Relative and Combined Effects of Ethanol and Protein Deficiency on Bone Manganese and Copper

Both manganese and copper may affect bone synthesis. Bone content of both metals can be altered in alcoholics, although controversy exists regarding this matter. To analyse the relative and combined effects of ethanol and a low protein diet on bone copper and manganese, and their relationships with bone structure and metabolism, including trabecular bone mass (TBM), osteoid area (OA), osteocalcin (OCN), insulin-like growth factor-1 (IGF-1), parathyroid hormone (PTH), urinary hydroxyproline (uHP) and vitamin D. Adult male Sprague-Dawley rats were divided into four groups. The control rats received a 18% protein-containing diet; a second group, an isocaloric, 2% protein-containing diet; a third one, an isocaloric, 36% ethanol-containing diet and a fourth, an isocaloric diet containing 2% protein and 36% ethanol. After sacrifice, TBM and OA were histomorphometrically assessed; bone and serum manganese and copper were determined by atomic absorption spectrophotometry, and serum OCN, IGF-1, PTH, uHP and vitamin D by radioimmunoassay. Ethanol-fed rats showed decreased TBM and bone manganese. Significant relationships existed between bone manganese and TBM, serum IGF-1 and OCN. Ethanol leads to a decrease in bone manganese, related to decreased bone mass and bone synthesis. No alterations were found in bone copper.     

Anthropometric profile, vertical jump, and throwing velocity in elite female handball players by playing positions

Women's handball is a sport, which has seen an accelerated development over the last decade. Although anthropometric and physical characteristics have been studied for male sports teams, in women's handball, studies are scarce. The aim of this study was twofold: first, to describe the anthropometric characteristics, throwing velocity, hand grip, and muscular power of the lower limbs in female handball players and second, to identify the possible differences in these parameters in terms of individual playing positions (center, back, wing, pivot, and goalkeeper). A total of 130 elite female Spanish handball players participated in the study (age 25.74 ± 4.84 years; playing experience 14.92 ± 4.88 years). Anthropometric assessment was performed for all the subjects following the International Society for the Advancement of Kinanthropometry protocols. Furthermore, all the subjects performed a vertical jump test (squat jump and countermovement jump). Hand grip and throwing velocity in several situations were also assessed. A 1-way analysis of variance and a Tukey post hoc test were used to study the differences among individual playing positions. Wings were less heavy, shorter, and showed a smaller arm span than did goalkeepers, backs and pivots (p ≤ 0.001). Additionally, pivots were heavier than centers. Backs and pivots exhibited higher muscular mass than did wings. Total players' somatotype was mesomorphy endomorphy (3.89-4.28-2.29). Centers showed higher throwing velocity levels than did wings in 9-m throws from just behind the line, with a goalkeeper. Backs exhibited higher hand-grip values than did wings. Statistical differences have been established between wings and other specific playing positions, especially with pivot and backs. Coaches can use this information to select players for the different specific positions.     

Phylogenetic and mixed Yule‐coalescent analyses reveal cryptic lineages within two South American marine snails of the genus Crepipatella (Gastropoda: Calyptraeidae)

The presence of sibling species within the marine gastropod genus Crepipatella has complicated the taxonomy of members of the group. Since the establishment of the genus, 15 species have been described, but recent studies have indicated that there are only five valid species, two of which inhabit the coasts of Chile, namely C. dilatata and C. fecunda. The two species are morphologically indistinguishable as adults, but can be differentiated on the basis of their encapsulated developmental stages. The primary aim of this study was to reconstruct phylogeny within the genus, and to establish species limits of C. dilatata and C. fecunda, using mitochondrial DNA data. To this end, we used maximum parsimony, maximum likelihood, and Bayesian inference to reconstruct phylogenies using 589 bp of the cytochrome oxidase I (COI) gene. The mtDNA phylogenies were then used as input in a general mixed Yule‐coalescent (GMYC) analysis to estimate species boundaries. In addition, quarter likelihood mapping was used to test a posteriori the confidence of inner branch patterns in the phylogenetic tree. Both DNA tree‐based and GMYC methods provide support for five isolated lineages within this species complex. Our data also suggest that Late Pleistocene and Holocene fragmentation and subsequent range expansion events may have shaped contemporary genetic patterns of Crepipatella in South America.     

Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF

Susceptibility testing of clinical isolates of anaerobic bacteria is not considered, often, mandatory in routine clinical practice and the treatments are empirically established. Thus, periodic monitoring of the susceptibility patterns of anaerobic bacteria is advisable. The aim of this study was to update on resistance of Bacteroides fragilis group in our Institution with special attention to carbapenems reporting metallo-beta-lactamase producing strains for the first time in Spain, and to compare fingerprinting analysis results obtained by using automated rep-PCR (DiversiLab System) and MALDI-TOF MS. A total of 830 non-duplicated clinical isolates of the B. fragilis group recovered from the years 2006 to 2010 were studied. B. fragilis was the most prevalent species (59.5%). The total susceptibility of B. fragilis group isolates were: penicillin, 13.3%; amoxicillin/clavulanic, 89.6%; piperacillin-tazobactam, 91.8%; cefoxitin, 65.8%; ertapenem, 95.9%; imipenem, 98.2%; clindamycin, 53.4% and metronidazole, 96.4%. The percentage of sensitive isolates did not change significantly over time for amoxicillin/clavulanic, cefoxitin, clindamycin and metronidazole. A slight increase in the rate of resistance to ertapenem and imipenem was observed. Imipenem resistance and carbapenemase production were detected for the first time in our laboratory in the year 2007. No other report of carbapenemase-producing B. fragilis in our country has been previously published. Six imipenem-resistant isolates were MBL-producing and PCR positive for cfiA gene. Four of them were PCR positive for IS-like immediately upstream cfiA gene and two of them were negative. Both, automated rep-PCR (DiversiLab) and MALDI-TOF MS, revealed a great genetic diversity among carbapenem-producing strains suggesting the acquisition of novel resistance genes more than clonal dissemination of them. Both methods seem to be useful tools for fast and accurate identification and strain typing of B. fragilis group in the daily laboratory routine. Because of the relevant increase observed in Bacteroides species isolated from blood cultures and the appearance of carbapenemase-producing strains in our Institution, we recommend to test the antimicrobial susceptibility of the isolates, at least in the most severe patients.     

Growth-dependent effects of dietary protein concentration and quality on the biomechanical properties of the diaphyseal rat femur

ObjectivesThis study compares the effects of feeding growing rats with increasing concentrations of casein (C) and wheat gluten (G), proteins that show different biological qualities, on the morphometrical and biomechanical properties of the femoral diaphysis.Materials and methodsFemale rats were fed with one of ten diets containing different concentrations (5–30%) of C and G between the 30th and 90th days of life (Control = C-20%). Biomechanical structural properties of the right femur middiaphysis were estimated using a 3-point bending mechanical test with calculation of some indicators of bone material properties.ResultsBody weight and length were affected by treatments, values being highest in rats fed the C-20% diet. G diets affected negatively both parameters. Changes in cross-sectional geometry (mid-diaphyseal cross-sectional and cortical areas, femoral volume, and rectangular moment of inertia) were positively related to the C content of the diet, while they were severely and negatively affected by G diets. Similar behaviors were observed in the bone structural properties (fracture load, yielding load, diaphyseal stiffness and elastic energy absorption). When values of strength and stiffness were normalized for body weight, the differences disappeared. The bone material quality indicators (elastic modulus, yielding stress, elastic energy absorption/volume) did not differ significantly among all studied groups. Femoral calcium concentration in ashes was not significantly different among groups.ConclusionThe clear differences in strength and stiffness of bone beams induced by dietary protein concentration and quality seemed to be the result of an induced subnormal gain in bone structural properties as a consequence of a correlative subnormal gain in bone growth and mass, yet not in bone material properties.     

Understanding the mechanisms of chilling injury in bell pepper fruits using the proteomic approach

In order to advance in the understanding of CI in pepper fruits, the cell ultrastructure alterations induced by CI and the physiological and metabolic changes have been studied along with the proteomic study. When stored at low temperatures bell pepper (Capsicum annuum) fruits exhibited visual CI symptoms and important alterations within the cell ultrastructure, since peroxisomes and starch grains were not detected and the structure of the chloroplast was seriously damaged in chilled tissues. Physiological and metabolic disorders were also observed in chilled fruits, such as higher ethylene production, increased MDA content, changes in sugar and organic acids and enzymatic activities. The comparative proteomic analysis between control and chilled fruits reveals that the main alterations induced by CI in bell pepper fruits are linked to redox homeostasis and carbohydrate metabolism. Thus, protein abundance in the ascorbate-glutathione cycle is altered and catalase is down-regulated. Key proteins from glycolysis, Calvin cycle and Krebs cycle are also inhibited in chilled fruits. Enolase and GAPDH are revealed as proteins that may play a key role in the development of chilling injury. This study also provides the first evidence at the protein level that cytosolic MDH is involved in abiotic stress.     

