Use of amine buffers for in vitro studies of bone resorption

Summary Responses of cultured fetal rat bones to parathyroid hormone (PTH) were compared in media gassed with 5% CO₂ and buffered with bicarbonate or in media buffered with the amine buffers HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)), Tris (tris(hydroxymethyl)aminomethane), or glycylglycine. PTH-induced bone resorption was markedly reduced in media buffered with the amine buffers and cultured in air in the absence of added bicarbonate. When the amine buffers were added to bicarbonate-buffered media, they decreased the responses of the bones to submaximal concentrations of PTH. But the response to supramaximal concentrations of PTH was usually not impaired. The results confirm our earlier studies indicating a CO₂ and/or bicarbonate requirement for optimal bone resorption. They also demonstrate that the amine buffers can inhibit the responses of bone to PTH even in the presence of CO₂ and bicarbonate. Supported by NIH research Grant #AM 11262. Supported by NIH Research Career Development Award #AM 70210.     

Polymorphism of carbon oxidation of drugs and clinical implications.

Eight volunteers previously phenotyped for their ability to hydroxylate debrisoquine (four extensive metabolisers (EM), four poor metabolisers (PM) were investigated for their metabolic handling of guanoxan and phenacetin. All three drugs are oxidised at carbon centres. Oxidative dealkylation of phenacetin was determined by measuring the rate of formation of paracetamol. The EM subjects excreted mostly metabolites of guanoxan (mean 29% of dose), whereas the PM group excreted large amounts of unchanged drug (48% of dose). The rate of formation of paracetamol was noticeably slower in the PM group, and, when analysed by minimum estimates of apparent first-order rate constants, the difference between the two phenotypes was significant. Thus the hydroxylation defect shown for debrisoquine metabolism carries over to the oxidative metabolism of phenacetin and guanoxan. Some 5% of the population are genetically defective hydroxylators of drugs. Thus methods for evaluating the metabolism of new drugs in respect of usage and side effects need to be revised.     

Preliminary study on the influence of fungal metabolites on germination of barley grains

The biological activity of metabolites in the filtrates of some moulds, previously isolated from grain, showed effective germ toxication of high germinating malting grain. Other fungal filtrates exhibited marked stimulative effect on germination. Both effects were inconsistently demonstrated by many fungal metabolites, irrespective of soaking duration. Some fungal metabolites induced rudimentary roots which resumed normal healthy growth when soaked in sterile water.

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Zusammenfassung Die biologische Aktivität der Metaboliten in den Filtraten von manchen Schimmelpilzen, die vorher von Getreidekörnern isoliert worden sind, zeigten wirkliche Keimvergiftigung der hoch keimenden Malzkörner. Andere Pilzfiltrate zeigten bedeutende stimulierende Wirkung auf die Keimung. Beide Wirkungen waren unstetig bei manchen Metaboliten der Pilze, ganz unabhängig von der Dauer des Einweichens. Manche Pilzmetaboliten haben rudimentäre Wurzeln zu einem normalen, gesunden Wachstum angeregt, wenn sie nachher in steriles Wasser eingeweicht worden sind

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Botany Department, Nottingham University, England.     

Manufacturing history of etanercept (Enbrel®): Consistency of product quality through major process revisions

Etanercept (ETN) (Enbrel®) is a soluble protein that binds to, and specifically inhibits, tumor necrosis factor (TNF), a proinflammatory cytokine. ETN is synthesized in Chinese hamster ovary cells by recombinant DNA technology as a fusion protein, with a fully human TNFRII ectodomain linked to the Fc portion of human IgG1. Successful manufacture of biologics, such as ETN, requires sophisticated process and product understanding, as well as meticulous control of operations to maintain product consistency. The objective of this evaluation was to show that the product profile of ETN drug substance (DS) has been consistent over the course of production. Multiple orthogonal biochemical analyses, which included evaluation of attributes indicative of product purity, potency, and quality, were assessed on >2,000 batches of ETN from three sites of DS manufacture, during the period 1998–2015. Based on the key quality attributes of product purity (assessed by hydrophobic interaction chromatography HPLC), binding activity (to TNF by ELISA), potency (inhibition of TNF-induced apoptosis by cell-based bioassay) and quality (N-linked oligosaccharide map), we show that the integrity of ETN DS has remained consistent over time. This consistency was maintained through three major enhancements to the initial process of manufacturing that were supported by detailed comparability assessments, and approved by the European Medicines Agency. Examination of results for all major quality attributes for ETN DS indicates a highly consistent process for over 18 years and throughout changes to the manufacturing process, without affecting safety and efficacy, as demonstrated across a wide range of clinical trials of ETN in multiple inflammatory diseases.     

