PURIFICATION AND ANALYSIS OF HUMAN ALPHA1-ANTITRYPSIN CONCENTRATE BY A NEW IMMUNOAFFINITY CHROMATOGRAPHY

Alpha₁-antitrypsin is a kind of plasma protein that requires a sequence of different fractionation steps to get generally. To report an effective process for isolating and purifying alpha₁-antitrypsin from Cohn Fraction IV based upon a new immunoaffinity chromatography medium, named “Alpha-1 Antitrypsin Select,” characterization of alpha₁-antitrypsin (α1-AT) was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and tandem mass spectroscopy. Total protein content was determined by the method of Bradford under visible light absorption at 595 nm. Pretreatment process and the immunoaffinity chromatography step achieved a 60.35 ± 1.39% yield. Thus, an overall 71.68 ± 1.32 fold increase in purity and a 41.88 ± 6.98% yield were obtained from plasma. The α1-AT had a specific activity of about 1.00–1.05 PU/mg. This technique will develop an effective process for isolating and purifying, with high purity and specific activity, alpha₁-antitrypsin from Cohn Fraction IV or human whole plasma, which could be an efficient and scaled-up method for alpha₁-antitrypsin products purification.     

Expression of tumor necrosis factor-alpha and vascular endothelial growth factor in different zones of fetal membranes: a possible relation to onset of labor

This study aimed to explore whether the altered expression of tumor necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF) and apoptotic changes in mid zone (MZ) and rupture zone (RZ) of fetal membranes (FM) are regulatory mechanisms associated with labor at term. Fifteen FM specimens were collected after vaginal deliveries and 13 specimens after elective caesarian section. Histological and immunohistochemical analysis were employed. Area percent of TNF-α and VEGF immunostaining and apoptotic index (AI) were evaluated using image analysis. The statistical data revealed significantly higher area % for TNF-α, VEGF immunoexpression and AI in labor compared to non-labor specimens (p < 0.0001). There was a significantly higher percentage of TNF-α immunoexpressed area in MZ compared with RZ in both groups (p < 0.0001). VEGF expression in RZ of both groups proved nearly double or triple the area % of expression relative to MZ with highly significant difference (p < 0.0001). quantitative analysis revealed near two fold increase in the AI in RZ (13.42 % ± 1.2 in labor; 11.20 % ± 0.96 in non-labor groups) when compared to MZ (7.20 % ± 0.6 in labor; 5.08 % ± 0.76 in non-labor groups) with highly significant zonal difference (p < 0.0001). Correlation analysis revealed significant correlation between apoptotic indices and area % of TNF-α (r = 0.575, p = 0.002 in non-labor; r = 0.652, p < 0.0001 in labor) and VEGF (r = 0.795, p < 0.0001 in non-labor; r = 0.668, p < 0.0001 in labor). In conclusion, Apoptosis may be regulated by TNF-α and VEGF expression in FM at labor. MZ is a step back from RZ and could participate actively in rupture of the FM during labor. TNF-α and VEGF increase with onset of labor and differentially expressed in the RZ and the MZ. These findings call for further study with tissue cultures or animal models.     

Do supervised weekly exercise programs maintain functional exercise capacity and quality of life, twelve months after pulmonary rehabilitation in COPD?

Background

Individuals with severe Z α1-antitrypsin (AAT) deficiency have a considerably increased risk of developing chronic obstructive lung disease (COPD). It has been hypothesized that compensatory increases in levels of other protease inhibitors mitigate the effects of this AAT deficiency. We analysed plasma levels of AAT, α1-antichymotrypsin (ACT) and secretory leukocyte protease inhibitor (SLPI) in healthy (asymptomatic) and COPD subjects with and without AAT deficiency.

