Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 degrees C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. (C) 2011 Elsevier B.V. All rights reserved.

Late evaluation of the relationship between morphological and functional renal changes and hypertension after non-operative treatment of high-grade renal injuries

Objective: To evaluate the anatomical and functional renal alterations and the association with post-traumatic arterial hypertension. Methods: The studied population included patients who sustained high grades renal injury (grades III to V) successfully non-operative management after staging by computed tomography over a 16-year period. Beyond the review of medical records, these patients were invited to the following protocol: clinical and laboratory evaluation, abdominal computed tomography, magnetic resonance angiography, DMSA renal scintigraphy, and ambulatory blood pressure monitoring. The hypertensive patients also were submitted to dynamic renal scintigraphy (Tc-99m EC), using captopril stimulation to verify renal vascular etiology. Results: Of the 31 patients, there were thirteen grade III, sixteen grade IV (nine lacerations, and seven vascular lesions), and two grade V injuries. All the patients were asymptomatic and an average follow up post-injury of 6.4 years. None had abnormal BUN or seric creatinine. The percentage of renal volume reduction correlates with the severity as defined by OIS. There was no evidence of renal artery stenosis in Magnetic Resonance angiography (MRA). DMSA scanning demonstrated a decline in percentage of total renal function corresponding to injury severity (42.2 +/- 5.5% for grade III, 35.3 +/- 12.8% for grade IV, 13.5 +/- 19.1 for grade V). Six patients (19.4%) had severe compromised function (< 30%). There was statistically significant difference in the decrease in renal function between parenchymal and vascular causes for grade IV injuries (p < 0.001). The 24-hour ambulatory blood pressure monitoring detected nine patients (29%) with post-traumatic hypertension. All the patients were male, mean 35.6 years, 77.8 % had a familial history of arterial hypertension, 66.7% had grade III renal injury, and average post-injury time was 7.8 years. Seven patients had negative captopril renography. Conclusions: Late results of renal function after conservative treatment of high grades renal injuries are favorable, except for patients with grades IV with vascular injuries and grade V renal injuries. Moreover, arterial hypertension does not correlate with the grade of renal injury or reduction of renal function.

Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes

A taxonomic and annotated functional description of microbial life was deduced from 53 Mb of metagenomic sequence retrieved from a planktonic fraction of the Neotropical high Andean (3,973 meters above sea level) acidic hot spring El Coquito (EC). A classification of unassembled metagenomic reads using different databases showed a high proportion of Gammaproteobacteria and Alphaproteobacteria (in total read affiliation), and through taxonomic affiliation of 16S rRNA gene fragments we observed the presence of Proteobacteria, micro-algae chloroplast and Firmicutes. Reads mapped against the genomes Acidiphilium cryptum JF-5, Legionella pneumophila str. Corby and Acidithiobacillus caldus revealed the presence of transposase-like sequences, potentially involved in horizontal gene transfer. Functional annotation and hierarchical comparison with different datasets obtained by pyrosequencing in different ecosystems showed that the microbial community also contained extensive DNA repair systems, possibly to cope with ultraviolet radiation at such high altitudes. Analysis of genes involved in the nitrogen cycle indicated the presence of dissimilatory nitrate reduction to N2 (narGHI, nirS, norBCDQ and nosZ), associated with Proteobacteria-like sequences. Genes involved in the sulfur cycle (cysDN, cysNC and aprA) indicated adenylsulfate and sulfite production that were affiliated to several bacterial species. In summary, metagenomic sequence data provided insight regarding the structure and possible functions of this hot spring microbial community, describing some groups potentially involved in the nitrogen and sulfur cycling in this environment. Citation: Jimenez DJ, Andreote FD, Chaves D, Montana JS, Osorio-Forero C, et al. (2012) Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes. PLoS ONE 7(12): e52069. doi:10.1371/journal.pone.0052069

Metabolic and functional effects of beta-hydroxy-beta-methylbutyrate (HMB) supplementation in skeletal muscle

