Analysis of the Alpha-1-Antitrypsin Deficient Alleles M3S, MZ, and ZZ by Biochemical and Molecular Methods: A Family Study

Deficiency of alpha-1-antitrypsin (α₁-AT, a major protease inhibitor controlling tissue degradation) is a genetic disorder transmitted in a codominant autosomal form. It has more than 100 genetically determined variants. This study attempted to determine the degree of association between serum α₁-AT levels and phenotypes and to provide a strategy for reliable laboratory evaluation of deficiencies. The study group consisted of a 38-year-old male proband with clinical features of emphysema, his first-degree relatives, and healthy controls. Family history revealed a four-generation pedigree. Genomic DNA was isolated from peripheral blood leukocytes. Alpha-1-AT levels were determined from human serum by immunonephelometry. Phenotypes were determined by isoelectric focusing of blood samples. DNA sequences of coding exons were analyzed by the amplification DNA technique and direct sequencing. Inheritance and plasma levels of the ZZ, MM, M3S, and MZ phenotypes were confirmed by the family study. In the family members with deficiencies, plasma concentrations were 22.55% ± 5.15 (ZZ), 84.18% ± 5.18 (M3S), and 61.06% ± 7.15 (MZ) of the normal MM level. We found a close association between α₁-AT level and genotype. A combination of genotyping, quantification, and phenotyping is the optimal strategy for the laboratory evaluation of α₁-AT deficiency.     

Isolation and Characterization of Alpha-1-Antitrrpsin from the Cohn Fraction Iv-1 of Human Plasma

Cohn Fraction IV-I from pooled human plasma was used as a starting material in the large-scale purification of alpha-1-antitrypsin (alpha-1-AT). Following ion-exchange, blospecific affinity and gel exclusion chromatographic procedures, material of high biological activity was obtained in 307percnt; overall yield. Homogeneity was demonstrated by acrylamide gel electrophoresis, immunoelectrophoresis, ultracentrifugation, gel filtration and end-group determination. The present preparation should be applicable to large scale industrial processing of alpha-1-AT with the potential for use in protein replacement therapy.     

Human alpha-chain globin messenger: prediction of a nucleotide sequence

The presence in human serum of inhibitory activity to rat liver insulin specific protease has been detected in an alpha₁ globulin preparation (Cohn Fraction IV₁). Separation into four components and partial purification (40 to 107 fold) has been achieved by heat denaturation of non-active protein, Sephadex G-100 gel filtration and ion-exchange chromatography upon QAE Sephadex. Each of the inhibitors was found to be competitive in nature. The molecular weight of the inhibitors is between 4,000–7,000 and the activity is destroyed for the most part by chymotrypsin.     

Developing procedures for the large-scale purification of human serum butyrylcholinesterase

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2× LD₅₀ of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05–0.2 M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.     

Expression,Purification, and Characterization of Recombinant Human α<Subscript>1</Subscript>-Antitrypsin Produced Using Silkworm–Baculovirus Expression System

Human α₁-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α₁-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~?15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.     

Preparation of concanavalin A free purified alpha-1-antitrypsin.

A simple technique is described to remove traces of concanavalin A (Con A) from human α-1-antitrypsin (α-1-AT) purified on commercially available Con A-Sepharose. The α-1-AT was fractionated from serum by ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, Con A-Sepharose, and activated thiol-Sepharose at 4°C with solution pH ranges of 7.4–7.6 in all steps. Contaminating Con A was easily removed by binding α-1-AT through the reactive sulfhydryl group to the activated thiol-Sepharose gel and washing away the contaminating Con A with a solution of methyl-α-d-glucopyranoside before final elution of bound α-1-AT. This simple procedure yields purified α-1-AT free of traces of Con A. The α-1-AT was obtained in overall yields of 40–48% from serum with an average molecular weight of 53,500 ± 3000 determined on 15% disc polyacrylamide gels containing sodium dodecyl sulfate (SDS). The isolated α-1-AT exhibited unaltered Pi M phenotype compared to serum α-1-AT but contained traces of several other serum proteins.     

