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Background Holding times for raw materials are relevant since they enable us to understand the allowable time that a raw material can be kept under ideal storage conditions before the start of the manufacturing process without its quality attributes being affected. The quantification of water activity can be used as an indicator of the microbiological, physicochemical, and organoleptic stability of a specimen, since low water activity retards autohydrolysis and microbiological growth. Objective The main purpose of this investigation was to test the stability of powdered raw materials for a maximum holding time of 8 days through water activity measurements. Thus microbiological, physicochemical, and organoleptic measurements were carried out in parallel and simultaneously to experimentally establish a relationship between the status of the water activity of processed raw materials and the microbiological, physicochemical, and organoleptic results. Methods The raw materials were stored for a maximum holding time of 8 days, in accordance with United States Pharmacopeia monographs. For all the raw materials tested, water activity measurements were performed using the dew point chilled-mirror method on days 0, 3, and 8. On days 0 and 8, microbiological, physicochemical, and organoleptic assessments were performed. Results It was established that under these storage conditions, the processed raw materials exhibited water activity below 0.60 during the entire holding time. However, there were statistically significant differences in water activity levels between days 0, 3, and 8 (ANOVA P  < 0.05). Despite observing statistically significant differences between days, the microbiological, physicochemical, and organoleptic features were within specification at those water activity levels below 0.60. Conclusions Water activity below 0.60 does not allow the growth of microorganisms, and the organoleptic and physicochemical features remain unperturbed. The results indicate that water activity can be used as an indicator of the microbiological load and chemical stability of the raw materials tested. Highlights This research provides evidence that corroborates that water activity status may be used as a reliable indicator for the microbiological burden and physicochemical features of pharmaceutical material during stability studies. [ABSTRACT FROM AUTHOR]

Mutations and polymorphisms in complement genes have been linked with numerous rare and prevalent disorders, implicating, dysregulation of complement in pathogenesis. The 3 common alleles of factor B (fB) encode Arg (fB[sub32R]), GIn (fB[sub32Q]), or Trp(fB[sub32w]) at position 32 in the Ba domain. The fB[sub32Q] allele is protective for age-related macular degeneration, the commonest cause of blindness in developed countries. Factor B variants were purified from plasma of homozygous individuals and were tested in hemolysis assays. The protective variant fB[sub32Q] had decreased activity compared with fB[sub32Q]. Biacore comparison revealed markedly different proenzyme formation; fB[sub32Q] bound C3b with 4-fold higher affinity, and formation of activated convertase was enhanced. Binding and functional differences were confirmed with recombinant f[sub32Q] and fB[sub32Q]; an intermediate affinity was revealed for fB[sub32Q]. To confirm contribution of Ba to binding, affinity of Ba for C3b was determined. Ba-fB[sub32Q] had 3-fold higher affinity compared with Ba-fB320. We demonstrate that the disease-protective effect of fB32Q is consequent on decreased potential to form convertase and amplify complement activation. Knowledge of the functional consequences of polymorphisms in complement activators and regulators will aid disease prediction and inform targeting of diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Age-related macular degeneration (AMD) and membranoproliferative glomerulonephritis type II (MPGN2) are dense deposit diseases that share a genetic association with complement genes and have complement proteins as important components of the dense deposits. Here, we present the case of a 64-year-old smoker male who developed both AMD and MPGN2 in his late 50s. The patient presented persistent low plasma levels of C3, factor H levels in the lower part of the normal range and C3NeF traces. Genetic analyses of the CFH, CFB, C3, CFHR1-CFHR3 and LOC387715/HTRA1 genes revealed that the patient was heterozygote for a novel missense mutation in exon 9 of CFH (c.1292 G>A) that results in a Cys431Tyr substitution in SCR7 of the factor H protein. In addition, he was homozygote for the His402 CFH allele, heterozygote for the Ser69 LOC387715 allele, homozygote for the Arg32 (BFS) CFB allele, heterozygote for the Gly102 (C3F) C3 allele and carried no deletion of the CFHR1/CFHR3 genes. Proteomic and functional analyses indicate absence in plasma of the factor H allele carrying the Cys431Tyr mutation. As a whole, these data recapitulate a prototypical complement genetic profile, including a partial factor H deficiency and the presence of major risk factors for AMD and MPGN2, which support the hypothesis that these dense deposit diseases have a common pathogenic mechanism involving dysregulation of the alternative pathway of complement activation. [Copyright &y& Elsevier]

A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering. [ABSTRACT FROM AUTHOR]

