Pollen substitutes increase honey bee haemolymph protein levels as much as or more than does pollen

Adequate substitutes for pollen are necessary for maintaining healthy bee colonies during periods of pollen dearth, but testing them objectively is both time consuming and expensive. We compared two commercial diets with bee collected pollen and acacia pod flour (used by beekeepers in some parts of Brazil) by measuring their effect on haemolymph protein contents of young bees exclusively fed on these diets, which is a fast and inexpensive assay. The commercial diets included a new, non-soy-based, pollen substitute diet (named Feed-Bee (R)) and a soy-based diet, named Bee-Pro (R). The diets were each given in patty form to groups of 100 Africanized honey bees in hoarding cages, maintained and fed from emergence until six days of age. Sucrose, in the form of sugar syrup, was used as a protein free control. Feed-Bee (R), Bee-Pro (R), pollen and acacia pod flour diets increased protein titers in the haemolymph by factors of 2.65, 2.51, 1.76 and 1.69, respectively, over protein titers in bees fed only sucrose solution. The bees fed Feed-Bee (R) and Bee-Pro (R) had their haemolymph significantly enriched in protein compared to the controls and those fed acacia pod flour and to titers slightly higher than those fed pollen. All four proteinaceous diets were significantly superior to sucrose alone.

Inhibitors of TACE and Caspase-1 as anti-inflammatory drugs

TNF-alpha neutralising agents such as Infliximab (Remicade(R)), Etanercept (Enbrel(R)) and the IL-1 receptor antagonist Anakinra (Kineret(R)), are currently used clinically for the treatment of many inflammatory diseases such as Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriatic arthritis and psoriasis. These protein preparations are expensive to manufacture and administer, need to be injected and can cause allergic reactions. An alternative approach to lowering the levels of TNF-alpha and IL-1 beta in inflammatory disease, is to inhibit the enzymes that generate these cytokines using cheaper small molecules. This paper is a broad overview of the progress that has been achieved so far, with respect to small molecule inhibitor design and pharmacological studies (in animals and humans), for the metalloprotease Tumour Necrosis Factor-alpha Converting Enzyme (TACE) and the cysteine protease Caspase-1 (Interieukin-1 beta Converting Enzyme, ICE). Inhibitors of these two enzymes are currently considered to be good therapeutic targets that have the potential to provide relatively inexpensive and orally bioavailable anti-inflammatory agents in the future.

Mycobacterium chelonae valve endocarditis resulting from contaminated biological prostheses

Objectives: A rapid-growing mycobacteria biological prosthetic valve (BPV) endocarditis related to prosthetic manufacturing process is described in Brazil. Methods: From 1999 to 2008, thirty-nine patients underwent BPV replacement due to culture-negative suspected endocarditis. All these cases had histological sections stained by Ziehl-Neelsen method. Clinical and microbiological data were reviewed in all acid-fast bacilli (AFB) positive cases. The 16S-23S internal transcribed sequence (ITS) was amplified using DNA extracted from paraffin-embedded samples, digested with restrictions enzymes and/or sequenced. Results: Eighteen AFB positive BPV (18/39)(46%) were implanted in 13 patients and were from the same manufacturer. Four of them were implanted in other hospitals. Thirteen BPV were histologically proven endocarditis and five showed a colonization pattern. The examination of six non-implanted ""sterile"" BPV from this manufacturer resulted in 5 AFB positive. Mycobacterium chelonae was the AFB identified by ITS restriction analysis and sequencing. Conclusions: Rapid-growing mycobacteria infections must be suspected and Ziehl-Neelsen stain always performed on histology of either early or late BPV endocarditis, particularly when blood cultures are negative. (C) 2010 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

46,XY DSD due to impaired androgen production

Disorders of androgen production can occur in all steps of testosterone biosynthesis and secretion carried out by the foetal Leydig cells as well as in the conversion of testosterone into dihydrotestosterone (DHT). The differentiation of Leydig cells from mesenchymal cells is the first walk for testosterone production. In 46,XY disorders of sex development (DSDs) due to Leydig cell hypoplasia, there is a failure in intrauterine and postnatal virilisation due to the paucity of interstitial Leydig cells to secrete testosterone. Enzymatic defects which impair the normal synthesis of testosterone from cholesterol and the conversion of testosterone to its active metabolite DHT are other causes of DSD due to impaired androgen production. Mutations in the genes that codify the enzymes acting in the steps from cholesterol to DHT have been identified in affected patients. Patients with 46,XY DSD secondary to defects in androgen production show a variable phenotype, strongly depending of the specific mutated gene. Often, these conditions are detected at birth due to the ambiguity of external genitalia but, in several patients, the extremely undervirilised genitalia postpone the diagnosis until late childhood or even adulthood. These patients should receive long-term care provided by multidisciplinary teams with experience in this clinical management. (C) 2009 Elsevier Ltd. All rights reserved.