Beta-hydroxy-beta-methylbutyrate (HMB) is a metabolite derived from leucine. The anti-catabolic effect of HMB is well documented but its effect upon skeletal muscle strength and fatigue is still uncertain. In the present study, male Wistar rats were supplemented with HMB (320 mg/kg per day) for 4 weeks. Placebo group received saline solution only. Muscle strength (twitch and tetanic force) and resistance to acute muscle fatigue of the gastrocnemius muscle were evaluated by direct electrical stimulation of the sciatic nerve. The content of ATP and glycogen in red and white portions of gastrocnemius muscle were also evaluated. The effect of HMB on citrate synthase (CS) activity was also investigated. Muscle tetanic force was increased by HMB supplementation. No change was observed in time to peak of contraction and relaxation time. Resistance to acute muscle fatigue during intense contractile activity was also improved after HMB supplementation. Glycogen content was increased in both white (by fivefold) and red (by fourfold) portions of gastrocnemius muscle. HMB supplementation also increased the ATP content in red (by twofold) and white (1.2-fold) portions of gastrocnemius muscle. CS activity was increased by twofold in red portion of gastrocnemius muscle. These results support the proposition that HMB supplementation have marked change in oxidative metabolism improving muscle strength generation and performance during intense contractions.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Overexpression of inducible 70-kDa heat shock protein in mouse improves structural and functional recovery of skeletal muscles from atrophy

Heat shock proteins play a key regulatory role in cellular defense. To investigate the role of the inducible 70-kDa heat shock protein (HSP70) in skeletal muscle atrophy and subsequent recovery, soleus (SOL) and extensor digitorum longus (EDL) muscles from overexpressing HSP70 transgenic mice were immobilized for 7 days and subsequently released from immobilization and evaluated after 7 days. Histological analysis showed that there was a decrease in cross-sectional area of type II myofiber from EDL and types I and II myofiber from SOL muscles at 7-day immobilization in both wild-type and HSP70 mice. At 7-day recovery, EDL and SOL myofibers from HSP70 mice, but not from wild-type mice, recovered their size. Muscle tetanic contraction decreased only in SOL muscles from wild-type mice at both 7-day immobilization and 7-day recovery; however, it was unaltered in the respective groups from HSP70 mice. Although no effect in a fatigue protocol was observed among groups, we noticed a better contractile performance of EDL muscles from overexpressing HSP70 groups as compared to their matched wild-type groups. The number of NCAM positive-satellite cells reduced after immobilization and recovery in both EDL and SOL muscles from wild-type mice, but it was unchanged in the muscles from HSP70 mice. These results suggest that HSP70 improves structural and functional recovery of skeletal muscle after disuse atrophy, and this effect might be associated with preservation of satellite cell amount.

Measure functional differential equations and functional dynamic equations on time scales

We study measure functional differential equations and clarify their relation to generalized ordinary differential equations. We show that functional dynamic equations on time scales represent a special case of measure functional differential equations. For both types of equations, we obtain results on the existence and uniqueness of solutions, continuous dependence, and periodic averaging.

Histologic and functional evaluation of lungs reconditioned by ex vivo lung perfusion

BACKGROUND: Only about 15% of donor lungs are considered suitable for transplantation (LTx). Ex vivo lung perfusion (EVLP) has been developed as a method to reassess and repair damaged lungs. We report our experience with EVLP in non-acceptable donor lungs and evaluate its ability to recondition these lungs. METHODS: We studied lungs from 16 brain-dead donors rejected for LTx. After harvesting, the lungs were stored at 4 degrees C for 10 hours and subjected to normothermic EVLP with Steen Solution (Vitro life, Goteborg, Sweden) for 60 minutes. For functional evaluation, the following variables were assessed: partial pressure of arterial oxygen (Pao(2)), pulmonary vascular resistance (PVR), and lung compliance (LC). For histologic assessment, lung biopsy was done before harvest and after EVLP. Tissue samples were examined under light microscopy. To detect and quantify apoptosis, terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling assay was used. RESULTS: Thirteen lima donors were refused for having impaired lung function. The mean Pao(2) obtained in the organ donor at the referring hospital was 193.7 mm Hg and rose to 489 mm Hg after EVLP. During EVLP, the mean PVR was 652.5 dynes/sec/cm(5) and the mean LC was 48 ml/cm H2O. There was no significant difference between the mean Lung Injury Score before harvest and after EVLP. There was a trend toward a reduction in the median number of apoptotic cells after EVLP. CONCLUSIONS: EVLP improved lung function (oxygenation capacity) of organs considered unsuitable for transplantation. Lung tissue structure did not deteriorate even after 1 hour of normothermic perfusion. J Heart Lung Transplant 2012;31:305-9 (C) 2012 International Society for Heart and Lung Transplantation. All rights reserved.