Methods

Studied groups included: 71 asymptomatic AAT-deficient subjects (ZZ, n = 48 and SZ, n = 23, age 31 ± 0.5) identified during Swedish neonatal screening for AAT deficiency between 1972 and 1974; age-matched controls (MM, n = 57, age 30.7 ± 0.6); older asymptomatic ZZ (n = 10); healthy MM (n = 20, age 53 ± 9.6); and COPD patients (ZZ, n = 10, age 47.4 ± 11 and MM, n = 10, age 59.4 ± 6.7). Plasma levels of SLPI, AAT and ACT were analysed using ELISA and immunoelectrophoresis.

Results

No significant difference was found in plasma ACT and SLPI levels between the healthy MM and the ZZ or SZ subjects in the studied groups. Independent of the genetic variant, subjects with COPD (n = 19) had elevated plasma levels of SLPI and ACT relative to controls (n = 153) (49.5 ± 7.2 vs 40.7 ± 9.1 ng/ml, p < 0.001 and 0.52 ± 0.19 vs 0.40 ± 0.1 mg/ml, p < 0.05, respectively).

Conclusion

Our findings show that plasma levels of ACT and SLPI are not elevated in subjects with genetic AAT deficiency compared MM controls and do not appear to compensate for the deficiency of plasma AAT.     

La déficience en alpha-1 antitrypsine: corrélation entre épreuves biochimiques et phénotypes dans une famille

Phenotypes (Pi) for serum alpha-1 antitrypsin (AAT) were determined in a family of 19 members spanning three generations and presenting a deficiency in this protein. The standard Fagerhol crossed immunoelectrophoresis procedure was used for this purpose. The individual phenotypes consisted of seven MM, three ZZ, five MZ, one SZ and three MS. AAT levels were obtained by radial immunodiffusion, trypsininhibitory capacity measurements and from cellulose acetate electrophoresis. Good correlation was found between the phenotype and the three biochemical methods for demonstrating the normal phenotype MM and the severe deficiency state ZZ. Some discrepancies were observed for the heterozygotes. It is concluded that these various assays are inaccurate for the interpretation of intermediate AAT concentrations. Since Pi typing is not suitable for use on a routine basis, it is suggested that this analysis should be performed by a specialized laboratory if intermediate deficiency is suspected.     

The changes of technetium-99m-labeled annexin-V in delayed anesthetic preconditioning during myocardial ischemia/reperfusion

This study was designed to use real-time imaging to test the hypothesis that delayed cardiac protection induced by volatile anesthetics inhibits apoptosis. Rats were divided into two groups. One group was exposed to 120 min of 33 % O₂ [control group (CON group)] and the other group was exposed to 2.5 % sevoflurane in 33 % O₂ for 120 min [sevoflurane group (SEVO group)]. Both groups were allowed to return to their cages for 24 h. After 24 h recovery, all rats underwent 30 min myocardial ischemia by occluding coronary artery followed by 2 h of reperfusion. After reperfusion, technetium-99m-labeled annexin-V was administered intravenously to identify apoptosis. Left ventricular samples were obtained to measure infarct size and radionuclide imaging and caspase-3. Radionuclide imaging indicated that apoptosis was reduced in SEVO group (0.78 % ± 0.82) when compared with the CON group (1.15 % ± 0.61), and the infarct size was also decreased in the SEVO group (40 % ± 7). The transferase dUTP nick end labeling (TUNEL)-positive cardiomyocytes in the SEVO group (16 % ± 6) were significantly decreased in the peri-infarct zone when compared with the CON group (28 % ± 4). After reperfusion, caspase-3 expression was significantly blunted in the SEVO group than in CON group (50 % ± 11 vs. 68 % ± 10, p < 0.05). This study used technetium-99m-labeled annexin-V of real-time imaging to detect cardiomyocyte apoptosis and the results were confirmed by the TUNEL assay and caspase-3 expression. We concluded that delayed volatile anesthetic preconditioning (APC) protects against I/R in vivo. The method of technetium-99m-labeled annexin-V of real-time imaging can be used to detect cardiomyocyte apoptosis in delayed APC during ischemia/reperfusion.     

The role of sialic acid and galactose residues in determining the survival of human plasma alpha-antitrypsin in the blood circulation.

