The critical phase inspection process: a benchmarking study in search of industries' best practices.

Benchmarking is the orderly process of measuring one's own products, services, and practices against those of companies recognized as leaders. Eli Lilly and Company's Quality Assurance Department formed the Critical Phase Inspection Team to benchmark the processes for selecting and conducting critical phase inspections and reporting inspection findings. The team developed a telephone survey that was conducted with 33 other quality assurance units across the country. Analysis of the phone survey responses resulted in the identification of 5 quality assurance units that we felt could provide valuable information to us on these activities. Site visits to these companies were arranged and information was shared. We present here the analysis and results of our benchmarking endeavor. Through the information sharing involved in the benchmarking process, namely, the telephone surveys and the site visits, fresh ideas emerged and new acquaintances were made. Comparisons and adaptations of our methods with others in the quality assurance business will lead us to breakthrough improvements that will allow us to improve our current processes.

Spectrophotometric assay and electrophoretic detection of trans-feruloyl esterase activity.

Wheat bran cell walls were subjected to mild acid hydrolysis and the major phenolic product was purified and identified as 5-O-(trans-feruloyl)-arabinofuranose. Sensitive continuous and stopped, microtiter plate-based spectrophotometric assays for trans-feruloyl esterase activity were developed using this compound as substrate. Procedures were also developed for the detection of trans-feruloyl esterase activities on gels following electrophoresis using this compound. These procedures are applicable to other natural feruloyl esters derived from plant cell walls by enzymatic hydrolysis. The extracellular trans-feruloyl esterases of Aspergillus niger 814 grown on 1% wheat bran were fractionated by anion-exchange chromatography and isoelectric focusing. These studies indicate that there are multiple forms of trans-feruloyl esterase but that most activity is associated with a major isozyme with a pI of 3.2.

Use of chemical fractionation and proton nuclear magnetic resonance to probe the physical structure of the primary plant cell wall.

Proton magnetic resonance has been used to monitor the microscopic physical properties of etiolated hypocotyl cell walls from Phaseolus vulgaris L. at all stages in a series of chemical fractionations with ammonium oxalate and potassium hydroxide. Solid echo measurements indicate that 75% of the polymers in the intact cell wall, including the cellulose and most of the hemicelluloses, are arranged such that there is almost complete restraint of molecular motion. The chemical fractionations generally altered the physical structures of the remaining cell wall components. Digestion with 0.25% ammonium oxalate/oxalic acid solubilized the pectin and increased the mobility of the hemicellulose I component. Extraction with 4% potassium hydroxide removed the hemicellulose I component and loosened the hemicellulose II. Further extraction with 24% potassium hydroxide removed the hemicellulose II and loosened some of the cellulose. The cellulose crystallinity, as monitored by Jeener echo measurements decreased from 83% to 63% during these fractionations. We conclude that, while hemicellulose I is firmly attached to hemicellulose II, it is not in a closely packed structure. Hemicellulose II is strongly bound to cellulose and has a much more closely packed structure.

Use of Per-C-Deuterated myo-Inositol for Study of Cell Wall Synthesis in Germinating Beans.

Cell wall polysaccharides of the hypocotyl and roots in germinating beans (Phaseolus vulgaris L.) were selectively labeled in arabinosyl, xylosyl, and galacturonosyl residues by per-C-deuterated myo-inositol, which was introduced through 72 hours of imbibition. Glucuronate residues remained unlabeled. Selected ion gas chromatography-mass spectrometry analysis revealed that deuterium was not redistributed in these three sugar residues or into other carbohydrate residues during this conversion, suggesting that the labeled residues are formed exclusively via the myo-inositol oxidation pathway and that no glucogenesis from myo-inositol takes place during this conversion. The presence of a significant level of deuterated arabinose, xylose, and galacturonate after just 72 hours of imbibitional uptake of per-C-deuterated myo-inositol indicated that the myo-inositol oxidation pathway has a predominant role in the biosynthesis of new cell walls.

myo-Inositol Synthesis from [1-H]Glucose in Phaseolus vulgaris L. during Early Stages of Germination.