Characterization of inflammatory responses to eccentric exercise in humans

Eccentric exercise commonly results in muscle damage. The primary sequence of events leading to exercise-induced muscle damage is believed to involve initial mechanical disruption of sarcomeres, followed by impaired excitation-contraction coupling and calcium signaling, and finally, activation of calcium-sensitive degradation pathways. Muscle damage is characterized by ultrastructural changes to muscle architecture, increased muscle proteins and enzymes in the bloodstream, loss of muscular strength and range of motion and muscle soreness. The inflammatory response to exercise-induced muscle damage is characterized by leukocyte infiltration and production of pro-inflammatory cytokines within damaged muscle tissue, systemic release of leukocytes and cytokines, in addition to alterations in leukocyte receptor expression and functional activity. Current evidence suggests that inflammatory responses to muscle damage are dependent on the type of eccentric exercise, previous eccentric loading (repeated bouts), age and gender. Circulating neutrophil counts and systemic cytokine responses are greater after eccentric exercise using a large muscle mass (e.g. downhill running, eccentric cycling) than after other types of eccentric exercise involving a smaller muscle mass. After an initial bout of eccentric exercise, circulating leukocyte counts and cell surface receptor expression are attenuated. Leukocyte and cytokine responses to eccentric exercise are impaired in elderly individuals, while cellular infiltration into skeletal muscle is greater in human females than males after eccentric exercise. Whether alterations in intracellular calcium homeostasis influence inflammatory responses to muscle damage is uncertain. Furthermore, the effects of antioxidant supplements are variable, and the limited data available indicates that anti-inflammatory drugs largely have no influence on inflammatory responses to eccentric exercise. In this review, we compare local versus systemic inflammatory responses, and discuss some of the possible mechanisms regulating the inflammatory responses to exercise-induced muscle damage in humans.

Extraadrenal 21-Hydroxylation by CYP2C19 and CYP3A4: Effect on 21-Hydroxylase Deficiency

Context: 21-Hydroxylase deficiency (21OHD) is caused by CYP21A2 gene mutations disrupting the adrenal 21-hydroxylase, P450c21. CYP21A2 mutations generally correlate well with the 21OHD phenotype, but some children with severe CYP21A2 mutations have residual 21-hydroxylase activity. Some hepatic P450 enzymes can 21-hydroxylate progesterone, but their physiological relevance in modifying 21OHD is not known. Objective: Wedetermined the ability of CYP2C19 and CYP3A4 to 21-hydroxylate progesterone and 17-hydroxyprogesterone (17OHP), determined the impact of the common P450 oxidoreductase (POR) variant A503V on these activities, and examined correlations between CYP2C19 variants and phenotype in patients with 21OHD. Methods: Bacterially expressed, N-terminally modified, C-His-tagged human P450c21, CYP2C19, and CYP3A4 were combined with bacterially expressed wild-type and A503V POR. The 21-hydroxylation of radiolabeled progesterone and 17OHP was assessed, and the Michaelis constant (Km) and maximum velocity (Vmax) of the reactions were measured. CYP2C19 was genotyped in 21OHD patients with genotypes predicting severe congenital adrenal hyperplasia. Results: Compared to P450c21, the Vmax/Km for 21-hydroxylation of progesterone by CYP2C19 and CYP3A4 were 17 and 10%, respectively. With both forms of POR, the Km for P450c21 was approximately 2.6 mu M, the Km for CYP2C19 was approximately 11 mu M, and the Km for CYP3A4 was approximately 110 mu M. Neither CYP2C19 nor CYP3A4 could 21-hydroxylate 17OHP. The CYP2C19 ultrametabolizer allele CYP2C19* 17 was homozygous in one of five patients with a 21OHD phenotype that was milder than predicted by the CYP21A2 genotype. Conclusions: CYP2C19 and CYP3A4 can 21-hydroxylate progesterone but not 17OHP, possibly ameliorating mineralocorticoid deficiency, but not glucocorticoid deficiency. Multiple enzymes probably contribute to extraadrenal 21-hydroxylation. (J Clin Endocrinol Metab 94: 89-95, 2009)

