-linked monomeric α-syn, we identified Gln¹⁰⁹ and Gln⁷⁹ as the major tTG substrates. We demonstrate that the altered electrophoretic mobility of the intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of tTG-catalyzed cross-linking of Gln¹⁰⁹ to lysine residues in the N terminus of α-syn, which leads to the formation of more compact monomers. Consistent with previous studies, we show that intramolecularly cross-linked α-syn forms off-pathway oligomers that are distinct from those formed by the wild-type protein and that do not convert to fibrils within the time scale of our experiments (3-5 days). We also show that membrane-bound α-syn is a substrate of tTG and that intramolecular cross-linking does not interfere with the ability of monomeric α-syn to adopt an α-helical conformation upon binding to synthetic membranes. These studies provide novel mechanistic insight into the sequence and structural basis of events that allow tTG to inhibit α-syn fibrillogenesis, and they shed light on the potential role of tTG-catalyzed cross-linking in modulating the physiological and pathogenic properties of α-syn.     

Tuning to natural stimulus dynamics in primary auditory cortex

The amplitude and pitch fluctuations of natural soundscapes often exhibit "1/f spectra", which means that large, abrupt changes in pitch or loudness occur proportionally less frequently in nature than gentle, gradual fluctuations. Furthermore, human listeners reportedly prefer 1/f distributed random melodies to melodies with faster (1/f0) or slower (1/f2) dynamics. One might therefore suspect that neurons in the central auditory system may be tuned to 1/f dynamics, particularly given that recent reports provide evidence for tuning to 1/f dynamics in primary visual cortex. To test whether neurons in primary auditory cortex (A1) are tuned to 1/f dynamics, we recorded responses to random tone complexes in which the fundamental frequency and the envelope were determined by statistically independent "1/f(gamma) random walks," with gamma set to values between 0.5 and 4. Many A1 neurons showed clear evidence of tuning and responded with higher firing rates to stimuli with gamma between 1 and 1.5. Response patterns elicited by 1/f(gamma) stimuli were more reproducible for values of gamma close to 1. These findings indicate that auditory cortex is indeed tuned to the 1/f dynamics commonly found in the statistical distributions of natural soundscapes.     

Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants

Graminaceous plants have evolved a unique mechanism to acquire iron through the secretion of a family of small molecules, called mugineic acid family phytosiderophores (MAs). All MAs are synthesized from l-Met, sharing the same pathway from l-Met to 2'-deoxymugineic acid (DMA). DMA is synthesized through the reduction of a 3'-keto intermediate by deoxymugineic acid synthase (DMAS). We have isolated DMAS genes from rice (OsDMAS1), barley (HvDMAS1), wheat (TaD-MAS1), and maize (ZmDMAS1). Their nucleotide sequences indicate that OsDMAS1 encodes a predicted polypeptide of 318 amino acids, whereas the other three orthologs all encode predicted polypeptides of 314 amino acids and are highly homologous (82-97.5%) to each other. The DMAS proteins belong to the aldo-keto reductase superfamily 4 (AKR4) but do not fall within the existing subfamilies of AKR4 and appear to constitute a new subfamily within the AKR4 group. All of the proteins showed DMA synthesis activity in vitro. Their enzymatic activities were highest at pH 8-9, consistent with the hypothesis that DMA is synthesized in subcellular vesicles. Northern blot analysis revealed that the expression of each of the above DMAS genes is up-regulated under iron-deficient conditions in root tissue, and that of the genes OsDMAS1 and TaDMAS1 is up-regulated in shoot tissue. OsDMAS1 promoter-GUS analysis in iron-sufficient roots showed that its expression is restricted to cells participating in long distance transport and that it is highly up-regulated in the entire root under iron-deficient conditions. In shoot tissue, OsDMAS1 promoter drove expression in vascular bundles specifically under iron-deficient conditions.     

Safety and Immunogenicity Study of Multiclade HIV-1 Adenoviral Vector Vaccine Alone or as Boost following a Multiclade HIV-1 DNA Vaccine in Africa

Background

We conducted a double-blind, randomized, placebo-controlled Phase I study of a recombinant replication-defective adenovirus type 5 (rAd5) vector expressing HIV-1 Gag and Pol from subtype B and Env from subtypes A, B and C, given alone or as boost following a DNA plasmid vaccine expressing the same HIV-1 proteins plus Nef, in 114 healthy HIV-uninfected African adults.

Methodology/Principal Findings

Volunteers were randomized to 4 groups receiving the rAd5 vaccine intramuscularly at dosage levels of 1×10¹⁰ or 1×10¹¹ particle units (PU) either alone or as boost following 3 injections of the DNA vaccine given at 4 mg/dose intramuscularly by needle-free injection using Biojector® 2000. Safety and immunogenicity were evaluated for 12 months. Both vaccines were well-tolerated. Overall, 62% and 86% of vaccine recipients in the rAd5 alone and DNA prime - rAd5 boost groups, respectively, responded to the HIV-1 proteins by an interferon-gamma (IFN-γ) ELISPOT. The frequency of immune responses was independent of rAd5 dosage levels. The highest frequency of responses after rAd5 alone was detected at 6 weeks; after DNA prime - rAd5 boost, at 6 months (end of study). At baseline, neutralizing antibodies against Ad5 were present in 81% of volunteers; the distribution was similar across the 4 groups. Pre-existing immunity to Ad5 did not appear to have a significant impact on reactogenicity or immune response rates to HIV antigens by IFN-γ ELISPOT. Binding antibodies against Env were detected in up to 100% recipients of DNA prime - rAd5 boost. One volunteer acquired HIV infection after the study ended, two years after receipt of rAd5 alone.

