Plasma Kallikrein and Angiotensin I-converting enzyme N- and C-terminal domain activities are modulated by the insertion/deletion polymorphism

Angiotensin I-converting enzyme (ACE) is recognized as one of the main effector molecules involved in blood pressure regulation. In the last few years some polymorphisms of ACE such as the insertion/deletion (I/D) polymorphism have been described, but their physiologic relevance is poorly understood. In addition, few studies investigated if the specific activity of ACE domain is related to the I/D polymorphism and if it can affect other systems. The aim of this study was to establish a biochemical and functional characterization of the I/D polymorphism and correlate this with the corresponding ACE activity. For this purpose, 119 male brazilian army recruits were genotyped and their ACE plasma activities evaluated from the C- and N-terminal catalytic domains using fluorescence resonance energy transfer (FRET) peptides, specific for the C-domain (Abz-LFK(Dnp)OH), N-domain (Abz-SDK(Dnp)P-OH) and both C- and N-domains (Abz-FRK(Dnp)P-OH). Plasma kallikrein activity was measured using Z-Phe-Arg-AMC as substrate and inhibited by selective plasma kallikrein inhibitor (PKSI). Some physiological parameters previously described related to the I/D polymorphism such as handgrip strength, blood pressure, heart rate and BMI were also evaluated. The genotype distribution was II n = 27, ID n = 64 and DD n = 28. Total plasma ACE activity of both domains in II individuals was significantly lower in comparison to ID and DD. This pattern was also observed for C- and N-domain activities. Difference between ID and DD subjects was observed only with the N-domain specific substrate. Blood pressure, heart rate, handgrip strength and BMI were similar among the genotypes. This polymorphism also affected the plasma kallikrein activity and DD group presents high activity level. Thus, our data demonstrate that the I/D ACE polymorphism affects differently both ACE domains without effects on handgrip strength. Moreover, this polymorphism influences the kallikrein-kinin system of normotensive individuals. (C) 2009 Elsevier Ltd. All rights reserved.

Aging and calorie restriction modulate yeast redox state, oxidized protein removal, and the ubiquitin-proteasome system

The ubiquitin-proteasome system governs the half-life of most cellular proteins. Calorie restriction (CR) extends the maximum life span of a variety of species and prevents oxidized protein accumulation. We studied the effects of CR on the ubiquitin-proteasome system and protein turnover in aging Saccharomyces cerevisiae. CR increased chronological life span as well as proteasome activity compared to control cells. The levels of protein carbonyls, a marker of protein oxidation, and those of polyubiquitinated proteins were modulated by CR. Controls, but not CR cells, exhibited a significant increase in oxidized proteins. In keeping with decreased proteasome activity, polyubiquitinated proteins were increased in young control cells compared to time-matched CR cells, but were profoundly decreased in aged control cells despite decreased proteasomal activity. This finding is related to a decreased polyubiquitination ability due to the impairment of the ubiquitin-activating enzyme in aged control cells, probably related to a more oxidative microenvironment. CR preserves the ubiquitin-proteasome system activity. Overall, we found that aging and CR modulate many aspects of protein modification and turnover. (C) 2011 Elsevier Inc. All rights reserved.

Relationship Between Cortisol Levels and Memory Performance may be Modulated by the Presence or Absence of Cognitive Impairment: Evidence from Healthy Elderly, Mild Cognitive Impairment and Alzheimer`s Disease Subjects

An inverted U-shape function between cortisol levels and memory performance has been reported in studies on both young animals and humans. Yet little is known about this relationship in normal aging or in older subjects with cognitive impairment. This issue is particularly significant since increased levels of cortisol have been reported in Alzheimer`s disease (AD). The present study examined the association between cortisol levels and visual memory performance in healthy subjects as well as in individuals presenting mild cognitive impairment (MCI) or AD. Salivary cortisol was measured in 40 healthy elderly subjects, 31 individuals with amnestic MCI, and 40 subjects with mild probable AD. Memory performance was evaluated using the Brief Cognitive Screening Battery. Higher cortisol levels were associated with better memory performance in healthy elderly (p = 0.005), while higher cortisol levels were correlated with poorer memory performance in MCI subjects (p = 0.011). No correlation between cortisol and memory was found in the AD group (p > 0.05). These results suggest that the relationship between cortisol levels and memory performance in the aging process could vary according to the presence or absence of cognitive impairment.