Extent and Mode of Action of Cationic Legume Proteins against Listeria monocytogenes and Salmonella Enteritidis

The methylated soybean protein and methylated chickpea protein (MSP and MCP) with isoelectric points around pI 8 were prepared by esterifying 83 % of their free carboxyl groups and tested for their interactions with Listeria monocytogenes and Salmonella Enteritidis. The two substances exhibited a concentration-dependent inhibitory action against the two studied bacteria with a minimum inhibitory concentration of about 100 μg/mL. The IC_50 % of the two proteins against L. monocytogenes (17 μg/mL) was comparable to penicillin but comparatively much lower (15 μg/mL) than that of penicillin (85 μg/mL) against S. Enteritidis. The two proteins could inhibit the growth of L. monocytogenes and S. Enteritidis by about 97 and 91 %, respectively, after 6–12 h of incubation at 37 °C. The constituting subunits of MSP (methylated 11S and methylated 7S) were both responsible for its antimicrobial action. Transmission electron microscopy of the protein-treated bacteria showed various signs of cellular deformation. The cationic proteins can electrostatically and hydrophobically interact with cell wall and cell membrane, producing large pores, pore channels and cell wall and cell membrane disintegration, engendering higher cell permeability leading finally to cell emptiness, lysis and death.     

Adjusting homestead feeding to requirements and nutrient intake of grazing goats on semi-arid, subtropical highland pastures

Intensive livestock grazing can largely deplete the natural fodder resources in semi-arid, subtropical highlands and together with the low nutritional quality of the pasture vegetation limit the growth and production of grazing animals. To evaluate the contribution of homestead feeding of grazing goats to rangeland conservation and animal nutrition, two researcher-managed on-farm trials were conducted in a mountain oasis of Northern Oman. Goats' feed intake on pasture in response to four rations containing different levels of locally available green fodder and concentrate feeds was determined in six male goats each (35 ± 10.2 kg body weight (BW)). Total feed intake was estimated using titanium dioxide as external fecal marker as well as the diet organic matter (OM) digestibility derived from fecal crude protein concentration. The nutritional quality of selected fodder plants on pasture was analyzed to determine the animals' nutrient and energy intake during grazing. The pasture vegetation accounted for 0.46 to 0.65 of the goats' total OM intake (87 to 107 g/kg0.75 BW), underlining the importance of this fodder resource for the husbandry system. However, metabolizable energy (7.2 MJ/kg OM) and phosphorus concentrations (1.4 g/kg OM) in the consumed pasture plants were low. Homestead feeding of nutrient and energy-rich by-products of the national fishery and date palm cultivation to grazing goats increased their daily OM intake (R2 = 0.36; P = 0.005) and covered their requirements for growth and production. While the OM intake on pasture was highest in animals fed a concentrate-based diet (P = 0.003), the daily intake of 21 g OM/kg0.75 BW of cultivated green fodder reduced the animals' feed intake on pasture (R2 = 0.44; P = 0.001). Adjusting homestead supplementation with locally available feedstuffs to the requirements of individual goats and to the nutritional quality of the pasture vegetation improves animal performance and eases the grazing pressure exerted on the natural vegetation. This management strategy therefore appears to be a valuable alternative to intensive livestock feeding in zero-grazing systems and may contribute to sustainable livestock production in ecologically fragile, semi-arid mountain regions.     

Isolation of diverse bioactive compounds from Euphorbia balsamifera: Cytotoxicity and antibacterial activity studies

Antibacterial and cytotoxic activities of Euphorbia balsamifera, fractions and pure compounds were evaluated. The cytotoxic assays for HCT116, HePG2 and MCF7 showed a significant IC₅₀: 54.7 and 76.2 µg/mL of non-polar fraction “n-hexane” against HCT116 and HePG2, respectively. Antibacterial results revealed that plant fractions exhibited significant potential against the tested pathogens than the total extract where n-butanol and ethyl acetate fractions showed significant antibacterial activity (P < 0.05) against tested bacterial strains. Isolation and structure determination of compounds from n-hexane and n-butanol fractions were performed. From n-hexane fraction, 29-nor-cycloartanol (1), lanost-8-en-3-ol (2a), cycloartanol (2b) and kampferol-3,4'-dimethyl ether (3) were isolated and structurally identified, along with 24 compounds were tentatively identified by GC–MS. From the polar n-butanol fraction, 4-O-β-D-glucopyranosyl-2-hydroxy-6-methoxyacetophenone (4), 4-O-α-L-rhamnosyl-(1 → 6)-β-D-glucopyranosyl-2-hydroxy-6methoxy-acetophenone (5), quercetin-3-O-glucopyranoside (6) and isoorientin (7) were assigned. Structures of the obtained compounds were determined by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Except compounds 1 and 5, all reported compounds announced antibacterial efficiency. Compound 2 showed selectively the highest activity against Enterococcus faecalis (22 ± 0.13 mm), meanwhile 4-O-β-D-glucopyranosyl-2-hydroxy-6-methoxyacetophenone (4) showed broadly the highest antibacterial activity with MIC of 1.15–1.88 mg/mL against the test Gram-positive and Gram-negative bacteria. Cytotoxic assays indicated that kampferol-3,4'-dimethyl ether (3) exhibited the highest activity with matching IC₅₀ values to doxorubicin; 111.46, 42.67 and 44.90 µM against HCT116, HePG2 and MCF7, respectively, however, it is toxic on retina normal cell line RPE1.     