Conclusions/Significance

The HIV-1 rAd5 vaccine, either alone or as a boost following HIV-1 DNA vaccine, was well-tolerated and immunogenic in African adults. DNA priming increased the frequency and magnitude of cellular and humoral immune responses, but there was no effect of rAd5 dosage on immunogenicity endpoints.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00124007","term_id":"NCT00124007"}}NCT00124007     

Intra-rater repeatability of gait parameters in healthy adults during self-paced treadmill-based virtual reality walking

Self-paced treadmill walking is becoming increasingly popular for the gait assessment and re-education, in both research and clinical settings. Its day-to-day repeatability is yet to be established. This study scrutinised the test-retest repeatability of key gait parameters, obtained from the Gait Real-time Analysis Interactive Lab (GRAIL) system. Twenty-three male able-bodied adults (age: 34.56 ± 5.12 years) completed two separate gait assessments on the GRAIL system, separated by 5 ± 3 days. Key gait kinematic, kinetic, and spatial-temporal parameters were analysed. The Intraclass-Correlation Coefficients (ICC), Standard Error Measurement (SEM), Minimum Detectable Change (MDC), and the 95% limits of agreements were calculated to evaluate the repeatability of these gait parameters. Day-to-day agreements were excellent (ICCs > 0.87) for spatial-temporal parameters with low MDC and SEM values, <0.153 and <0.055, respectively. The repeatability was higher for joint kinetic than kinematic parameters, as reflected in small values of SEM (<0.13 Nm/kg and <3.4°) and MDC (<0.335 Nm/kg and <9.44°). The obtained values of all parameters fell within the 95% limits of agreement. Our findings demonstrate the repeatability of the GRAIL system available in our laboratory. The SEM and MDC values can be used to assist researchers and clinicians to distinguish ‘real’ changes in gait performance over time.     

Therapeutic effects of Typha elephantina leave’s extract against paracetamol induced renal injury in rabbits

Present study focuses on ameliorative potential of Typha elephantina leave’s aqueous (TE.AQ) extract against Paracetamol (PCM) induced toxicity in rabbits. We fed the male rabbits with 300 mg PCM in alone and in combination with TE.AQ at different doses i.e. (100, 200 and 300 mg/kg body weight) or silymarin (100 mg/kg) daily for 21 days. PCM in alone significantly (P < 0.5) increased serum urea, uric acid, creatinine, total protein, albumin, globulin and blood urea nitrogen. Serum sodium, potassium and magnesium level were high. The glutathione, radical scavenging activity and Thiobarbituric acid reactive substances were significantly reduced. Treatment with TE.AQ at dose rate 300 mg/kg body weight and Silymarin significantly ameliorated all the parameters when compared with PCM administered group. The 100 and 200 mg of TE.AQ showed no significant effects. The histopathological examination confirmed the therapeutic potential of TE.AQ. These results established the presence of natural antioxidants in Typha elephantina leaves.     

Dimethylglycine Provides Salt and Temperature Stress Protection to Bacillus subtilis

Glycine betaine is a potent osmotic and thermal stress protectant of many microorganisms. Its synthesis from glycine results in the formation of the intermediates monomethylglycine (sarcosine) and dimethylglycine (DMG), and these compounds are also produced when it is catabolized. Bacillus subtilis does not produce sarcosine or DMG, and it cannot metabolize these compounds. Here we have studied the potential of sarcosine and DMG to protect B. subtilis against osmotic, heat, and cold stress. Sarcosine, a compatible solute that possesses considerable protein-stabilizing properties, did not serve as a stress protectant of B. subtilis. DMG, on the other hand, proved to be only moderately effective as an osmotic stress protectant, but it exhibited good heat stress-relieving and excellent cold stress-relieving properties. DMG is imported into B. subtilis cells primarily under osmotic and temperature stress conditions via OpuA, a member of the ABC family of transporters. Ligand-binding studies with the extracellular solute receptor (OpuAC) of the OpuA system showed that OpuAC possesses a moderate affinity for DMG, with a K_d value of approximate 172 μM; its K_d for glycine betaine is about 26 μM. Docking studies using the crystal structures of the OpuAC protein with the sulfur analog of DMG, dimethylsulfonioacetate, as a template suggest a model of how the DMG molecule can be stably accommodated within the aromatic cage of the OpuAC ligand-binding pocket. Collectively, our data show that the ability to acquire DMG from exogenous sources under stressful environmental conditions helps the B. subtilis cell to cope with growth-restricting osmotic and temperature challenges.     

Angiotensin II induced catabolic effect and muscle atrophy are redox dependent

Angiotensin II (Ang II) causes skeletal muscle wasting via an increase in muscle catabolism. To determine whether the wasting effects of Ang II were related to its ability to increase NADPH oxidase-derived reactive oxygen species (ROS) we infused wild-type C57BL/6J or p47^phox−/− mice with vehicle or Ang II for 7 days. Superoxide production was increased 2.4-fold in the skeletal muscle of Ang II infused mice, and this increase was prevented in p47^phox−/− mice. Apocynin treatment prevented Ang II-induced superoxide production in skeletal muscle, consistent with Ang II increasing NADPH oxidase derived ROS. Ang II induced loss of body and skeletal muscle weight in C57BL/6J mice, whereas the reduction was significantly attenuated in p47^phox−/− animals. The reduction of skeletal muscle weight caused by Ang II was associated with an increase of proteasome activity, and this increase was completely prevented in the skeletal muscle of p47^phox−/− mice. In conclusion, Ang II-induced skeletal muscle wasting is in part dependent on NADPH oxidase derived ROS.