Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-beta superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34-38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.

Modulation of Bone Morphogenetic Protein-9 Expression and Processing by Insulin, Glucose, and Glucocorticoids: Possible Candidate for Hepatic Insulin-Sensitizing Substance

Bone morphogenetic protein 9 (BMP-9), a member of the TGF-beta superfamily predominantly expressed in nonparenchymal liver cells, has been demonstrated to improve glucose homeostasis in diabetic mice. Along with this therapeutic effect, BMP-9 was proposed as a candidate for the hepatic insulin-sensitizing substance ( HISS). Whether BMP-9 plays a physiological role in glucose homeostasis is still unknown. In the present study, we show that BMP-9 expression and processing is severely reduced in the liver of insulin-resistant rats. BMP-9 expression and processing was directly stimulated by in situ exposition of the liver to the combination of glucose and insulin and oral glucose in overnight fasted rats. Additionally, prolonged fasting ( 72 h) abrogated refeeding-induced BMP-9 expression and processing. Previous exposition to dexamethasone, a known inductor of insulin resistance, reduced BMP-9 processing stimulated by the combination of insulin and glucose. Finally, we show that neutralization of BMP-9 with an anti-BMP-9 antibody induces glucose intolerance and insulin resistance in 12-h fasted rats. Collectively, the present results demonstrate that BMP-9 plays an important role in the control of glucose homeostasis of the normal rat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resistance. BMP-9 regulation according to the feeding status and the presence of diabetogenic factors reinforces the hypothesis that BMP-9 might exert the role of HISS in glucose homeostasis physiology. ( Endocrinology 149: 6326-6335, 2008)

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

Effect of meloxicam and diclofenac sodium on peri-implant bone healing in rats

Background: This study evaluated the effects of diclofenac sodium and meloxicam on peri-implant bone healing. Methods: Thirty male rats were divided into three groups: the control group (CG) received no drug; the diclofenac sodium group (DSG) received 1.07 mg/kg twice a day for 5 days; and the meloxicam group (MG) received 0.2 mg/kg daily for 5 days. A screw-shaped titanium implant was placed in the tibia. Fluorochromes, oxytetracycline (OxT), calcein (CA), and alizarin (AL), were injected at 7, 14, and 21 days, respectively, after implantation, and the animals were sacrificed 28 days after implant placement. The percentages of OxT-, CA-, and AL-labeled bone as well as the percentages of bone-to-implant contact (BIC), cortical bone area (CBA), and trabecular bone area (TBA) within the implant threads were evaluated. Results: Bone healing was delayed in the DSG during the first 14 days after implant placement (OxT-labeled bone: DSG: 5.3% +/- 7.3% versus CG: 13.2% +/- 9.8%, P= 0.002, and versus MG: 14.4% +/- 13.1%, P = 0.05). The percentages of BIC (DSG: 49.6% +/- 21.9%; MG: 67.1% +/- 22.8%; and CG: 68.1% +/- 22.8%) and CBA (DSG: 63.7% +/- 21.2%; MG: 82.7% +/- 12.4%; CG: 84.9% +/- 10.6%) were lower in the DSG compared to the MG and CG (P<0.001). The percentage of TBA was significantly greater in the DSG compared to the MG and CG (DSG: 36.3% +/- 21.2% versus MG: 17.3% +/- 12.7% and versus CG: 15.1% +/- 10.6%; P<0.001). Conclusion: Diclofenac sodium seemed to delay peri-implant bone healing and to decrease BIC, whereas meloxicam had no negative effect on peri-implant bone healing.

Influence of rhBMP-2 on bone formation and osseointegration in different implant systems after sinus-floor elevation. An in vivo study on sheep

Background Several studies have reported certain bone morphogenic proteins (BMPs) to have positive effects on bone generation Although some investigators have studied the effects of human recombinant BMP (rhBMP-2) in sinus augmentation in sheep, none of these studies looked at the placement of implants at the time of sinus augmentation Furthermore, no literature could be found to report on the impact that different implant systems, as well as the positioning of the implants had on bone formation if rhBMP-2 was utilized in sinus-lift procedures Purpose The aim of this study was to compare sinus augmentation with rhBMP-2 on a poly-D, L-lactic-co-glycolic acid gelatine (PLPG) sponge with sinus augmentation with autologous pelvic cancellous bone in the maxillary sinus during the placement of different dental Implants Materials and methods Nine adult female sheep were submitted to bilateral sinus-floor elevation In one side (test group) the sinus lift was performed with rhBMP-2 on a PLPG-sponge, while the contralateral side served as the control by using cancellous bone from the iliac crest Three different implants (Branemark (R), 31 (R) and Straumann (R)) were inserted either simultaneously with the sinus augmentation or as a two staged procedure 6 weeks later The animals were sacrificed at 6 and 12 weeks for histological and histomorphometrical evaluations during which bone-to-implant contact (BIC) and bone density (BD) were evaluated Results BD and BIC were significantly higher at 12 weeks in the test group if the Implants were placed at the time of the sinus lift (p < 0 05) No difference was observed between the different implant systems or positions Conclusions The use of rhBMP-2 with PLPG-sponge increased BIC as well as BD in the augmented sinuses if compared to autologous bone Different implant systems and positions of the implants had no effect on BIC or BD (C) 2010 European Association for Cranio-Maxillo-Facial Surgery