Radiolabeled d-[1-(3)H]glucose was fed by imbibition under sterile conditions to bean (Phaseolus vulgaris L.) seeds. After 72 and 96 hours of feeding, the (3)H was located in uronic acid and pentose residues as well as hexose residues of cell wall polysaccharides in growing hypocotyl and root. Free myo-inositol present in cotyledons, hypocotyl, and root also contained (3)H, showing that de novo synthesis of myo-inositol from [1-(3)H]glucose did occur during the first 72 hours of germination. More than 90% of the labeled, free myo-inositol was present in the cotyledons. The (3)H percentage in trifluoroacetic acid-soluble arabinose residues of cell wall polysaccharides from 72-hour-old bean hypocotyls was only half of their mole percentage. On the other hand, (3)H percentages in hexose residues were higher than their mole percentages. The results suggest that myo-inositol is synthesized from reserve sugars during the very early stages of germination, and that the newly synthesized myo-inositol, as well as that stored in cotyledons, can be used for the construction of new hypocotyl and root cell wall polysaccharides after conversion into uronic acids and pentoses via the myo-inositol oxidation pathway.

The permeability of plant cell walls as measured by gel filtration chromatography.

The permeability of plant cell walls to macromolecules may limit the ability of enzymes to alter the biochemical and physical properties of the wall. Proteins of molecular weight up to 60,000 can permeate a substantial portion of the cell wall. Measurements of wall permeability in which cells are exposed to hypertonic solutions of macromolecules may seriously underestimate wall permeability.

Extracellular glycoprotein from virulent and avirulent Cryptococcus species.

Two virulent strains of Cryptococcus neoformans and two nonvirulent forms (C. albidus and C. laurentii) were grown in liquid culture to produce maximal capsule formation. A glycoprotein was isolated from the culture medium and was homogeneous as determined by cellulose acetate electrophoresis and anion-exchange chromatography. The amino acid, neutral sugar, amino sugar, uronic acid, and O-acetyl compositions and the infrared spectra of the glycoprotein were determined. The product of the C. neoformans strains contained more mannose and uronic acid than did that from the nonpathogenic strains. O-acetyl groups were absent from glycoprotein of the two nonpathogens.

Partial purification and some properties of a cholinesterase from bush bean (Phaseolus vulgaris L.) roots.

A cholinesterase was partially purified from bush bean (Phaseolus vulgaris L.) roots by using acridinium-based ligand affinity chromatography. The procedure gave a 78-fold increase in specific activity, although at least three inactive contaminants remained. The enzyme activity was maximal against acetyl esters of choline and was inhibited by neostigmine. Di-isopropyl phosphorofluoridate completely inhibited activity at concentrations greater than 0.1 mM. The catalytic centre activity was 2 X 10(-4) times that of electric eel acetylcholinesterase. Cholinesterase activity appeared as a peak (s = 4.2 +/- 0.1 S) after isokinetic sedimentation. The Stokes radius was 4.00 nm and the apparent molecular weight was 72700 +/- 1900. The smallest active and native form of the enzyme appeared to be a monomer. This contrasts with animal acetylcholinesterases, in which the smallest active and native forms are multimeric.

Isolation and properties of a ferredoxin from leaves of Sambucus racemosa L.

Ferredoxin was isolated in good yield from leaves of Sambucus racemosa L. by the following procedure: (1) homogenization in buffered acetone-water (1:1v/v); (2) ion-exchange chromatography on several columns of DEAE-cellulose; and (3) purification by gel filtration with Sephadex G-75. The ultraviolet and visible spectrum showed maxima at 277, 331, 423, and 466 nm. The electron paramagnetic resonance spectrum was centered around g = 1.957. The protein sustained an initial photoreduction rate of 86 mumol NADP per milligram chlorophyll per hour. The amino acid composition was found to be Lys 5, His 2, Arg1, Asx11, Thr5, Ser7, Glx17, Pro6, Gly7, Ala6-7, Cys4, Val8, Ile5, Leu7, Tyr3, Phe2, and Trp1. The molecule had a molecular weight of 10 700 and contained two atoms of iron. The amino-terminal residue was alanine. These properties are highly similar to those of other angiosperm ferredoxins. Sambucus ferredoxin was found to be most closely related to that of Leucaena.