Abstract: The surface carboxylic groups of penicillin G acylase and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylenediamine via the 1-ethyl-3-(dimethylamino-propyl) carbodiimide coupling method. Then, both proteins were immobilized on glyoxyl agarose. In both cases, the immobilization of the chemically modified enzymes improved the enzyme stability compared to the stability of the immobilized but non-modified enzyme (by a four-fold factor in the case of PGA and a 20-fold factor in the case of GA). The chemical modification presented a deleterious effect on soluble enzyme stability. Therefore, the improved stability should be related to a higher multipoint covalent attachment, involving both the lysine amino groups and also the new amino groups chemically introduced on the enzyme. Moreover, the lower pK a of the new amino groups permitted to immobilize the enzyme under milder conditions. In fact, the aminated proteins could be immobilized even at pH 9, while the non-modified enzymes could only be immobilized at pH over 10. [Copyright &y& Elsevier]

Abstract: A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%). [Copyright &y& Elsevier]

Generation of the alternative pathway C3-convertase, the central amplification enzyme of the complement cascade, initiates by the binding of factor B (f B) to C3b to form the proconvertase, C3bB. C3bB is subsequently cleaved by factor D (fD) at a single site in f B, producing Ba and Bb fragments. Ba dissociates from the complex, while Bb remains bound to C3b, forming the active alternative pathway convertase, C3bBb. Using single-particle electron micros- copy we have determined the 3-dimensional structures of the C3bB and the C3bBb complexes at ≈27Å resolution. The C3bB structure shows that fB undergoes a dramatic conformational change upon binding to C3b. However, the C3b-bound fB structure was easily interpreted after independently fitting the atomic structures of the isolated Bb and Ba fragments. Interestingly, the divalent cation-binding site in the von Willebrand type A domain in Bb faces the C345C domain of C3b, whereas the serine-protease domain of Bb points outwards. The structure also shows that the Ba fragment interacts with C3b separately from Bb at the level of the α'NT and CUB domains. Within this conformation, the long and flexible linker between Bb and Ba is likely exposed and accessible for cleavage by fD to form the active convertase, C3bBb. The architecture of the C3bB and C3bBb complexes reveals that C3b could promote cleavage and activation of fB by actively displacing the Ba domain from the von Willebrand type A domain in free f B. These structures provide a structural basis to understand fundamental aspects of the activation and regulation of the alternative pathway C3-convertase. [ABSTRACT FROM AUTHOR]

Abstract: At alkaline pHs, glyoxyl agarose is able to immobilize most of the proteins contained in a crude extract. However, due to its special immobilization features, at pH 7.0 only proteins that contain at least two exposed low pK amino groups in the same plane were immobilized (β-galactosidase from Escherichia coli, catalase from bovine liver, and IgG from rabbit). However, with many other proteins, even multimeric ones, immobilization could not be achieved (e.g.: glucose oxidase from Aspergillus niger and Penicillium vitale; catalase from Micococcus sp., A. niger and bovine liver; alcohol oxidase from Pichia pastoris, Hansenula sp. and Candida boidinii, β-galactosidase from Thermus sp., etc.). Elution of the attached proteins under mild conditions was not simple, if the number of protein-support bonds was very high, only boiling in SDS allowed the elution of the proteins. However, using glyoxyl agarose 4BCL with only 20μmol of aldheyde groups/g of support, proteins could be fully eluted by competition with amino compounds (e.g., Tris buffer). In this first approach, we have tried to take advantage of this specific immobilization at pH 7.0 to purify multimeric proteins, using a β-galactosidase from E. coli as a model. The enzyme could be eluted from the support using Tris–HCl buffer as eluting agent, with a high yield (80%) and a high purification factor (32). [Copyright &y& Elsevier]

Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C(3923ΔDG), which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H₂O) by spontaneous thioester hydrolysis, C(3923ΔDG) generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C(3b923ΔDG) and C3(H₂O)(923ΔDG) were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase-restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments. [ABSTRACT FROM AUTHOR]

Abstract: This paper reports that penicillin G acylase (PGA) is able to hydrolyze prochiral diethyl and dimethyl phenylmalonate, although activity is higher with the dimethyl ester. The process has two very interesting features: (i) the enzyme is fully specific and did not recognize the monoester as substrate, very likely due to the presence of a charged carboxylic acid in alpha-position, permitting to fully transform the diester in the chiral monoester and (ii) the reaction is almost fully asymmetric, producing (+)-methyl phenylmalonate. Although the activity of PGA against this substrate is lower than against other substrates of PGA (e.g., 20-fold lower than when using (+)-methyl mandelate), the use of a highly loaded biocatalyst (80mg of PGA per gram of support) permitted to obtain around 80U/g of biocatalyst, permitting to produce 100mM (+)-methyl phenylmalonate using 1g of biocatalyst per 100ml of reaction volume in only 5h. [Copyright &y& Elsevier]