The relationship between social support and functional capacity in elderly persons with cognitive alterations

The objective of this study was to identify the relationship between social support and the functional capacity of elderly persons with cognitive alterations. It is a descriptive, cross-sectional and quantitative study. The subjects were 101 elderly persons registered in Family Health Centers whose performance in the Mini-Exam for Mental Status was below a certain specified level in a previous study. The Medical Outcomes Study questionnaire, Katz Index and Pfeffer Questionnaire were applied. The dimensions of material, affective, emotional, informational and positive social interaction support resulted in an average final score of 74.32 points, indicating a better level of material and affective support in relation to the other dimensions of support. There was a statistically significant correlation between emotional support and the Katz Index. Knowledge about this relationship favors the development of a nursing care pathway for the elderly which is capable of maintaining their functional capacity and ensuring satisfactory social relations.

SPATIO-TEMPORAL AND FUNCTIONAL STRUCTURE OF THE AMPHIPOD COMMUNITIES OFF SANTOS, SOUTHWESTERN ATLANTIC

The amphipod fauna was employed to investigate a bottom environmental gradient in the continental shelf adjacent to Santos Bay. The constant flow of less saline water from the estuarine complex of the Santos and Sao Vicente rivers besides the seasonal intrusion of the cold saline South Atlantic Central Water (SACW) bring a highly dynamic water regime to the area. Density, distribution, diversity and functional structure of the communities were studied on a depth gradient from 10 to 100 m on two cruises in contrasting seasons, winter 2005 and summer 2006. Twenty-one sediment samples were taken with a 0.09m(2) box corer. Temperature and salinity were measured at each station and an additional surface sediment sample was obtained with the box corer for granulometric and chemical analyses. Sixty species were collected on each survey and higher density values were found in summer. A priori one-way Analysis of Similarities (ANOSIM) indicated the existence of three different groups of amphipods related to the depth gradient: the Coastal group, the Mixed Zone group and the Deep Zone group. The Coastal Zone in both cruises was inhabited by a community presenting low diversity and density, besides high dominance of the infaunal tube-dweller Ampelisca paria; the area around 30 m presented the highest values of all the ecological indicators and the species showed several life styles; the outer area, situated between 50 and 100 m depth in the SACW domain, presented a community characterized by lower diversity and high biomass and density values. A season-depth ANOSIM showed the influence of depth and season for the Coastal and Mixed Zone groups whereas no seasonal difference was obtained for the Deep Zone group. The synergistic effect of the SACW and depth in the first place, followed by physical changes in substrate, seem to be the main factors controlling the fauna's distribution. In addition, the estuarine waters from Santos Bay apparently had no effect on the establishment of the environmental gradient observed on the adjacent shelf. Diversity, distribution, functional groups and trophic conditions of superficial sediments are discussed in the light of the main oceanographic processes present on the southern Brazilian shelf.

Exercise training improves neurovascular control and functional capacity in heart failure patients regardless of age

Background: Exercise training is a non-pharmacological strategy for treatment of heart failure. Exercise training improves functional capacity and quality of life in patients. Moreover, exercise training reduces muscle sympathetic nerve activity (MSNA) and peripheral vasoconstriction. However, most of these studies have been conducted in middle-aged patients. Thus, the effects of exercise training in older patients are much less understood. The present study was undertaken to investigate whether exercise training improves functional capacity, muscular sympathetic activation and muscular blood flow in older heart failure patients, as it does in middle-aged heart failure patients. Design: Fifty-two consecutive outpatients with heart failure from the database of the Unit of Cardiovascular Rehabilitation and Physiology Exercise were divided by age (middle-aged, defined as 45-59 years, and older, defined as 60-75 years) and exercise status (trained and untrained). Methods: MSNA was recorded directly from the peroneal nerve using the microneurography technique. Forearm Blood Flow (FBF) was measured by venous occlusion plethysmography. Functional capacity was evaluated by cardiopulmonary exercise test. Results: Exercise training significantly and similarly increased FBF and peak VO2 in middle-aged and older heart failure patients. In addition, exercise training significantly and similarly reduced MSNA and forearm vascular resistance in these patients. No significant changes were found in untrained patients. Conclusion: Exercise training improves neurovascular control and functional capacity in heart failure patients regardless of age.

WBC count and functional changes induced by co-administration of clofazimine and clarithromycin, in single and multiple doses, in Wistar rats

Clofazimine and clarithromycin are used to treat leprosy and infections caused by Mycobacterium avium complex. Little data on the toxicity of co-administration of these two drugs are available. Here we evaluated the potential adverse effects of polytherapy with these two drugs in male Wistar rats by determining WBCs counts and other blood cell counts, neutrophilic phagocytosis, and burst oxidative, by flow cytometry. We observed an increase in WBCs, in multiple-dose regimens, and in polymorphonuclear cells, in both single- clarithromycin only and multiple dose regimens. We also observed a reduction in mononuclear cell counts in single and multiple doses. The drugs seem to reverse the mononuclear and polymorphonuclear cell ratio. An increase in oxidative burst was observed in animals treated with the drugs administered either individually or combined. In conclusion, clofazimine and clarithromycin change WBCs counts. Our results may contribute for a better understanding of the mechanisms related to the effects of co-administrating the two drugs.