Angiotensin receptor blockade improves the net balance of cardiac Ca(2+) handling-related proteins in sympathetic hyperactivity-induced heart failure

Aims: The clinical benefits of angiotensin II type 1 (AT1) receptor blockers (ARB) in heart failure (HF) include cardiac anti-remodeling and improved ventricular function. However, the cellular mechanisms underlying the benefits of ARB on ventricular function need to be better clarified. In the present manuscript, we evaluated the effects of AT1 receptor blockade on the net balance of Ca(2+) handling proteins in hearts of mice lacking alpha(2A) and alpha(2C) adrenoceptors (alpha(2A)/alpha(2C)ARKO), which develop sympathetic hyperactivity (SH) induced-HF. Main methods: A cohort of male wild-type (WT) and congenic alpha(2A)/alpha(2C)ARKO mice in a C57BL6/J genetic background (5-7 mo of age) was randomly assigned to receive either placebo or ARB (Losartan, 10 mg/kg for 8wks). Ventricular function (VF) was assessed by echocardiography, and cardiac myocyte width and ventricular fibrosis by a computer-assisted morphometric system. Sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), phospholamban (PLN), phospho-Ser(16)-PLN, phospho-Thr(17)-PLN, phosphatase 1 (PP1), Na(+)-Ca(2+) exchanger (NCX), Ca(2+)/calmodulin-dependent protein kinase 11 (CaMKII) and phospho-Thr(286)-CaMKII were analyzed by Western blot. Key findings: alpha(2A)/alpha(2C)ARKO mice displayed ventricular dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis paralleled by decreased SERCA2 and increased phospho-Thr(17)-PLN, CaMKII, phospho-Thr(286)-CaMKII and NCX levels. ARB induced anti-cardiac remodeling effect and improved VF in alpha(2A)/alpha(2C)ARKO associated with increased SERCA2 and phospho-Ser(16)-PLN levels, and SERCA2:NCX ratio. Additionally, ARB decreased phospho-Thr(17)-PLN levels as well as reestablished NCX, CaMKII and phospho-Thr(286)-CaMKII toward WT levels. Significance: Altogether, these data provide new insights on intracellular Ca(2+) regulatory mechanisms underlying improved ventricular function by ARB therapy in HF. (c) 2011 Elsevier Inc. All rights reserved.

SJL dystrophic mice express a significant amount of human muscle proteins following systemic delivery of human adipose-derived stromal cells without immunosuppression

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence of or defective muscular proteins. The murine model for limb-girdle muscular dystrophy 2B (LGMD2B), the SJL mice, carries a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal. The mice show muscle weakness that begins at 4-6 weeks and is nearly complete by 8 months of age. The possibility of restoring the defective muscle protein and improving muscular performance by cell therapy is a promising approach for the treatment of LGMDs or other forms of progressive muscular dystrophies. Here we have injected human adipose stromal cells (hASCs) into the SJL mice, without immunosuppression, aiming to assess their ability to engraft into recipient dystrophic muscle after systemic delivery; form chimeric human/mouse muscle fibers; express human muscle proteins in the dystrophic host and improve muscular performance. We show for the first time that hASCs are not rejected after systemic injection even without immunosuppression, are able to fuse with the host muscle, express a significant amount of human muscle proteins, and improve motor ability of injected animals. These results may have important applications for future therapy in patients with different forms of muscular dystrophies.

Response of Wolfiporia cocos to iron availability: alterations in growth, expression of cellular proteins, Fe3+-reducing activity and Fe3+-chelators production

Aims: The main objective of this study was to evaluate the behaviour of the brown-rot fungus Wolfiporia cocos under differential iron availability. Methods and Results: W. cocos was grown under three differential iron conditions. Growth, catecholate and hydroxamate production, and mycelial and extracellular Fe3+-reducing activities were determined. Iron starvation slowed fungal growth and accelerated pH decline. Some mycelial proteins of low molecular weight were repressed under iron restriction, whereas others of high molecular weight showed positive iron regulation. Mycelial ferrireductase activity decreased as culture aged, while Fe3+-reducing activity of low molecular reductants constantly increased. Hydroxamates production suffered only limited iron repression, whereas catecholates production showed to be more iron repressible. Conclusions: W. cocos seems to possess more than one type of iron acquisition mechanism; one involving secretion of organic acids and ferrireductases and/or extracellular reductants, and another relying on secretion of catecholates and hydroxamates chelators. Significance and Impact of the Study: This paper is the first to report the kinetic study of brown-rot fungus grown under differential iron availability, and the information provided here contributes to address more traditional problems in protecting wood from brown decay, and also makes a contribution in the general area of the physiology of brown-rot fungi.