Comparative Study of Antibiofilm Activity of Copper Oxide and Iron Oxide Nanoparticles Against Multidrug Resistant Biofilm Forming Uropathogens

The effectiveness of the metal oxide nanoparticles viz. CuO and Fe₂O₃ as antibacterial agents against multidrug resistant biofilm forming bacteria was evaluated. CuO nanoparticles were also experimented for antibiofilm and time kill assay. The CuO displayed maximum antibacterial activity with zone of inhibition of (22 ± 1) mm against methicillin resistant Staphylococcus aureus (MRSA) followed by Escherichia coli (18 ± 1) mm. The Fe₂O₃ showed the zone of inhibition against MRSA of (14 ± 1) mm followed by E. coli (12 ± 1) mm. CuO proved to be more toxic than Fe₂O₃ nanoparticles showing significantly high antibacterial activity and found to possess dose dependent antibiofilm properties.     

The isolation of alpha-1-protease inhibitor by a unique procedure designed for industrial application

Individuals who are congenitally deficient in the human plasma protein α₁-protease inhibitor (α₁PI, which is also called α₁-antitrypsin) usually develop chronic obstructive lung disease as a consequence of improperly regulated granulocyte elastase. In this report, a unique, facile one- or two-step method is presented for the large-scale isolation of α₁PI for potential therapeutic use. The method takes advantage of the unusual disulfide bond in α₁PI, which consists of a single cysteine residue in the polypeptide chain bound to a free pendant cysteine. In contrast to other circulating plasma proteins, the disulfide bridge in α₁PI does not add to its structural stability. Therefore, if an α₁PI-containing solution of plasma proteins is precipitated out in the presence of reductant, much more extensive separation of contaminating proteins will be achieved than in the absence of such reductant. We have used Cohn Fraction IV-1, a relatively unused side product in albumin and gamma globulin production, as our starting material. After activation of the α₁PI in basic media, partial purification is achieved with successive additions of Aerosil (a fumed silica), dithiothreitol, and ammonium sulfate. From 90 to 95% of the contaminating proteins are precipitated by this single procedure, resulting in a product that is ～70% pure. DEAE-cellulose chromatography can be used as an additional purification step, and this results in a product that is nearly homogenous. Overall yield is ～45%. The method is simple, inexpensive, and reproducible and is directly applicable to large-scale industrial processing.     

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.     

Assessment of the effect of candidate anti-inflammatory treatments on the interaction between meningococci and inflammatory cellsin vitro in a whole blood model

A wide range of immunomodulating agents are now available which may be of benefit in reducing inflammatory cell activation in meningococcal sepsis. In order to facilitate selection of candidate anti-inflammatory agents for clinical trials, we have used an in vitro whole blood model to evaluate the effects on meningococcal induced neutrophil and monocyte activation, of dexamethasone, prostacyclin, pentoxifylline and a human IgM anti-lipid A monoclonal antibody (HA-1A). Known concentrations of heat and penicillin killed meningococci were added to whole blood and the time course of cellular activation was determined. Using elastase-α ₁-antitrypsin (elastase-α ₁-AT) and TNFα production as markers of neutrophil and monocyte activation respectively, plasma levels of elastase-α ₁-AT and TNFα were found to increase in a dose-dependant manner. Elastase-α ₁-AT was detected early, with most release occurring between 15–30 min whereas TNFα was detected later, between 120–180 min. Dexamethasone, prostacyclin and pentoxifylline caused a dose dependant inhibition of TNFα release but had no effect on elastase release. HA-1A had no effect on either TNFα or elastase release. This model may be useful in determining the sequence of inflammatory cell activation and in selecting candidate anti-inflammatory agents for evaluation in clinical trials.     

Catabolism of desialylated human plasma alpha1-antitrypsin and its trypsin complex in the rat.