Muscle Atrophy and Functional Deficits of Knee Extensors and Flexors in People With Chronic Stroke

Background. Further clarification is needed with regard to the degree of atrophy in individual muscle groups and its possible relationship to joint torque deficit poststroke. Objective. The purpose of this study was to investigate quadriceps and hamstring muscle volume and strength deficits of the knee extensors and flexors in people with chronic hemiparesis compared with a control group. Design. This was a cross-sectional study. Methods. Thirteen individuals with hemiparesis due to chronic stroke (hemiparetic group) and 13 individuals who were healthy (control group) participated in this study. Motor function, quadriceps and hamstring muscle volume, and maximal concentric and eccentric contractions of the knee extensors and flexors were assessed. Results. Only the quadriceps muscle of the paretic limb showed reduced muscle volume (24%) compared with the contralateral (nonparetic) limb. There were no differences in muscle volume between the hemiparetic and control groups. The peak torque of the paretic-limb knee extensors and flexors was reduced in both contraction modes and velocities compared with the nonparetic limb (36%-67%) and with the control group (49%-75%). The nonparetic limb also showed decreased extensor and flexor peak torque compared with the control group (17%-23%). Power showed similar deficits in strength (12%-78%). There were significant correlations between motor function and strength deficits (.54-.67). Limitations. Magnetic resonance imaging coil length did not allow measurement of the proximal region of the thigh. Conclusions. There were different responses between quadriceps and hamstring muscle volumes in the paretic limb that had quadriceps muscle atrophy only. However, both paretic and nonparetic limbs showed knee extensor and flexor torque and power reduction.

Anatomical and functional evaluation of tympanoplasty using a transitory natural latex biomembrane implant from the rubber tree Hevea brasiliensis

PURPOSE: To compare the role of transitory latex and sylastic (R) implants in tympanoplasty on the closure of tympanic perforations. METHODS: A randomized double-blind prospective study was conducted on 107 patients with chronic otitis media submitted to underlay tympanoplasty and divided at random into three groups: control with no transitory implant, latex membrane group, and sylastic (R) membrane group. RESULTS: Greater graft vascularization occurred in the latex membrane group (p<0.05). Good biocompatibility was obtained with the use of the latex and silicone implants, with no effect on the occurrence of infection, otorrhea or otorragy. CONCLUSION: The use of a transitory latex implant induced greater graft vascularization, with a biocompatible interaction with the tissue of the human tympanic membrane.

Effects of a combined strengthening, stretching and functional training program versus usual-care on gait biomechanics and foot function for diabetic neuropathy: a randomized controlled trial

Background: Polyneuropathy is a complication of diabetes mellitus that has been very challenging for clinicians. It results in high public health costs and has a huge impact on patients' quality of life. Preventive interventions are still the most important approach to avoid plantar ulceration and amputation, which is the most devastating endpoint of the disease. Some therapeutic interventions improve gait quality, confidence, and quality of life; however, there is no evidence yet of an effective physical therapy treatment for recovering musculoskeletal function and foot rollover during gait that could potentially redistribute plantar pressure and reduce the risk of ulcer formation. Methods/Design: A randomised, controlled trial, with blind assessment, was designed to study the effect of a physiotherapy intervention on foot rollover during gait, range of motion, muscle strength and function of the foot and ankle, and balance confidence. The main outcome is plantar pressure during foot rollover, and the secondary outcomes are kinetic and kinematic parameters of gait, neuropathy signs and symptoms, foot and ankle range of motion and function, muscle strength, and balance confidence. The intervention is carried out for 12 weeks, twice a week, for 40-60 min each session. The follow-up period is 24 weeks from the baseline condition. Discussion: Herein, we present a more comprehensive and specific physiotherapy approach for foot and ankle function, by choosing simple tasks, focusing on recovering range of motion, strength, and functionality of the joints most impaired by diabetic polyneuropathy. In addition, this intervention aims to transfer these peripheral gains to the functional and more complex task of foot rollover during gait, in order to reduce risk of ulceration. If it shows any benefit, this protocol can be used in clinical practice and can be indicated as complementary treatment for this disease.