Human plasma α₁-antitrypsin (α₁-AT) is a glycoprotein known to contain terminal sialic acids (N-acetylneuraminic acids) in the carbohydrate units. These residues were converted to a radioactive seven-carbon analog (NANA-7) by sequential periodate oxidiation and tritiated borohydride reduction. Modified α₁-AT prepartions, namely, (a) periodate oxidized α₁-AT, (b) asialo α₁-AT (neuraminidase-treated α₁-AT), (c) (NANA 7)-α₁-AT (periodate-oxidized, -tritiated, borohydride-reduced α₁-AT), (d) (NANA-7)-α₁ AT (partially desialylated by neuraminidase), and (e) partially desialylated (NANA 7)-α₁-AT oxidized with galactose oxidase, all retained the following properties attributable to native α₁-AT: trypsin-inhibitory and chymotrypsin-inhibitory activities, immunological reactivity to antibody against native α₁-AT, and the ability to bind to concanavalin A-Sepharose 4-B columns. After intravenous injection of intact (NANA-7)-α₁-AT into rats, the labeled material had a circulating half-life of 18 h. When (NANA-7)-α₁-AT was partially desialylated (four residues of NANA-7 out of a total of six were removed, thus exposing an equivalent number of galactose residues at the terminal positions) by neuraminidase, injection into rats of this material resulted in a rapid and almost complete disappearance of the label from the circulation in 30 min. There was a concomitant accumulation of radioactivity in the liver. The rate of this rapid transfer depended on the presence of intact galactose residues as the terminal, nonreducing sugar in the carbohydrate units. Galactose oxidase treatment of the partially desialylated (NANA-7)-α₂-AT, which presumably oxidized the primary alcohol of galactose at C-6 to an aldehyde group, caused a reversion of its survival time in the circulation to that of the intact (NANA-7)-α₁-AT.     

Ulva lactuca and U. flexuosa (Chlorophyta,Ulvophyceae) cultivation in Brazilian tropical waters: recruitment,growth, and ulvan yield

Ulva spp. are used in a wide range of commercial applications, including bioremediation, food, bioenergy, pharmaceuticals, and agriculture. The sulfated polysaccharide ulvan obtained from Ulva spp. is of interest for triggering plant defenses against disease. However, the cultivation of Ulva spp. is still in its infancy. This study verified the feasibility of cultivating Ulva lactuca and Ulva flexuosa at two sites on the tropical Brazilian coast. We investigated the following: (a) methods to induce sporulation, (b) comparison of seeding ropes inoculated in vitro versus seeding at sea over 40 days, (c) production and harvest cycles at 15 and 30 days, (d) growth productivity of U. flexuosa at sea and in outdoor tanks, and (e) comparison of ulvan yields from biomass cultivated in tanks and the sea. High nutrient treatment was the most efficient method to induce sporulation (7,540?±?3,133 spores m^?1). Sea-based cultivation of U. flexuosa was only successful at one site. Seeding of ropes in vitro was more efficient than seeding at sea (0.31?±?0.20 g m^?2 day^?1), and 15-day harvest cycles were most efficient (20.1?±?1.8 % day^?1; 0.46?±?0.11 g m^?2 day^?1). Despite differences in plant growth in tanks (27.9?±?4.4 % day^?1) and at sea (20.1?±?1.8 % day^?1), the dry biomass and ulvan yields (17.7?±?5.0 %) did not differ between these systems. Cultivation of U. flexuosa was feasible at sea using in vitro seeding with a production cycle of 15 days in Brazilian tropical waters and tanks with high irradiance and enriched seawater.     