An optimized model for rat liver perfusion studies

Conditions which influence the viability, integrity, and extraction efficiency of the isolated perfused rat liver were examined to establish optimal conditions for subsequent work in reperfusion injury studies including the choice of buffer, use of oncotic agents, hematocrit, perfusion flow rate, and pressure. Rat livers were perfused with MOPS-buffered Ringer solution with or without erythrocytes. Perfusates were collected and analyzed for blood gases, electrolytes, enzymes, radioactivity in MID studies, and lignocaine in extraction studies. Liver tissue was sampled for histological examinations, and wet:dry weight of the liver was also determined. MOPS-buffered Ringer solution was found to be superior to Krebs bicarbonate buffer, in terms of pH control and buffering capacity, especially during any prolonged period of liver perfusion. A pH of 7.2 is chosen for perfusion since this is the physiological pH of the portal blood. The presence of albumin was important as an oncotic agent, particularly when erythrocytes were used in the perfusate. Perfusion pressure, resistance, and vascular volume are how-dependent and the inclusion of erythrocytes in the perfusate substantially altered the flow characteristics for perfusion pressure and resistance but not vascular volume. Lignocaine extraction was relatively flow-independent. Perfusion injury as defined by enzyme release and tissue fine structure was closely related to the supply of O-2. The optimal conditions for liver perfusion depend upon an adequate supply of oxygen. This can be achieved by using either erythrocyte-free perfusate at a how rate greater than 6 ml/min/g liver or a 20% erythrocyte-containing perfusate at 2 ml/min/g. (C) 1996 Academic Press, Inc.

Tobacco Smoke Induces Ventricular Remodeling Associated with an Increase in NADPH Oxidase Activity

Background: Recent studies have assessed the direct effects of smoking on cardiac remodeling and function. However, the mechanisms of these alterations remain unknown. The aim of this study was to investigate de role of cardiac NADPH oxidase and antioxidant enzyme system on ventricular remodeling induced by tobacco smoke. Methods: Male Wistar rats that weighed 200-230 g were divided into a control group (C) and an experimental group that was exposed to tobacco smoke for a period of two months (ETS). After the two-month exposure period, morphological, biochemical and functional analyses were performed. Results: The myocyte cross-sectional area and left ventricle end-diastolic dimension was increased 16.2% and 33.7%, respectively, in the ETS group. The interstitial collagen volume fraction was also higher in ETS group compared to the controls. In addition to these morphological changes, the ejection fraction and fractional shortening were decreased in the ETS group. Importantly, these alterations were related to augmented heart oxidative stress, which was characterized by an increase in NADPH oxidase activity, increased levels of lipid hydroperoxide and depletion of antioxidant enzymes (e.g., catalase, superoxide dismutase and glutathione peroxidase). In addition, cardiac levels of IFN-gamma, TNF-alpha and IL-10 were not different between the groups. Conclusion: Cardiac alterations that are induced by smoking are associated with increased NADPH oxidase activity, suggesting that this pathway plays a role in the ventricular remodeling induced by exposure to tobacco smoke. Copyright (C) 2011 S. Karger AG, Basel

Super-CHO - A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6)cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6)cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Ratio of Metalloproteinase 2 to Tissue Inhibitor of Metalloproteinase 2 in Medullary Thyroid Carcinoma

Objectives: To develop an index for the ratio of metalloproteinase 2 (MMP-2) to its tissue inhibitor (TIMP-2) in immunostained medullary thyroid carcinoma specimens and to correlate it with clinical and pathologic prognostic factors. Metalloproteinases, enzymes related to the degradation of the extracellular matrix, take part in carcinogenesis and have been associated with the prognosis of neoplasias. Nevertheless, medullary carcinoma is rarely considered in research analysis. Researchers tend to favor the ratio of enzymes to their inhibitors over the absolute concentrations of these enzymes. Design: Retrospective study of surgical samples. Setting: Head and Neck Surgery and Endocrinology Departments, Universidade de Sao Paulo Medical School Hospital. Patients: Surgical specimens from 33 patients who had been observed for a mean of 76.8 months (range, 4-201 months) were immunohistochemically stained for MMP-2 and TIMP-2. Only patients whose clinical and pathologic data were complete and whose specimens were preserved were included in the study. Main Outcome Measures: The ratio between the expressions of MMP-2 and TIMP-2 was based on a staining index (immunostaining extent and intensity) of each of the markers. Results: Proportionally large expressions of TIMP-2 over MMP-2 correlated with low occurrences of positive findings on initial cervical examination for the presence of thyroid nodules and/or lymphadenopathy (P = .02) and cervical lymph node metastases (P < .001), conditions correlated with prognosis. A correlation with cure at the end of follow-up (P = .01) was also observed. (P < .05 was considered statistically significant.) Conclusion: The ratio of MMP-2 to TIMP-2 expression is an additional and novel prognostic predictor of the outcome of medullary carcinoma treated surgically.