Uncovering suitable reference proteins for expression studies in human adipose tissue with relevance to obesity

Background

Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals.

Methodology and Findings

To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples.

Conclusions

ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study.     

Randomized controlled trial of recombinant human granulocyte-macrophage colony-stimulating factor for the treatment of chronic hepatitis c

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered subcutaneously to 45 chronic hepatitis C patients, randomly assigned to receive 0.5, 1 or 2 microg GM-CSF/kg b.w. daily/6 weeks (n=30), or no treatment (n=15). Alanine transaminase (ALT) values normalized in four out of 10 (40%) patients administered 2 microg GM-CSF [1 cleared hepatitis C virus (HCV) RNA] but in none given 0.5 or 1 microg or untreated controls (P=0.0079). Following 4 weeks of rest, patients received 5 million units of interferon (IFN)alpha2b every other day/6 months, alone (n=30), or combined with 2 microg GM-CSF/daily for 3 months (n=15). At treatment end, ALT levels in patients administered the combination normalized more frequently than in those given monotherapy (73% vs 47%, P=0.089). Viraemia decreased significantly in 11/15 (73%) patients administered GM-CSF/IFNalpha2b combination (mean log HCV RNA copies/ml+/-SEM: 4.13+/-0.40 vs 5.29+/-0.23;P=0.011), and in 20/30 (67%) receiving IFNalpha2b monotherapy (4.27+/-0.28 vs 5. 31+/-0.14;P=0.004); 27% and 20% of patients given the combination and monotherapy, respectively, cleared HCV RNA. One patient in each regime had a sustained response after 12 months. 2', 5'-Oligoadenylate synthetase activity (2-5AS) increased during GM-CSF therapy (P=0.033 with the 2 microg dose). 2-5AS increased more in the GM-CSF/IFN-alpha2b combination than with IFNalpha2b monotherapy (P<0.02). GM-CSF provoked a skin reaction at the injection site, accompanied by moderate and reversible rises in eosinophil and leucocyte counts. In summary, daily s.c. GM-CSF administration is safe and shows effects against HCV; the GM-CSF/IFNalpha2b combination has an additional-but transient-antiviral activity in chronic hepatitis C.     

Human metastasis regulator protein H-prune is a short-chain exopolyphosphatase

The DHH superfamily human protein h-prune, a binding partner of the metastasis suppressor nm23-H1, is frequently overexpressed in metastatic cancers. From an evolutionary perspective, h-prune is very close to eukaryotic exopolyphosphatases. Here, we show for the first time that h-prune efficiently hydrolyzes short-chain polyphosphates (k cat of 3-40 s (-1)), including inorganic tripoly- and tetrapolyphosphates and nucleoside 5'-tetraphosphates. Long-chain inorganic polyphosphates (>or=25 phosphate residues) are converted more slowly, whereas pyrophosphate and nucleoside triphosphates are not hydrolyzed. The reaction requires a divalent metal cofactor, such as Mg (2+), Co (2+), or Mn (2+), which activates both the enzyme and substrate. Notably, the exopolyphosphatase activity of h-prune is suppressed by nm23-H1, long-chain polyphosphates and pyrophosphate, which may be potential physiological regulators. Nucleoside triphosphates, diadenosine hexaphosphate, cAMP, and dipyridamole (inhibitor of phosphodiesterase) do not affect this activity. Mutation of seven single residues corresponding to those found in the active site of yeast exopolyphosphatase led to a severe decrease in h-prune activity, whereas one variant enzyme exhibited enhanced activity. Our results collectively suggest that prune is the missing exopolyphosphatase in animals and support the hypothesis that the metastatic effects of h-prune are modulated by inorganic polyphosphates, which are increasingly recognized as critical regulators in cells.