Human plasma α₁-antitrypsin (α₁-AT) was labeled with either ³H [³H-labeled NANA (N-acetyl-neuraminic acid)-7] residues in the carbohydrate moiety) or ¹⁴C (?-N-methyl-[¹⁴C]lysyl residues in the protein backbone) or with both isotopes in the corresponding residues. After intravenous injection into rats of the doubly labeled partially (50%) desialylated (methyl-[¹⁴C]·[³H]NANA-7)-α₁-AT, the rates of disappearance from the plasma of both isotopes were very rapid and yielded essentially the same circulatory half-life of 5 min. The rapid disappearance of the doubly labeled glycoprotein from the plasma was accompanied by concomitant fast and equal accumulations of ¹⁴C and ³H in the liver which constituted about 70% of the administered dose 15 min after the injection. The asialo (methyl-[¹⁴C])-α₁-AT·trypsin complex or methyl-[¹⁴C]-α₁-AT·trypsin complex had a plasma survival time (45 min) that was intermediate between methyl-[¹⁴C]-α₁-AT and its desialylated derivative. These complexes were removed from the plasma by the liver (45% of the injected dose 60 min after injection), although not as rapidly as asialo (methyl-[¹⁴C])-α₁-AT. Blockade of the reticuloendothelial (Kupffer) cells by simultaneous injection of heat-denatured albumin inhibited the liver uptake of the inhibitor·trypsin complexes but not that of the uncomplexed asialo α₁-AT. Radioactive ?-N,N-dimethyllysine, ?-N-monomethyllysine, methionine, choline, and betaine were separated and identified from the trichloro-acetic acid-soluble fraction of rat livers 25 min after injection of asialo (methyl-[¹⁴C])-α₁-AT.     

Radioactive labeling of alpha1-antitrypsin, trypsin, and chymotrypsin by reductive methylation: properties of the labeled derivatives.

Human plasma α₁-antitrypsin (α₁-AT), bovine trypsin, and α-chymotrypsin were labeled with either ¹⁴C or ³H by reductive methylation. The labeled inhibitor retained the capacity to inactivate and to form 1:1 molar complexes with either the unlabeled or labeled trypsin and α-chymotrypsin. After intravenous injection of reductively methylated α₁-AT into rats, the labeled glycoprotein showed a circulating half-life of 12 h. When the N-acetylneuraminic acid residues were removed from the labeled α₁-AT by neuraminidase in vitro, injection into rats of this product resulted in a rapid (half-life of about 5 min) and almost complete disappearance of the label from the circulation in 30 min. There was a concomitant accumulation of radioactivity in the liver of over 75% of the injected dose. The reductively methylated radioactively labeled trypsin and chymotrypsin experienced no loss of enzymatic activities. They showed the ability to form complexes in vivo with the two major plasma inhibitors, namely, α₁-AT and α₂-macroglobulin. High-voltage paper electrophoretic separation of acid hydrolysates of the labeled proteins revealed that ?-N-monomethyllysine and ?N,N-dimethyllysine are the only residues found to be radioactive.     

One-step purification of rat plasma α1-acid glycoprotein by antibody affinity chromatography: Application to normal and inflamed rat sera

Most purification procedures used previously to isolate α₁-acid glycoprotein (AGP) from plasma can lead to some alterations in its carbohydrate moiety. An immunoaffinity chromatographic method is proposed for purifying in one step rat plasma AGP without any detectable modification of its glycan moiety. Crossed immunoaffinoelectrophoresis with concanavalin A before and after purification showed identical patterns, suggesting no glycan selection during the purification. In the same way no desialylation occurred during the purification step. This immunoaffinity chromatographic procedure provided evidence of a decreased level of fucosyl residues in turpentine oil rat plasma AGP compared with normal rat plasma AGP.     

Affinity chromatography in the separation of human alpha 1-antitrypsin (alpha 1-AT) and antithrombin-III (AT-III).

The two antiproteases alpha 1-antitrypsin (alpha 1-AT) and antithrombin-III (AT-III) have been purified simultaneously from human plasma. Purification procedure consisted of gel filtration on Sephadex G-200 after initial processing of plasma, followed by ion exchange chromatography on DEAE-Sephadex A50 and DEAE-Cellulose, at a pH of 9.0 and pH 8.3 respectively. The two proteins could not be separated by any of these procedures including a lower pH (7.4) in ion exchange chromatography. Affinity chromatography on heparin-Sepharose separated the proteins since alpha 1-AT did not bind to the matrix. Alpha 1-AT unbound to the heparin-Sepharose was subsequently purified through con A-Sepharose affinity column. The final yield of both the proteins was about 20%. The molecular weight estimated on SDS electrophoresis for AT-III and alpha 1-AT was 63,000 and 50,000, respectively.     