A fructan: fructan fructosyltransferase activity from Lolium rigidum

Fructan:fructan fructosyltransferase (FFT) activity was purified about 300-fold from leaves of Lolium rigidura Gaudin by a combination of affinity chromatography, gel filtration, anion exchange and isoelectric focusing. The FFT activity was free of sucrose:sucrose fructosyltransferase and invertase activities. It had an apparent pI of 4.7 as determined by isoelectric focusing, and a molecular mass of about 50000 (gel filtration). The FFT activity utilized the trisaccharides 1-kestose and 6(G)-kestose as sole substrates, but was not able to use 6-kestose as sole substrate. The FFT activity was not saturated when assayed at concentrations of 1-kestose, 6(G)-kestose or (1,1)-kestotetraose of up to 400 mM The rate of reaction of the FFT activity was most rapid when assayed with 1-kestose and was less rapid when assayed with 6(G)-kestose, (1,1)-kestotetraose or (1,1,1)-kestopentaose. The FFT activity when assayed at a relatively high concentration of enzyme activity (approximately equivalent to about half the activity in crude extracts per gram fresh mass) did not synthesize fructan of degree of polymerization > 6, even during extended assays of up to 10 h. When assayed with a combination of 1-kestose and uniformly labelled [C-14]sucrose as substrates, the major reaction was the transfer of a fructosyl residue from 1-kestose to sucrose resulting in the re-synthesis of 1-kestose. Tetrasaccharide and 6(G)-kestose were also synthesized. When assayed with 6(G)-kestose and [C-14]sucrose as substrates, the major reaction of the FFT activity was the synthesis of tetrasaccharide. However, some synthesis of 1-kestose and re-synthesis of 6(G)-kestose also occurred. When 6, kestose was the sole substrate for the FFT activity, synthesis of tetrasaccharide was 2.7 to 3.4-fold slower than when 1-kestose was used as the sole substrate. Owing to differences in the fructan:sucrose fructosyltransferase activity of the FFT with each of the trisaccharides, net synthesis of tetrasaccharide by the FFT was altered significantly in the presence of sucrose. The magnitude of this effect depended on the concentration of the trisaccharides. In the presence of sucrose, 6(G)-kestose could be a substrate of equivalent importance to 1-kestose for synthesis of tetrasaccharide.

Aerobic Exercise Improves Reverse Cholesterol Transport in Cholesteryl Ester Transfer Protein Transgenic Mice

We analyzed the effect of a 6-week aerobic exercise training program on the in vivo macrophage reverse cholesterol transport (RCT) in human cholesteryl ester transfer protein (CETP) transgenic (CETP-tg) mice. Male CETP-tg mice were randomly assigned to a sedentary group or a carefully supervised exercise training group (treadmill 15 m/min, 30 min sessions, five sessions per week). The levels of plasma lipids were determined by enzymatic methods, and the lipoprotein profile was determined by fast protein liquid chromatography (FPLC). CETP activity was determined by measuring the transfer rate of (14)C-cholesterol from HDL to apo-B containing lipoproteins, using plasma from CETP-tg mice as a source of CETP. The reverse cholesterol transport was determined in vivo by measuring the [(3)H]-cholesterol recovery in plasma and feces (24 and 48 h) and in the liver (48 h) following a peritoneal injection of [(3)H]-cholesterol labeled J774-macrophages into both sedentary and exercise trained mice. The protein levels of liver receptors were determined by immunoblot, and the mRNA levels for liver enzymes were measured using RT-PCR. Exercise training did not significantly affect the levels of plasma lipids or CETP activity. The HDL fraction assessed by FPLC was higher in exercise-trained compared to sedentary mice. In comparison to the sedentary group, a greater recovery of [(3)H]-cholesterol from the injected macrophages was found in the plasma, liver and feces of exercise-trained animals. The latter occurred even with a reduction in the liver CYP7A1 mRNA level in exercised trained animals. Exercise training increased the liver LDL receptor and ABCA-1 protein levels, although the SR-BI protein content was unchanged. The RCT benefit in CETP-tg mice elicited by exercise training helps to elucidate the role of exercise in the prevention of atherosclerosis in humans.