Hypotensive response to debrisoquine and hydroxylation phenotype

Seven human volunteers with normal blood pressure, who had been phenotyped according to their ability to effect the 4-hydroxylation of the antihypertensive drug debrisoquine, each took orally a 20mg tablet of the drug. Lying and standing blood pressure were measured at hourly intervals up to 8h post-dosing. The four extensive metabolizers (EM phenotype) showed little or no response to the drug, whilst the three non-metabolizers (NM phenotype) demonstrated a statistically significant orthostatic hypotensive effect compared to the EM group. Dose-dependent metabolism was also observed with the NM subjects. It is concluded that the genetic factor which controls the metabolism of debrisoquine is also largely responsible for the differences in response seen between the two debrisoquine hydroxylator phenotypes.     

Prognostic value of infiltrating immune cells in clear cell renal cell carcinoma (ccRCC)

In this study, we attempted to evaluate the prognostic value of infiltrating immune/stromal cells in clear cell renal cell carcinoma (ccRCC), by using the immune scores and stromal scores based on the “Estimation of STromal and Immune cells in MAlignant Tumours using Expression data” algorithm to represent the levels of infiltrating immune cells and stromal cells. We found that the infiltrating immune cells were associated with poor prognosis of ccRCC. To assess the role of infiltrating immune cells in ccRCC cells, first, we performed differentially expressed genes analysis and functional analysis for validation. The results showed that the underlying mechanism by which infiltrating immune cells promoted cancer progression involved in regulating the nuclear division, angiogenesis, and immune response. Next, we investigated the relationship between infiltrating immune cells and mutations in ccRCC cells. We found that the infiltrating immune cells have certain effects on genetic mutations. In conclusion, infiltrating immune cells within the tumor microenvironment can be used to predict prognosis in ccRCC.     

The Combination of Amoxicillin-Clavulanic Acid and Ketoconazole in the Treatment of Madurella mycetomatis Eumycetoma and Staphylococcus aureus Co-infection

Eumycetoma is a chronic progressive disabling and destructive inflammatory disease which is commonly caused by the fungus Madurella mycetomatis. It is characterized by the formation of multiple discharging sinuses. It is usually treated by antifungal agents but it is assumed that the therapeutic efficiency of these agents is reduced by the co-existence of Staphylococcus aureus co-infection developing in these sinuses. This prospective study was conducted to investigate the safety, efficacy and clinical outcome of combined antibiotic and antifungal therapy in eumycetoma patients with superimposed Staphylococcus aureus infection. The study enrolled 337 patients with confirmed M. mycetomatis eumycetoma and S. aureus co-infection. Patients were allocated into three groups; 142 patients received amoxicillin-clavulanic acid and ketoconazole, 93 patients received ciprofloxacin and ketoconazole and 102 patients received ketoconazole only. The study showed that, patients who received amoxicillin-clavulanic acid and ketoconazole treatment had an overall better clinical outcome compared to those who had combined ciprofloxacin and ketoconazole or to those who received ketoconazole only. In this study, 60.6% of the combined amoxicillin-clavulanic acid/ketoconazole group showed complete or partial clinical response to treatment compared to 30.1% in the ciprofloxacin/ketoconazole group and 36.3% in the ketoconazole only group. The study also showed that 64.5% of the patients in the ciprofloxacin/ketoconazole group and 59.8% in the ketoconazole only group had progressive disease and poor outcome. This study showed that the combination of amoxicillin-clavulanic acid and ketoconazole treatment is safe and offers good clinical outcome and it is therefore recommended to treat eumycetoma patients with Staphylococcus aureus co-infection.