Chemical modifications of lysyl and arginyl residues of human plasma α1-antitrypsin

Reactions of human plasma α₁-antitrypsin (α₁-AT) with reagents known to modify the lysyl residues [citraconic anhydride, acetic anhydride, 2,4,6-trinitrobenzenesulfonic acid (TNBS)] and arginyl residues [1,2-cyclohexanedione (CHD) and phenylglyoxal (PGO)] in proteins have been studied. Native and modified human plasma α₁-AT preparations were tested for their inhibitory activities against trypsin and α-chymotrypsin. TNBS was utilized to modify and quantitate free amino groups (?-NH₂ groups of lysine residues) in human plasma α₁-AT. The number of lysine residues determined by the TNBS spectrophotometric procedure agreed well with that found by amino acid analyses. Both the trypsin-inhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed by modification with TNBS. CHD was employed to modify the arginyl residues of α₁-AT. Neither the trypsin-inhibitory nor the chymotrypsin-inhibitory activity of α₁-AT was affected by modification of its arginyl residues. Amino acid analyses of the CHD-treated α₁AT revealed that only the arginine residues were modified. PGO was also utilized for the modification of the arginyl residues in α₁-AT. Both the trypsininhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed after modification. However, amino acid analyses showed that not only the arginyl, but also the lysyl residues of the PGO-treated inhibitor were modified. The side reaction of PGO with the lysyl residues could explain the loss of inhibitory activities. Reaction of a α₁-AT with citraconic anhydride resulted in an extensive modification of the amino groups accompanied by a 100% loss in inhibitory activity against both trypsin and α-chymotrypsin. Comparable results were observed when acetic anhydride was utilized as the acylating reagent. With the exception of the citraconylated α₁AT, all of the other chemically modified α₁-AT derivatives studied presently retained their immunological reactivities against antisera to native α₁-AT. Regeneration of about 60% of the PGO-blocked arginyl residues in α₁-AT did not lead to any recovery of the proteinase inhibitory activities. Full recovery of trypsin-inhibitory and immunological activities were achieved when about 50% of the citraconylated amino groups were deblocked. The CHD-treated α₁-AT still retained the capacity to form complexes with both trypsin and chymotrypsin. On the other hand, the other chemically modified α₁-AT derivatives have completely lost the ability to form complexes with the enzymes. Recovery of the ability to form complexes with the enzymes was, however, recovered when about 50% of the citraconylyl groups was removed from the α₁-AT molecule. Based on these modification studies, it is concluded that α₁-AT is a lysyl inhibitor type (i.e., the reactive site is Lys-X bond) and that the interaction of α₁-AT with trypsin or chymotrypsin very likely involves or requires the same site as in the case of the soybean trypsin inhibitor (Kunitz).     

Epidemiology of alpha1-antitrypsin deficiency in the Netherlands

Summary During a 3-year period, newborns in the eastern part of the Netherlands were investigated for alpha₁-antitrypsin deficiency. Electroimmunoassay was used for screening, followed by Pi typing in suspected cases. In all 95 033 newborns were screened, and a mean frequency of deficiency (phenotypes PiZ, PiSZ, and PiS) of 8.00 in 10 000 was found.The distribution of deficient Pi types over the area was remarkably uneven, Pi type Z being more predominant north and Pi type S south of the Rhine. Cluster areas of alpha₁-antitrypsin deficiency, with frequencies of up to 59.6 in 10 000 liver births, occurred mainly in small rural communities. In urbanized areas the frequency of deficiency was lower than the mean.     