Smad5 regulates Akt2 expression and insulin-induced glucose uptake in L6 myotubes

Insulin-induced glucose uptake by skeletal muscle results from Akt2 activation and is severely impaired during insulin resistance Recently, we and others have demonstrated that BMP9 improves glucose homeostasis in diabetic and non-diabetic rodents. However, the mechanism by which BMP9 modulates insulin action remains unknown. Here we demonstrate that Smad5. a transcription factor activated by BMP9, and Akt2. are upregulated in differentiated L6 myotubes. Smad5, rather than Smad1/8, is downregulated ""in vivo"" and ""in vitro"" by dexamethasone Smad5 knockdown decreased Akt2 expression and serine phosphorylation and insulin-induced glucose uptake, and increased the expression of the lipid phosphatase Ship2. Additionally, binding of Smad5 to Akt2 gene is decreased in dexamethasone-treated rats and Increased in L6 myotubes compared to myoblasts The present study indicates that Smad5 regulates glucose uptake in skeletal muscle by controlling Akt2 expression and phosphorylation These finding reveals Smad5 as a potential target for the therapeutic of type 2 diabetes. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Role of alpha 7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

TAF15 and the leukemia-associated fusion protein TAF15-CIZ/NMP4 are cleaved by caspases-3 and-7

Caspases are central players in proteolytic pathways that regulate cellular processes Such as apoptosis and differentiation. To accelerate the discovery of novel caspase substrates we developed a method combining in silico screening and in vitro validation. With this approach, we identified TAH15 as a novel caspase Substrate in a trial Study. We find that TAF15 was specifically cleaved by caspases-3 and -7. Site-directed mutagenesis revealed the consensus sequence (106)DQPD/Y(110) as the only site recognized by these caspases. Surprisingly, TAF15 was cleaved at more than one site in staurosporine-treated Jurkat cells. In addition, we generated two oncogenic TAF15-CIZ/NMP4-fused proteins which have been found in acute myeloid leukemia and demonstrate that caspases-3 and -7 cleave the fusion proteins at one single site. Broad application of this combination approach should expedite identification of novel caspase-interacting proteins and provide new insights into the regulation of caspase pathways leading to cell death in normal and cancer cells. (C) 2009 Elsevier Inc. All rights reserved.

NEONATAL HANDLING AND THE MATERNAL ODOR PREFERENCE IN RAT PUPS: INVOLVEMENT OF MONOAMINES AND CYCLIC AMP RESPONSE ELEMENT-BINDING PROTEIN PATHWAY IN THE OLFACTORY BULB

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic-pituitary-adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Influence of thermoplastic extrusion on the nutritive value of bovine rumen protein

This Study investigated the impact of thermoplastic extrusion on the nutritive quality of bovine rumen protein. Proximal composition, amino acid profile and in vivo true protein digestibility among rats were determined in raw (RBR) and extruded (EBR) rumen. Raw and extruded bovine rumen presented high percentages of protein (more than 95% on dry basis). Neither raw nor extruded proteins had any limiting amino acid, and the RBR and EBR amino acid scores were, respectively, 1.28 (leucine) and 1.25 (methionine plus cystine). Extrusion reduced significantly true protein digestibility from 97.7% to 93.1% (p < 0.001), but protein digestibility-corrected amino acid scores for both proteins (RBR and EBR) were 100%. Animal growth presented comparable profiles using raw and extruded rumen. In conclusion, thermoplastic extrusion did not affect the protein quality of bovine rumen, and this does not hinder the use of this material as a food ingredient. (C) 2009 Elsevier Ltd. Ail rights reserved.

Assessment of production efficiency, physicochemical properties and storage stability of spray-dried chlorophyllide, a natural food colourant, using gum Arabic, maltodextrin and soy protein isolate-based carrier systems

P>The aim of this research was to study spray drying as potential action to protect chlorophyllide from environmental conditions for shelf-life extension and characterisation of the powders. Six formulations were prepared with 7.5 and 10 g of carrier agents [gum Arabic (GA), maltodextrin (MA) and soybean protein isolate (SPI)]/100 mL of chlorophyllide solutions. The powders were evaluated for morphological characteristics (SEM), particle size, water activity, moisture, density, hygroscopicity, cold water solubility, sorption isotherms, colour and stability, during 90 days. All the powders were highly soluble, with solubility values around 97%. A significant lower hygroscopicity was observed for GA powders, whilst the lower X(m) values obtained by GAB equation fitting of the sorption isotherms was observed for the 7.5 g MA/100 mL samples. All formulations, but the 1 (7.5 g SPI/100 mL of chlorophyllide), provided excellent stability to the chlorophyllide during 90 days of storage even at room temperature.

