pH-Sensitive Binding of Cytochrome c to the Inner Mitochondrial Membrane. Implications for the Participation of the Protein in Cell Respiration and Apoptosis

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pK(a) values and site L containing ionizable groups with pK(aobs),values around 7.0. This protein feature implies that cytochrome c can participate in the fusion of mitochondria and have its detachment from the inner membrane regulated by cell acidosis and alkalosis. In this study, We demonstrated that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration: a high-affinity and low-efficiency pi-I-independent binding (microscopic dissociation constant K(sapp2), similar to 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pK(a) of similar to 6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on K(sapp1), was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed K(sapp1) values at pH 7.2 and 6.2, respectively. Recombinant mutated cytochrome c H26N and H33N also restored the respiration of the cytochrome c-depleted mitoplast in a pH-dependent manner. Consistently, the detachment of cytochrome c from nondepleted mitoplasts was favored by alkalinization, suggesting that site Lionization influences the participation of cytochrome c in the respiratory chain and apoptosis.

Claudin-7 down-regulation is an important feature in oral squamous cell carcinoma

Aims: Claudins, a large family of essential tight junction (TJ) proteins, are abnormally regulated in human carcinomas, especially claudin-7. The aim of this study was to investigate claudin-7 expression and alterations in oral squamous cell carcinoma (OSCC). Methods and results: Expression of claudin-7 was analysed in 132 cases of OSCC organized in a tissue microarray. Claudin-7 mRNA transcript was evaluated using real-time polymerase chain reaction and the methylation status of the promoter was also assessed. Claudin-7 was negative in 58.3% of the cases. Loss of claudin-7 protein expression was associated with recurrence (P = 0.019), tumour size (P = 0.014), clinical stage of OSCC (P = 0.055) and disease-free survival (P = 0.015). Down-regulation of the claudin-7 mRNA transcripts was observed in 78% of the cases, in accordance with immunoexpression. Analysis of the methylation status of the promoter region of claudin-7 revealed that treatment of O28 cells (that did not express claudin-7 mRNA transcripts) with 5-Aza-2`-Deoxycytidine (5-Aza-dC) led to the re-expression of claudin-7 mRNA transcript. Conclusion: Loss of claudin-7 expression is associated with important subcellular processes in OSCC with impact on clinical parameters.

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), SmA, is a vaccine candidate against, S. mansoni and F hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine. (C) 2008 Elsevier B.V. All rights reserved.

Absence of Melatonin Induces Night-Time Hepatic Insulin Resistance and Increased Gluconeogenesis Due to Stimulation of Nocturnal Unfolded Protein Response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-alpha serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. (Endocrinology 152: 1253-1263, 2011)

SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (similar to 50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (similar to 30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

Impaired Vasodilator Activity in Deoxycorticosterone Acetate-Salt Hypertension Is Associated With Increased Protein O-GlcNAcylation

Hyperglycemia, which increases O-linked beta-N-acetylglucosamine (O-GlcNAc) proteins, leads to changes in vascular reactivity. Because vascular dysfunction is a key feature of arterial hypertension, we hypothesized that vessels from deoxycorticosterone acetate and salt (DOCA-salt) rats exhibit increased O-GlcNAc proteins, which is associated with increased reactivity to constrictor stimuli. Aortas from DOCA rats exhibited increased contraction to phenylephrine (E(max) [mN] = 17.6 +/- 4 versus 10.7 +/- 2 control; n = 6) and decreased relaxation to acetylcholine (47.6 +/- 6% versus 73.2 +/- 10% control; n = 8) versus arteries from uninephrectomized rats. O- GlcNAc protein content was increased in aortas from DOCA rats (arbitrary units = 3.8 +/- 0.3 versus 2.3 +/- 0.3 control; n = 5). PugNAc (O- GlcNAcase inhibitor; 100 mu mol/L; 24 hours) increased vascular O- GlcNAc proteins, augmented phenylephrine vascular reactivity (18.2 +/- 2 versus 10.7 +/- 3 vehicle; n = 6), and decreased acetylcholine dilation in uninephrectomized (41.4 +/- 6 versus 73.2 +/- 3 vehicle; n = 6) but not in DOCA rats (phenylephrine, 16.5 +/- 3 versus 18.6 +/- 3 vehicle, n = 6; acetylcholine, 44.7 +/- 8 versus 47.6 +/- 7 vehicle, n = 6). PugNAc did not change total vascular endothelial nitric oxide synthase levels, but reduced endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) phosphorylation (P < 0.05). Aortas from DOCA rats also exhibited decreased levels of endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) (P < 0.05) but no changes in total endothelial nitric oxide synthase or Akt. Vascular O-GlcNAc-modified endothelial nitric oxide synthase was increased in DOCA rats. Blood glucose was similar in DOCA and uninephrectomized rats. Expression of O- GlcNAc transferase, glutamine: fructose-6-phosphate amidotransferase, and O- GlcNAcase, enzymes that directly modulate O-GlcNAcylation, was decreased in arteries from DOCA rats (P < 0.05). This is the first study showing that O-GlcNAcylation modulates vascular reactivity in normoglycemic conditions and that vascular O- GlcNAc proteins are increased in DOCA-salt hypertension. Modulation of increased vascular O-GlcNAcylation may represent a novel therapeutic approach in mineralocorticoid hypertension. (Hypertension. 2009; 53: 166-174.)