Low-intensity Ultrasound Increases FAK, ERK-1/2, and IRS-1 Expression of Intact Rat Bones in a Noncumulative Manner

Background Low-intensity pulsed ultrasound stimulation (LIPUS) reportedly increases osteogenesis in fracture models but fails in intact bone, suggesting LIPUS does not act on mechanotransduction and growth factor pathways of intact bone. Questions/Purposes We asked whether daily 20-minute LIPUS applied to intact tibias would act on bone proteins involved in mechanotransduction (focal adhesion kinase [FAK], and extracellular signal-regulated kinase-1/2 [ERK-1/2]), and growth factor signaling (insulin receptor substrate-1 [IRS-1]) pathways at 7, 14, and 21 days of treatment. Methods Immunoblotting was performed to detect FAK, ERK-1/2, and IRS-1 expression and activation from the stimulated intact tibias at 7, 14, and 21 days of daily 20-minute LIPUS. Results LIPUS increased FAK expression (at 7 days), ERK-1/2 (at 14 days), and IRS-1 (at 7 days), but expression decreased 7 days later, indicating a noncumulative effect of LIPUS. As only FAK expression was detected at 21 days, these observations suggest LIPUS influences nuclear reactions that may be modulated by a major cellular mechanism preferentially inhibiting IRS-1 expression and not FAK expression. Increased ERK-1/2 expression at 14 days suggests the differing mechanisms for promoting ERK-1/2, FAK, and IRS-1 syntheses. IRS-1 expression behaved similarly to FAK expression; therefore, LIPUS may modulate growth factor pathways. LIPUS increased sustained FAK and ERK-1/2 activation, but not IRS-1, suggesting sustained ERK-1/2 activation is not the result of mechanically induced growth factor activation. Conclusions LIPUS acts on mechanotransduction and growth factor pathways in intact bone in a noncumulative manner. Clinical relevance These data suggest LIPUS applied to intact bone acts on proteins involved in osteogenesis.

Evaluation of the effect of ammonium carbamate on the stability of proteins

BACKGROUND: The use of the volatile salt ammonium carbamate in protein downstream processing has recently been proposed. The main advantage of using volatile salts is that they can be removed from precipitates and liquid effluents through pressure reduction or temperature increase. Although previous studies showed that ammonium carbamate is efficient as a precipitant agent, there was evidence of denaturation in some enzymes. In this work, the effect of ammonium carbamate on the stability of five enzymes was evaluated. RESULTS: Activity assays showed that alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), lysozyme (1,4-beta-N-acetylmuramoylhydrolase, EC 3.2.1.17) and lipase (triacyl glycerol acyl hydrolase, EC 3.1.1.3) did not undergo activity loss in ammonium carbamate solutions with concentrations from 1.0 to 5.0 mol kg(-1), whereas cellulase complex (1,4-(1,3 : 14)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) and peroxidase (hydrogen peroxide oxidoreductase, EC 1.11.1.7) showed an average activity loss of 55% and 44%, respectively. Precipitation assays did not show enzyme denaturation or phase separation for alpha-amylase and lipase, while celullase and peroxidase precipitated with some activity reduction. Analysis of similar experiments with ammonium and sodium sulfate did not affect the activity of enzymes. CONCLUSION: Celullase and peroxidase were denatured by ammonium carbamate. While more systematic studies are not available, care must be taken in designing a protein precipitation with this salt. The results suggest that the generally accepted idea that salts that denature proteins tend to solubilize them does not hold for ammonium carbamate. (C) 2010 Society of Chemical Industry

On the solubility of proteins as a function of pH: Mathematical development and application

Thermodynamic relations between the solubility of a protein and the solution pH are presented in this work. The hypotheses behind the development are that the protein chemical potential in liquid phase can be described by Henry`s law and that the solid-liquid equilibrium is established only between neutral molecules. The mathematical development results in an analytical expression of the solubility curve, as a function of the ionization equilibrium constants, the pH and the solubility at the isoelectric point. It is shown that the same equation can be obtained either by directly calculating the fraction of neutral protein molecules or by integrating the curve of the protein average charge. The methodology was successfully applied to the description of the solubility of porcine insulin as a function of pH at three different temperatures and of bovine beta-lactoglobulin at four different ionic strengths. (C) 2011 Elsevier B.V. All rights reserved.

Phase-Locked Loop design applied to frequency-modulated atomic force microscope

The atomic force microscope (AFM) introduced the surface investigation with true atomic resolution. In the frequency modulation technique (FM-AFM) both the amplitude and the frequency of oscillation of the micro-cantilever must be kept constant even in the presence of tip-surface interaction forces. For that reason, the proper design of the Phase-Locked Loop (PLL) used in FM-AFM is vital to system performance. Here, the mathematical model of the FM-AFM control system is derived considering high order PLL In addition a method to design stable third-order Phase-Locked Loops is presented. (C) 2010 Elsevier B.V. All rights reserved.