Effect of arginine on the development of the pectoralis muscle and the diameter and the protein:deoxyribonucleic acid rate of its skeletal myofibers in broilers

This work aimed at evaluating the effects of the supplementation of starter diet with Arg on breast muscle development in broilers and the activation of satellite cells and the aggregation of myofibrillar protein. Male Cobb chicks (n = 990) were randomly assigned to 1 of 5 treatments in a complete random design. Measurements of 33 chicks per treatment were made in 6 repetitions. The treatments consisted of a basal diet with 1.390% digestible Arg (without supplementation) and 4 dietary levels of Arg (1.490, 1.590, 1.690, and 1.790%) with Arg:Lys ratios of 1.103, 1.183, 1.262, 1.341, and 1.421, respectively. Arginine supplementation was used only in the starter phase (1 to 21 d). Dietary supplementation with Arg had a positive effect (P < 0.05) on breast and breast fillet weight on d 7 and 21 and on myofiber diameter on d 14 and 21. However, no effect was observed (P > 0.05) on the protein: DNA ratio, which demonstrates that Arg does not interfere with the mitotic activity of the satellite cells. Independently from mechanism, Arg affected muscle growth in the starter phase positively. Dietary supplementation with Arg in the starter phase had no effect (P > 0.05) on the carcass yield of broilers on d 42. Diet supplementation with Arg at levels above the ones recommended for the starter phase may be necessary for improved muscle development in broilers.

Effects of dietary urea levels on milk protein fractions of Holstein cows

The aim of this study was to evaluate the effects of substituting soybean meal for urea on milk protein fractions (casein, whey protein and non-protein nitrogen) of dairy cows in three dietary levels. Nine mid-lactation Holstein cows were used in a 3 x 3 Latin square arrangement, composed of 3 treatments, 3 periods of 21 days each, and 3 squares. The treatments consisted of three different diets fed to lactating cows, which were randomly assigned to three groups of three animals: (A) no urea inclusion, providing 100% of crude protein (CP), rumen undegradable protein (RUP) and rumen degradable protein (RDP) requirements, using soybean meal and sugarcane as roughage; (B) urea inclusion at 7.5 g/kg DM in partial substitution of soybean meal CP equivalent; (C) urea inclusion at 15 g/kg DM in partial substitution of soybean meal CP equivalent. Rations were isoenergetic and isonitrogenous-1 60 g/kg DM of crude protein and 6.40 MJ/kg DM of net energy for lactation. When the data were analyzed by simple polynomial regression, no differences were observed among treatments in relation to milk CP content, true protein, casein, whey protein, non-casein and non-protein nitrogen, or urea. The milk true protein:crude protein and casein:true protein ratios were not influenced by substituting soybean meal for urea in the diet. Based on the results it can be concluded that the addition of urea up to 15 g/kg of diet dry matter in substitution of soybean meal did not alter milk protein concentration casein, whey protein and its non-protein fractions, when fed to lactating dairy cows. (c) 2007 Elsevier B.V. All rights reserved.

Lifetime Overproduction of Circulating Angiotensin-(1-7) Attenuates Deoxycorticosterone Acetate-Salt Hypertension-Induced Cardiac Dysfunction and Remodeling

We evaluated the development of arterial hypertension, cardiac function, and collagen deposition, as well as the level of components of the renin-angiotensin system in the heart of transgenic rats that overexpress an angiotensin (Ang)-(1-7)-producing fusion protein, TGR(A1-7)3292 (TG), which induces a lifetime increase in circulating levels of this peptide. After 30 days of the induction of the deoxycorticosterone acetate (DOCA)-salt hypertension model, DOCA-TG rats were hypertensive but presented a lower systolic arterial pressure in comparison with DOCA-Sprague-Dawley (SD) rats. In contrast to DOCA-SD rats that presented left ventricle (LV) hypertrophy and diastolic dysfunction, DOCA-TG rats did not develop cardiac hypertrophy or changes in ventricular function. In addition, DOCA-TG rats showed attenuation in mRNA expression for collagen type I and III compared with the increased levels of DOCA-SD rats. Ang II plasma and LV levels were reduced in SD and TG hypertensive rats in comparison with normotensive animals. DOCA-TG rats presented a reduction in plasma Ang-(1-7) levels; however, there was a great increase in Ang-(1-7) (approximate to 3-fold) accompanied by a decrease in mRNA expression of both angiotensin-converting enzyme and angiotensin-converting enzyme 2 in the LV. The mRNA expression of Mas and Ang II type 1 receptors in the LV was not significantly changed in DOCA-SD or DOCA-TG rats. This study showed that TG rats with increased circulating levels of Ang-(1-7) are protected against cardiac dysfunction and fibrosis and also present an attenuated increase in blood pressure after DOCA-salt hypertension. In addition, DOCA-TG rats showed an important local increase in Ang-(1-7) levels in the LV, which might have contributed to the attenuation of cardiac dysfunction and prefibrotic lesions. (Hypertension. 2010;55:889-896.)

Separating the wheat from the chaff: unbiased filtering of background tandem mass spectra improves protein identification

Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.