Quantitative microanalysis of cell walls of Saprolegnia diclina Humphrey and Tremella mesenterica Fries.

Cell walls of the fungi Saprolegnia declina Humphrey and Tremella mesenterica Fries were analyzed quantitatively. Particular attention was paid to the hydrolysis and analysis of neutral sugars, amino sugars and amino acids. These components, together with total lipids, total uronic acids and the ashed residue, accounted for more than 90% by weight of the original dry cell wall preparation. There were substantial losses of amino acids during hydrolysis; however, analytical recovery approached 100% when total protein was calculated from the total nitrogen analysis. The analytical procedures were reproducible (+/- 3% for amino acids and amino sugars, and +/- 5-10% for other components) when applied to individual cell wall preparations. However, even under carefully standardized conditions, different cell wall preparations from the same species showed variable composition. Glucose was the predominant neutral sugar in the cell wall polymers of both species. The amino acid compositions were remarkable in that neither species contained detectable levels of cyst(e)ine. Hydroxyproline was detected in both species. The report from Tremella mesenterica is the first for this amino acid from the cell wall of a Basidiomycete.

Gibberellic-Acid-induced cell elongation in lettuce hypocotyls.

Gibberellic acid (10 muM) causes lettuce hypocotyl cells to elongate by 400-500% more than water controls in 72 hr. Kinetic data indicate that whereas in water controls cell elongation occurs between 24 and 48 hr, in gibberellic-acid-treated material it starts at 8 hr and continues to 72 hr. Dry weight of the cell wall shows a corresponding increase with cell elongation. Two-hour pulse labeling with [(14)C]glucose, however, indicates a peak in the incorporation of label in the wall fraction at 8 hr, when growth has only just begun, and a progressive decline later, when elongation is occurring at maximum rate. The peak coincides with extensive dictyosomal activity, proliferation of endoplasmic reticulum and polyribosomes, and connections between the endoplasmic reticulum and plasmalemma in both water- and gibberellic-acid-treated hypocotyls. At later times, the cells contain only a thin layer of cytoplasm and no special cytological features are observed. These observations indicate that, although cell growth in lettuce hypocotyls is accompanied by wall synthesis, nevertheless the cells undergo their most rapid polysaccharide and protein synthesis prior to extension growth. They also explain the earlier reported "enhanced sensitivity" of lettuce hypocotyls to gibberellic acid application at 8 hr after the beginning of the experiment.

An inducible hydrolase from Aspergillus niger, acting on carbon-carbon bonds, for phlorrhizin and other C-acylated phenols.

1. An inducible enzyme catalysing the hydrolysis of phloretin to form phloroglucinol and phloretic acid has been extracted from the acetone-dried powders of the mycelial felts of an Aspergillus niger strain grown in the presence of phlorrhizin. The enzyme was partially purified by treatment with protamine sulphate, ammonium sulphate fractionation, negative adsorption on tricalcium phosphate gel, and DEAE-cellulose column chromatography. 2. The hydrolytic activity on phloretin appeared to be maximal at about pH9.6. However, the characteristics of the enzyme were studied at pH7.2, because of the lability of the product, phloroglucinol, under alkaline conditions. 3. The apparent K(m) value at pH7.2 was about 0.3-0.4mm for phloretin and 0.15mm for 3'-methylphloracetophenone. 4. Maximum activity of the enzyme was obtained without the addition of any cofactor or metal ion. The involvement of thiol groups in the reaction was demonstrated by the potent inhibitory action of both heavy-metal ions and p-chloromercuribenzoate. 5. The enzyme showed a rather broad substrate specificity, and some other C-acylated phenols related to phloretin were hydrolysed. It was found that 3'-methylphloracetophenone, phloracetophenone and 2',4,4'-trihydroxydihydrochalcone were attacked more efficiently than phloretin. We propose the systematic name C-acylphenol acylhydrolase for the enzyme. This enzyme belongs to EC group 3.7.1.