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

The study reported here is a classical bottom-up proteomic approach where proteins from wasp venom were extracted and separated by 2-DE; the individual protein spots were proteolytically digested and subsequently identified by using tandem mass spectrometry and database query with the protein search engine MASCOT. Eighty-four venom proteins belonging to 12 different molecular functions were identified. These proteins were classified into three groups; the first is constituted of typical venom proteins: antigens-5, hyaluronidases, phospholipases, heat shock proteins, metalloproteinases, metalloproteinase-desintegrin like proteins, serine proteinases, proteinase inhibitors, vascular endothelial growth factor-related protein, arginine kinases, Sol i-II and -II like proteins, alpha-glucosidase, and superoxide dismutases. The second contained proteins structurally related to the muscles that involves the venom reservoir. The third group, associated with the housekeeping of cells from venom glands, was composed of enzymes, membrane proteins of different types, and transcriptional factors. The composition of P. paulista venom permits us to hypothesize about a general envenoming mechanism based on five actions: (i) diffusion of venom through the tissues and to the blood, (ii) tissue, (iii) hemolysis, (iv) inflammation, and (v) allergy-played by antigen-5, PLA1, hyaluronidase, HSP 60, HSP 90, and arginine kinases.

Menstrual and hormonal alterations in juvenile dermatomyositis

Objective To evaluate age at menarche, menstrual cycles and hormone profile in juvenile dermatomyositis (JDM) patients and controls. Methods Twelve consecutive JDM patients were compared to 24 age-matched healthy subjects. Age at menarche and age of maternal menarche were recorded. Menstrual cycle was evaluated prospectively for 6 consecutive months and the mean cycle length and flow were calculated. The hormone profile was collected on the last menstrual cycle. Demographic data, clinical features, muscle enzymes, JDM scores and treatment were analysed. Results The median of current age of JDM patients and controls was similar (18 vs. 17 years, p=0.99). The median age at menarche of the JDM patients was higher than in the control group (13 vs. 11 years, p=0.02) whereas the median age of maternal menarche was alike in both groups (12 vs. 13 years, p=0.67). Menstrual disturbances were not observed, except for one patient who had longer length of menstrual cycle. The median of follicle stimulating hormone (FSH) was significantly higher in JDM patients compared to controls (4.5 vs. 3.0 IU/L, p=0.02) and none of them had premature ovarian failure (POF). The median of progesterone was significantly lower in JDM patients (0.3 vs. 0.7 ng/mL, p=0.01) with a higher frequency of decreased progesterone compared to controls (75% vs. 29%, p=0.01). Conclusions Our study identifies in JDM patients delayed menarche with normal cycles and low follicular reserve. The decreased progesterone levels may suggest an underlying subclinical corpus luteum dysfunction in this disease.

Intake of trans Fatty Acids Causes Nonalcoholic Steatohepatitis and Reduces Adipose Tissue Fat Content

We investigated the effects of dietary trans fatty acids, PUFA, and SEA on body and liver fat content, liver histology, and mRNA of enzymes involved in fatty acid metabolism. LDL receptor knockout weaning male mice were fed for 16 wk with diets containing 40% energy as either trans fatty acids (TRANS), PUFA, or SEA. Afterwards, subcutaneous and epididymal fat were weighed and histological markers of nonalcoholic fatty liver disease (NAFLD) were assessed according to the Histological Scoring System for NAFLD. PPAR alpha, PPAR gamma, microsomal triglyceride transfer protein (MTP), carnitine palmitoyl transferase 1 (CPT-1), and sterol regulatory element binding protein-1c (SREBP-1c) mRNA were measured by quantitative RT-PCR. Food intake was similar in the 3 groups, although mice fed the TRANS diet gained less weight than those receiving the PUFA diet. Compared with the PUFA- and SEA-fed mice, TRANS-fed mice had greater plasma total cholesterol (TC) and triglyceride (TG) concentrations, less epididymal and subcutaneous fat, larger livers with nonalcoholic steatohepatitis (NASH)-like lesions, and greater liver TC and TG concentrations. Macrosteatosis in TRANS-fed mice was associated with a higher homeostasis model assessment of insulin resistance (HOMA(IR)) index and upregulated mRNA related to hepatic fatty acid synthesis (SREBP-1 c and PPAR gamma) and to downregulated MTP mRNA. Diet consumption did not alter hepatic mRNA related to fatty acid oxidation (PPAR alpha and CPT-1). In conclusion, compared with PUFA- and SFA-fed mice, TRANS-fed mice had less adiposity, impaired glucose tolerance characterized by greater HOMA(IR) index, and NASH-like lesions due to greater hepatic lipogenesis. These results demonstrate the role of trans fatty acid intake on the development of key features of metabolic syndrome. J. Nutr. 140: 1127-1132, 2010.