Protein profile and alpha-lactalbumin concentration in the milk of standard and transgenic goats expressing recombinant human butyrylcholinesterase

The expression of recombinant proteins of pharmaceutical interest in the milk of transgenic farm animals can result in phenotypes exhibiting compromised lactation performance, as a result of the extraordinary demand placed on the mammary gland. In this study, we investigated differences in the protein composition of milk from control and transgenic goats expressing recombinant human butyrylcholinesterase. In Experiment 1, the milk was characterized by gel electrophoresis and liquid chromatography/mass spectrometry in order to identify protein bands that were uniquely visible in the transgenic milk and/or at differing band densities compared with controls. Differences in protein content were additionally evaluated by computer assisted band densitometry. Proteins identified in the transgenic milk only included serum proteins (i.e. complement component 3b, ceruloplasmin), a cytoskeleton protein (i.e. actin) and a stress-induced protein (94 kDA glucose-regulated protein). Proteins exhibiting evident differences in band density between the transgenic and control groups included immunoglobulins, serum albumin, β-lactoglobulin and α-lactalbumin. These results were found to be indicative of compromised epithelial tight junctions, premature mammary cell death, and protein synthesis stress resulting from transgene expression. In Experiment 2, the concentration of α-lactalbumin was determined using the IDRing^® assay and was found to be significantly reduced on day 1 of lactation in transgenic goats (4.33 ± 0.97 vs. 2.24 ± 0.25 mg/ml, P < 0.01), but was not different from non-transgenic controls by day 30 (0.99 ± 0.46 vs. 0.90 ± 0.11 mg/ml, P > 0.05). We concluded that a decreased/delayed expression of the α-lactalbumin gene may be the cause for the delayed start of milk production observed in this herd of transgenic goats.     

Bacillus cihuensis sp. nov., isolated from rhizosphere soil of a plant in the Cihu area of Taiwan

A Gram-positive, moderately halotolerant, rod-shaped, spore forming bacterium, designated strain FJAT-14515^T was isolated from a soil sample in Cihu area, Taoyuan County, Taiwan. The strain grew at 10–35 °C (optimum at 30 °C), pH 5.7–9.0 (optimum at pH 7.0) and at salinities of 0–5 % (w/v) NaCl (optimum at 1 % w/v). The diagnostic diamino acid of the peptidoglycan of the isolated strain was meso-diaminopimelic acid and major respiratory isoprenoid quinone was MK-7. Major cellular fatty acids were anteiso-C_15:0 (40.6 %), iso-C_15:0 (20.7 %) and the DNA G+C content of strain FJAT-14515^T was 37.1 mol %. A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain FJAT-14515^T belongs to the genus Bacillus, and was most closely related to the reference strains of Bacillus muralis DSM 16288^T (97.6 %) and Bacillus simplex DSM 1321^T (97.5 %). Levels of DNA–DNA relatedness between strain FJAT-14515^T and the reference strains of B. muralis DSM 16288^T and B. simplex DSM 1321^T were 27.9 % ± 3.32 and 44.1 % ± 0.57, respectively. Therefore, on the basis of phenotypic, chemotaxonomic and genotypic properties, strain FJAT-14515^T represents a novel species of the genus Bacillus, for which the name Bacillus cihuensis sp. nov. is proposed. The type strain is FJAT-14515^T (=DSM 25969^T = CGMCC 1.12697^T).     

Optimising microencapsulated formulation stability of Bacillus thuringiensis subsp. kurstaki (Bt-KD2) against ultraviolet condition using response surface methodology

In this paper, microencapsulated formulation of Bacillus thuringiensis subsp. kurstaki were prepared by the emulsion gelling method. Stability of formulation under ultraviolet (UV) radiation by spore viability and mortality teston Ephestia kuehniella Zeller larvae are investigated. Response surface methodology was used for optimising the formulations. Calcium Chloride (0.1 and 0.3 M) and sodium alginate (2 and 3% w/w) were used in the formulations, and stirring speed was selected between 1500 and 2000 rpm. Morphology of the microcapsule was evaluated by light microscopy. The optimal values for the variables were: sodium alginate = 2.15%, CaCl₂ = 0.24 M, speed = 1606 rpm and spore viability, mortality and diameter of 68% ± 1.73, 85% ± 1.5 and 9μ ± 1.3, respectively; while the predicted values by the software were 70% for spore viability, 87% for mortality and 8.3 μ for diameter.     