Aflatoxins,discolouration and insect damage in dried cowpea and pigeon pea in Malawi and the effectiveness of flotation/washing operation in eliminating the aflatoxins

Aflatoxin contamination and biodeterioration were examined in 302 samples of dry cowpeas and pigeon peas that were randomly purchased from 9 districts of the Southern Region of Malawi during July and November 2015. Further, the impact of flotation/washing on aflatoxin levels on the pulses was elucidated. Aflatoxin analyses involved immunoaffinity column (IAC) clean-up and HPLC quantification with fluorescence detection (FLD) while legume biodeterioration assessments were done by visual inspection. Aflatoxins were frequently detected in cowpea (24%, max., 66 μg/kg) and pigeon pea (22%, max., 80 μg/kg) samples that were collected in the month of July. Lower aflatoxin incidence of 15% in cowpeas (max., 470 μg/kg) and 14% in pigeon peas (max., 377 μg/kg) was recorded in the November collection. Overall, aflatoxin levels were significantly higher in the pulses that were collected in November. However, there were no significant differences in the total aflatoxin (aflatoxin B₁ (AFB₁) + AFB₂ + AFG₁ + AFG₂) levels between the two types of pulses. Remarkably, in 76.2% of the aflatoxin positive cowpea and in 41.7% of the aflatoxin positive pigeon pea samples, aflatoxin G₁ concentration exceeded aflatoxin B_1. Insect damage percentage averaged at 18.1 ± 18.2% (mean ± SD) in the cowpeas and 16.1 ± 19.4% in pigeon peas. Mean discolouration percentage (number of pulses) of the cowpeas and pigeon peas was found to be at 6.7 ± 4.9 and 8.7 ± 6.2%, respectively. Washing and discarding the buoyant fraction was highly efficient in reducing aflatoxin levels; only 5.2 ± 11.1% of the initial aflatoxin level was found in the cleaned samples. In conclusion, cowpeas and pigeon peas sold on the local market in Malawi may constitute a hazard especially if floatation/washing step is skipped.     

Evaluation of growth and phycobiliprotein composition of cyanobacteria isolates cultivated in different nitrogen sources

Phycobiliproteins, light-harvesting pigments found in cyanobacteria and in some eukaryotic algae, have numerous commercial applications in food, cosmetic, and pharmaceutical industries. Colorant production from cyanobacteria offers advantages over their production from higher plants, as cyanobacteria have fast growth rate and high photosynthetic efficiency and require less space. In this study, three cyanobacteria strains were studied for phycobiliprotein production and the influence of sodium nitrate, potassium nitrate and ammonium chloride on the growth and phycobiliprotein composition of the strains were evaluated. In the batch culture period of 12 days, Phormidium sp. and Pseudoscillatoria sp. were able to utilize all tested nitrogen sources; however, ammonium chloride was the best nitrogen source for both strains to achieve maximum growth rate μ?=?0.284?±?0.03 and μ?=?0.274?±?0.13 day^?1, chlorophyll a 16.2?±? 0.5 and 12.2?±? 0.2 mg L^?1, and phycobiliprotein contents 19.38?±?0.09 and 19.99?±?0.14% of dry weight, whereas, for Arthrospira platensis, the highest growth rate of μ?=?0.304?±?0.0 day^?1, chlorophyll a 19.1?±?0.5 mg L^?1, and phycobiliprotein content of 22.27?±?0.21% of dry weight were achieved with sodium nitrate. The phycocyanin from the lyophilized cyanobacterial biomass was extracted using calcium chloride and food grade purity (A₆₂₀/A₂₈₀ ratio >?0.7) was achieved. Furthermore, phycocyanin was purified using two-step chromatographic method and the analytical grade purity (A₆₂₀/A₂₈₀ ratio >?4) was attained. SDS-PAGE demonstrated the purity and presence of two bands corresponding to α- and β-subunits of the C-phycocyanin. The results showed that Phormidium sp. and Pseudoscillatoria sp. could be good candidates for phycocyanin production.     

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.     

Separation and characterization of two bovine alpha1-fetoprotein molecular variants by concanavalin A-Sepharose chromatography.

Two molecular variants of bovine alpha₁-fetoprotein were separated by affinity chromatography of fetal calf serum on a concanavalin A-Sepharose column. Radialimmunodiffusion assay of bovine alpha₁-fetoprotein revealed that 29% of the alpha₁-fetoprotein in fetal serum lacked concanavalin A-binding activity whilst 71% of the alpha₁-fetoprotein was capable of binding to the lectin. These two bovine alpha₁-fetoprotein variants show antigenic identity suggesting that the polypeptide chain rather than the carbohydrate moiety of the alpha₁-fetoprotein molecule is the antigenic determinant.