Digital subtraction radiographic analysis of the combination of bioabsorbable membrane and bovine morphogenetic protein pool in human periodontal infrabony defects

OBJECTIVES: This study assessed the bone density gain and its relationship with the periodontal clinical parameters in a case series of a regenerative therapy procedure. MATERIAL AND METHODS: Using a split-mouth study design, 10 pairs of infrabony defects from 15 patients were treated with a pool of bovine bone morphogenetic proteins associated with collagen membrane (test sites) or collagen membrane only (control sites). The periodontal healing was clinically and radiographically monitored for six months. Standardized pre-surgical and 6-month postoperative radiographs were digitized for digital subtraction analysis, which showed relative bone density gain in both groups of 0.034 ± 0.423 and 0.105 ± 0.423 in the test and control group, respectively (p>0.05). RESULTS: As regards the area size of bone density change, the influence of the therapy was detected in 2.5 mm² in the test group and 2 mm² in the control group (p>0.05). Additionally, no correlation was observed between the favorable clinical results and the bone density gain measured by digital subtraction radiography (p>0.05). CONCLUSIONS: The findings of this study suggest that the clinical benefit of the regenerative therapy observed did not come with significant bone density gains. Long-term evaluation may lead to a different conclusions.

Effects of the condylar process fracture on facial symmetry in rats submitted to protein undernutrition

PURPOSE: To investigate the facial symmetry of rats submitted to experimental mandibular condyle fracture and with protein undernutrition (8% of protein) by means of cephalometric measurements. METHODS: Forty-five adult Wistar rats were distributed in three groups: fracture group, submitted to condylar fracture with no changes in diet; undernourished fracture group, submitted to hypoproteic diet and condylar fracture; undernourished group, kept until the end of experiment, without condylar fracture. Displaced fractures of the right condyle were induced under general anesthesia. The specimens were submitted to axial radiographic incidence, and cephalometric mensurations were made using a computer system. The values obtained were subjected to statistical analyses among the groups and between the sides in each group. RESULTS: There was significative decrease of the values of serum proteins and albumin in the undernourished fracture group. There was deviation of the median line of the mandible relative to the median line of the maxilla, significative to undernutrition fracture group, as well as asymmetry of the maxilla and mandible, in special in the final period of experiment. CONCLUSION: The mandibular condyle fracture in rats with proteic undernutrition induced an asymmetry of the mandible, also leading to consequences in the maxilla.

Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.

Induction of Zenk protein expression within the nucleus taeniae of the amygdala of pigeons following tone and shock stimulation

In this study, we evaluated the expression of the Zenk protein within the nucleus taeniae of the pigeon’s amygdala (TnA) after training in a classical aversive conditioning, in order to improve our understanding of its functional role in birds. Thirty-two 18-month-old adult male pigeons (Columba livia), weighing on average 350 g, were trained under different conditions: with tone-shock associations (experimental group; EG); with shock-alone presentations (shock group; SG); with tone-alone presentations (tone group; TG); with exposure to the training chamber without stimulation (context group; CG), and with daily handling (naive group; NG). The number of immunoreactive nuclei was counted in the whole TnA region and is reported as density of Zenk-positive nuclei. This density of Zenk-positive cells in the TnA was significantly greater for the EG, SG and TG than for the CG and NG (P < 0.05). The data indicate an expression of Zenk in the TnA that was driven by experience, supporting the role of this brain area as a critical element for neural processing of aversive stimuli as well as meaningful novel stimuli.

Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

Effects of nitrate and phosphate availabilities on growth, photosynthesis and pigment and protein contents in colour strains of Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. (Gigartinales, Rhodophyta)

In Brazil, Hypnea musciformis is the main raw material for carrageenan production and the knowledge of nitrogen and phosphorus metabolism in algae is critical for the success of cultivation because these elements can limit seaweed productivity. Thus, the objective of this study was to evaluate the effects of nitrate (zero to 100 μM) and nitrate plus phosphate (zero to 25 μM) availabilities on the growth, the contents of photosynthetic pigments (phycobiliproteins and chlorophyll a) and proteins, and the photosynthesis and respiration of the brown (BR) and light green (LG) strains of H. musciformis. The results revealed metabolic differences between the colour strains of H. musciformis for nitrogen metabolism: upon nitrate addition, the LG strain stored nitrogen mainly as proteins, while the BR strain stored it as proteins and pigments. Moreover, the respiration of the LG strain and the photosynthesis of the BR strain increased with nitrate concentrations, indicating that the BR strain fixed more photosynthetic carbon than the LG strain.

Mineral and vitamin content of beef, chicken, and turkey hydrolysates mineral and vitamin content of protein hydrolysates

The purpose of this study was to assess the concentration of vitamins and minerals in meat protein hydrolysates. Calcium, phosphorus and iron were analyzed by inductively coupled-plasma atomic emission spectrophotometry; vitamin C was analyzed by the reduction of cupric ions and vitamins B1 and B2 by fluorescence. Regarding minerals, the beef hydrolysate (BH) had more iron than the turkey hydrolysate (TH) and the chicken hydrolysate (CH); TH had a little more phosphorus. BH had the largest amount of vitamin C, and similar amounts of vitamins B1 and B2. The amount of these nutrients found in the hydrolysates suggests that it is possible to use them to enrich special dietary formulations.

Productive performance and milk protein fraction composition of dairy cows supplemented with sodium monensin

The objective of this work was to evaluate the levels of sodium monensin on lactating cows and their effects on productive performance and milk protein fraction composition. It was used 12 Holstein cows, distributed in four balanced 3 × 3 Latin squares, and fed three diets: one control without monensin, and two diets with monensin at the levels of 24 or 48 mg/kg DM added to the concentrate. Milk production was daily measured throughout the entire experimental period. The samples used for analysis of milk composition were collected on two alternated days from the two daily milking. Non-protein nitrogen, total nitrogen and non-casein nitrogen contents were directly evaluated in the milk, and casein, whey protein and true protein contents were indirectly determined. The use of monensin in the rations reduced dry matter and nutrient intake, especially when diet with 48 mg/kg of dry matter was given. The ration with 24 mg/kg of DM increased milk production, with or without correction, and also fat and lactose yield, and it improved productive efficiency. The levels of monensin in the ratios did not influence contents of milk crude protein, non-protein nitrogen, non-casein nitrogen, true protein, casein, casein/true protein ratio, whey protein, and of all those fractions expressed as percentage of crude protein. The utilization of monensin in the ratio at the dose of 24 mg/kg of DM influences positively the productive performance of lactating cows, and it does not influence the composition of milk protein fractions.

Productive performance and composition of milk protein fraction in dairy cows supplemented with fat sources

The objective of this study was to evaluate the use of fat sources in rations for lactating cows on the productive performance and composition of milk protein fraction. Twelve Holstein cows were used, grouped in three balanced 4 × 4 Latin squares, fed with the following rations: control; refined soybean oil; whole raw soybean; and calcium salts of unsaturated fatty acid (Megalac-E). Dry matter and nutrient intake, and daily milk production were evaluated. The samples used to analyze milk composition were collected in two alternate days and were obtained from two daily milking. Milk composition and total nitrogen, non-protein nitrogen and non-casein nitrogen ratios were analyzed. The casein, serum protein and true protein ratios were obtained by difference. Dry matter and nutrient intakes were lower when cows received the diet containing calcium salts of fatty acids, in relation to the control diet. Among the diets with fat sources, the one with whole raw soybean and calcium salts decreased milk production. There was no effect of fat sources added to the diet on crude protein, non-protein nitrogen, non-casein nitrogen, true protein, casein, casein/milk true protein ratio and serum protein. Similarly, the experimental diets did not influence the protein fractions when expressed in percentage of milk crude protein. The utilization of fat sources in diets changes milk production and composition of lactating cows, but does not influence the composition of milk protein fractions.