Effect of geographical location and stochastic weather variation on life cycle assessment of biodiesel production from camelina in the northwestern USA

Purpose

The effect of regional factors on life cycle assessment (LCA) of camelina seed production and camelina methyl ester production was assessed in this study. While general conclusions from LCA studies point to lower environmental impacts of biofuels, it has been shown in many studies that the environmental impacts are dependent on location, production practices, and even local weather variations.

Methods

A cradle-to-farm gate and well-to-pump approaches were used to conduct the LCA. To demonstrate the impact of agro-climatic and management factors (weather condition, soil characteristics, and management practices) on the overall emissions for four different regions including Corvallis, OR, Pendleton, OR, Pullman, WA, and Sheridan, WY, field emissions were simulated using the DeNitrification-DeComposition (DNDC) model. openLCA v.1.4.2 software was used to quantify the environmental impacts of camelina seed and camelina methyl ester production.

Results and discussion

The results showed that greenhouse gas (GHG) emissions during camelina production in different regions vary between 49.39 and 472.51 kg CO₂-eq./ha due to differences in agro-climatic and weather variations. The GHG emissions for 1 kg of camelina produced in Corvallis, Pendleton, Pullman, and Sheridan were 0.76 ± 11, 0.55 ± 10, 0.47 ± 18, and 1.26 ± 6 % kg CO₂-eq., respectively. The GHG emissions for 1000 MJ of camelina biodiesel using camelina produced in Corvallis, Pendleton, Pullman, and Sheridan were 53.60 ± 5, 48.87 ± 5, 44.33 ± 7, and 78.88 ± 4 % kg CO₂-eq., respectively. Other impact categories such as acidification and ecotoxicity for 1000 MJ of camelina biodiesel varied across the regions by 43 and 103 %, respectively.

Conclusions

It can be concluded that process-based crop models such as DNDC in conjunction with Monte Carlo analysis are helpful tools to quantitatively estimate the influence of regional factors on field emissions which consequently can provide information about the expected variability in LCA results.

     

Conservation,ex vitro direct regeneration,and genetic uniformity assessment of alginate-encapsulated nodal cuttings of <Emphasis Type="Italic">Sphagneticola calendulacea</Emphasis> (L.) Pruski

A well-organized procedure was established for the conservation and distribution of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] for the first time, using alginate-encapsulated nodal segments (NSs) as synthetic seeds. The ideal beads were obtained through a combination of 2.5% sodium alginate and 75 mM calcium chloride with 84.40 ± 2.20% rate of shoot emergence. The maximum regeneration (88.84 ± 2.24%) from synthetic seeds was achieved on liquid 1/2Murashige and Skoog (MS) medium in comparison to its other formulations. Furthermore, superior frequency (91.09 ± 2.24%) of complete plantlet (having both shoots and roots) formation was achieved when synthetic seeds were cultured on liquid 1/2MS (1.5% sucrose) fortified with 1.0 mg L^?1 N₆-benzyladenine plus 0.25 mg L^?1 α-naphthalene acetic acid. Synthetic seeds could be effectively stored at low temperature (8 °C) up to 90 days with a survival rate of 52.38 ± 3.06%, whereas higher temperature (25 °C) did not support regeneration after 75 days of storage. The plantlets were successfully acclimatized to natural conditions with ~ 89% survival frequency. To by-pass the time-consuming in vitro culture step after encapsulation, synthetic seeds were directly regrown into complete plantlets ex vitro on sand, soil, and vermicompost (1:1:1; w/w). Regeneration rate of 42.22 ± 2.22% was attained when NSs were pretreated on 1/2MS medium containing 4.0 mg L^?1 indole-3-acetic acid for 24 h in dark, prior to encapsulation. The random amplified polymorphic DNA and intersimple sequence repeat fingerprinting profiles demonstrated genetic uniformity amongst the regenerated plantlets, in vitro mother plant, as well as in vivo wild plant.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