Nutritional Quality of Sorghum Seeds: Storage Proteins and Amino Acids

One sorghum commercial genotype (MASSA 03) and nine ICRISAT high-lysine genotypes from India were analyzed for storage protein content, distribution profile, and soluble amino acid concentrations. Storage proteins fraction were extracted and separated by SDS-PAGE. Soluble amino acids contents were determined by HPLC. Variations in intensity and appearance and disappearance of protein bands were observed among the sorghum genotypes suggesting genetic variability. Amino acid profile also indicated large variations in the amino acid concentrations. The high lysine and threonine soluble concentrations observed in the seeds of the sorghum genotypes encouraged the use of these genotypes as potential food source due to the better balanced amino acids profile.

TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5

The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved beta-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.

Conservation of dual-targeted proteins in Arabidopsis and rice points to a similar pattern of gene-family evolution

Gene duplication followed by acquisition of specific targeting information and dual targeting were evolutionary strategies enabling organelles to cope with overlapping functions. We examined the evolutionary trend of dual-targeted single-gene products in Arabidopsis and rice genomes. The number of paralogous proteins encoded by gene families and the dual-targeted orthologous proteins were analysed. The number of dual-targeted proteins and the corresponding gene-family sizes were similar in Arabidopsis and rice irrespective of genome sizes. We show that dual targeting of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase was maintained despite occurrence of whole-genome duplications in Arabidopsis and rice as well as a polyploidization followed by a diploidization event (gene loss) in the latter.

Effect of the Partial Substitution of Soy Proteins by Highly Methyl-esterified Pectin on Chemical and Sensory Characteristics of Sausages

Pectin can be used as a natural emulsifier in food formulations. In this study, textured soybean protein (TSP), used as an emulsifier in commercial sausages, was partially replaced by a mixture containing pectin and isolated soybean proteins, which were either extruded (EXT) or not extruded (MIX), and the chemical and sensory characteristics of samples were evaluated after 60 days of storage at 4 degrees C. Responses such as oxidation measured by PV and TBARS, hardness, color, pH and sensory characteristics were compared with those of a commercial sausage (CON). The mixture containing highly methyl-esterified pectin, textured soybean proteins and isolated soybean proteins, as emulsifier agent, reduced the hardness (EXT: 21.69 +/- 0.98 and MIX: 20.17 +/- 2.76 N) and the pH (EXT: 5.46 +/- 0.03 and MIX: 5.29 +/- 0.01) of the samples and increased the concentration of peroxides (EXT: 0.10 +/- 0.01 and MIX: 0.15 +/- 0.01 meq/kg) when compared with samples formulated only with TSP (28.57 +/- 2.54 N, pH of 6.92 +/- 0.04 and PV = 0.07 +/- 0.01 meq/kg). These effects were likely caused by the anionic character of the emulsifier. However, no sensory difference was observed between the sausages containing highly methyl-esterified pectin, textured soybean proteins and isolated soybean proteins submitted to the extrusion process (EXT) and the control sausages, suggesting that the formulation proposed in this study can be a potential alternative for the further development of sausages that have functional properties or are free of artificial additives.

Relationships between gene polymorphisms of folate-related proteins and vitamins and metabolites in pregnant women and neonates

Background: The methylenetetrahydrofolate reductase (MTHFR), glutamate carboxypeptidase II (GCPII) and reduced folate carrier (RFC1) gene polymorphisms were associated with folate status. We investigated the effects of these polymorphisms on serum folate (SF) and folate-related metabolites in mothers and their neonates. Methods: Cobalamin (Cbl), SF, total homocysteine (tHcy), methylmalonic acid (MMA), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in 275 healthy women and their neonates. MTHFR C677T, GCPII C1561T and RFC1 A80G polymorphisms were determined by PCR-RFLP. Results: Maternal tHcy was affected individually by MTHFR C677T and GCPII C1561T polymorphisms and by combined genotypes MTHFR 677TT/GCPII 1561CC and MTHFR 677TT/RFC1 80AG. The MTHFR and RFC1 polymorphisms were not associated with variations in vitamins or SAM, SAH and MMA in neonates. Neonatal tHcy was predicted directly by maternal tHcy and inversely by maternal SF, neonatal Cbl and neonatal RFC1 80G allele (AG+GG genotypes). Maternal MMA and SAM/SAH were predicted by creatinine and Cbl, respectively. Neonatal MMA was predicted by maternal MMA and GCPII 1561T allele (CT+TT genotypes) and by neonatal Cbl. Conclusions: Maternal tHcy was affected by MTHFR C677T, RFC1 A80G and GCPII C1561T polymorphisms. Maternal GCPII C1561T variant was associated with neonatal MMA. Neonatal RFC1 A80G polymorphism influenced tHcy in neonates. (C) 2008 Elsevier B.V. All rights reserved.