Targeting the click beetle Agriotes obscurus with entomopathogens as a concept for wireworm biocontrol

Applications of Metarhizium brunneum Petch (Ascomycota: Hypocreales: Clavicipitaceae) isolate LRC112 conidia caused high mortality to Agriotes obscurus L. (Coleoptera: Elateridae) click beetles in field trials. Banded conidiated rice (4.4 × 10¹⁴ conidia ha^?1) and conidia dust (5.0 × 10¹³ conidia ha^?1) resulted in 93.3 % ± 7.3 and 91.3 % ± 3.0 mortality after 18 days, while aqueous conidia suspension spray (5.0 × 10¹³ conidia ha^?1) with and without 80 g ha^?1 spinosad resulted in 68.2 % ± 17.7 and 52.6 % ± 17.4 mortality. Differences in results between 2012 and 2013 were attributed to rainfall, with pronounced effects in 2012 (rain beginning 35 h post treatment) and minimal effects in 2013 (rain beginning at 4 h). In another field experiment, beetles dosed with 1.49 × 10⁷ ± 5.08 × 10⁶ conidia per beetle retained 4.6 % of conidia after seven days while conidia viability on beetle bodies remained unchanged. The results inferred opportunities for controlling click beetles using fungal entomopathogens, and for horizontal transmission of the inoculum.     

Embryogenesis,plant regeneration and cardiac glycoside determination in Digitalis ferruginea subsp. ferruginea L

The present study reports, for the first time, an efficient in vitro plant regeneration protocol for Digitalis ferruginea subsp. ferruginea L. (rusty foxglove). We have used different concentrations of gibberellic acid (GA₃) on Murashige and Skoog (MS) medium to assess the germination frequency of seeds. High frequency of germination was achieved on MS medium with 1.0 mg l^?1 GA₃. 6-Benzylaminopurine (BAP) combined with α-naphtaleneacetic acid (NAA) or 2, 4-dichlorophenoxy acetic acid (2, 4-D) in the induction MS medium induced both somatic embryogensis and shoot organogenesis. The highest percentage of callus growth (85 %) was obtained when hypocotyl explants were cultured on MS medium containing 0.5 mg l^?1 2, 4-D plus 1.0 mg l^?1 BAP. The maximum mean number of somatic embryos (7.3 ± 1.3 embryos) or shoots (12.0 ± 1.1 shoots) per callus was obtained when medium contained 0.25 mg l^?1 NAA plus 1.0 mg l^?1 BAP or 0.5 mg l^?1 NAA plus 2.0 mg l^?1 BAP. The regenerated shoots easily rooted on MS medium. Higher amounts of lanatoside C [13.2 ± 0.5 mg 100 g^?1 dry weight (dw)] and digoxin (2.93 ± 0.31 mg 100 g^?1 dw) accumulation were obtained when shoots were obtained by indirect regeneration. We also investigated derivatives of cardenolides, i.e., digitoxigenin (730 ± 180 mg 100 g^?1 dw), gitoxigenin (50 ± 20 mg 100 g^?1 dw) and digoxigenin (490 ± 170 mg 100 g^?1 dw) from natural samples.     

The purification of human serum 1 -antitrypsin by affinity chromatography on concanavalin A

α₁-Antitrypsin (α₁-AT) has been isolated from human serum by a two-step procedure which involves chromatography on DEAE-Sephadex followed by affinity chromatography on insolubilized concanavalin A. This protein appeared to be homogeneous when examined by electrophoresis on cellulose acetate and double immunodiffusion; minor contaminants, however, were detected by gel electrophoresis and immunoelectrophoresis. This procedure is readily adaptable to the large-scale purification of α₁-AT and should facilitate further studies on the physicochemical and biological properties of α₁-AT and its genetic variants.     

Anti-diabetic xanthones from the bark of Garcinia xanthochymus

An ethyl acetate extract the bark of Garcinia xanthochymus exhibited strong inhibition towards α-glucosidase and PTP1B with IC₅₀ values of 0.3 ± 0.1 μg/mL and 2.3 ± 0.4 μg/mL, respectively. Chemical constituents of the extract were therefore examined, and two new compounds, xanthochymusxanthones A (1) and B (2), along with ten known xanthones (3–12), were isolated. Their structures were determined using spectroscopic methods, mainly 1D and 2D NMR. Inhibitory activity of the isolated compounds was then tested, and subelliptenone F (12) showed significant effect towards α-glucosidase with IC₅₀ value of 4.1 ± 0.3 μM (compared with acarbose, IC₅₀ = 900.0 ± 3.0 μM) whilst xanthochymusxanthone B (2) exhibited remarkable activity towards PTP1B with IC₅₀ value of 8.0 ± 0.6 μM (compared with RK682, IC₅₀ = 4.4 ± 0.3 μM).     

Association between the rs6950982 polymorphism near the SERPINE1 gene and blood pressure and lipid parameters in a high-cardiovascular-risk population: interaction with Mediterranean diet

The SERPINE1 (serpin peptidase inhibitor, clade E, member 1) gene, better known by its previous symbol PAI-1 (plasminogen activator inhibitor 1), has been associated with cardiovascular phenotypes with differing results. Our aim was to examine the association between the rs6950982 (G > A) near the SERPINE1 gene, blood pressure (BP) and plasma lipid concentrations as well as the modulation of the polymorphism effects by adherence to Mediterranean diet (AMD). We studied 945 high-cardiovascular-risk subjects. Biochemical, clinical, dietary and genetic data (rs6950982) were obtained. We also determined the common rs1799768 (4G/5G), for checking independent effects. AMD was measured by a validated questionnaire, and four groups were considered. rs6950982 (A > G) and rs1799768 (4G/5G) were only in moderate–low linkage disequilibrium (D′ = 0.719; r ² = 0.167). The most significant associations we obtained were with rs6950982 (A > G). In males, the G allele was nominally associated with higher diastolic BP (AA: 81.5 ± 10.9, AG: 82.1 ± 11.4, GG: 85.7 ± 10.5 mmHg; P _additive = 0.030) and systolic BP (AA + AG: 141.4 ± 6.9 mmHg vs. GG: 149.8 ± 8.0 mmHg; P _recessive = 0.036). In the whole population, the rs6950982 was also associated with plasma lipids. Subject with the G allele presented higher total cholesterol (P _additive = 0.016, P _recessive = 0.011), low-density lipoprotein cholesterol (P _additive = 0.032, P _recessive = 0.031) and triglycerides (P _additive = 0.040, P _recessive = 0.029). AMD modulated the effect of rs6950982 on triglyceride concentrations (P for interaction = 0.036). Greater AMD reduced the higher triglyceride concentrations in GG subjects. No significant interactions were found for the other parameters. The rs6950982 was associated with higher BP in men and higher triglycerides in the whole population, this association being modulated by AMD.     

N-(o-carboxybenzyl) chitosans: Novel chelating polyampholytes

The imine formed by chitosan with phthalaldehydic acid was reduced with sodium cyanoborohydride and the resulting N-(o-carboxybenzyl) chitosan (NCBC) was insolubilised with ethanol and acetone and obtained as a white, free-flowing powder, soluble in both acidic and alkaline solutions. A sample of NCBC with the following degrees of substitution: acetamido 42%±4%, N-(o-carboxybenzyl) amine 43%±3%, free amine 15%±1% and containing 16%±1% moisture, was characterised by IR and UV-Vis. spectrometry, titration and viscometry. The isoelectric point was 6·8; the pK_a values were 5·7 and 8·0. NCBC could be determined by UV-Vis. spectrophotometry in aqueous solutions at 274 nm; maximum viscosity of the solutions was observed at pH 4. Upon addition of NCBC to transition metal ion solutions (0·1–0·5 mm) chelation and insolubilisation took place immediately. The dependence of the collection percentage on pH, NCBC and metal ion concentrations was studied for nine metal ions.