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PubMed1986

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.

1-Deoxynojirimycin↗
PubMed1984

On the synthesis of a 32P-labelled Edman reagent for the sensitive identification of amino-acid derivatives.

A phosphorus-32 containing derivative of phenylisothiocyanate was prepared to increase the sensitivity of amino-acid sequence determination. The respective compound 2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinane 2-oxide showed about the same reactivity, stability, and polarity as the Edman reagent itself. A repetitive yield of 94% was obtained in the stepwise degradation of insulin B chain using a solid phase sequencer. The synthesis of this radioactive reagent was achieved within 5 h but with a specific activity of 1 Ci/mol. Eight amino acids were reacted with the 32P-labelled reagent and identified by autoradiography after two dimensional thin-layer chromatography.

Amino Acid Sequence↗
PubMed2000

Ego Function Assessment of substance abusers: standardization and reliability.

This study examined the reliability of Ego Function Assessment in a self-report inventory which yields 12 function measures. 89 substance abusers in an urban treatment facility completed the inventory; 81 completed the inventory again after 2 wk. Test protocols of the first administration were assessed for interitem consistency utilizing a hierarchical algorithm which revised scales to yield optimal alphas. Retest scores were used to evaluate the stability of the 12 scales over 2 wk. Standardization, reliability, and stability data are presented for the original and revised scales. The possibility of response sets in the items are explored, and data patterns are discussed in terms of state vs trait issues in ego functioning.

Adult↗
PubMed2005

Short form of Sarason's Social Support Questionnaire: French adaptation and validation.

The original short form of Sarason's Social Support Questionnaire measures two dimensions of social support, availability and satisfaction. The factorial structure of the social support was assessed in a French sample along with the association between the dimensions of social support and personality traits such as Extraversion, Neuroticism, and Depression. A 6-item French version was administered to 648 subjects: 348 university freshmen (228 females, 120 males) and 304 unemployed men. A principal component analysis followed by oblique rotations was undertaken on each sample. This yielded two factors, Availability and Satisfaction. These results sustained the stability of the factorial structure from one culture to another. Validity and reliability were satisfactory. Moreover, these two dimensions were correlated with two personality scales (high extraversion, low depression). Such results agree with previous results establishing relations between social support and salutogenic dimensions of personality.

Adolescent↗
PubMed1997

The effects of "decelerated" rehabilitation following anterior cruciate ligament reconstruction on a hyperelastic female adolescent: a case study.

Current concepts in postoperative anterior cruciate ligament (ACL) reconstruction management include participation in an "accelerated" rehabilitation program. There are no published reports examining the effects of accelerated or conservative rehabilitation on subjects with generalized ligamentous hyperelasticity. The purpose of this case study was to examine the effects of a conservative or "decelerated" rehabilitation program on the functional outcome of a hyperelastic female adolescent athlete following ACL reconstruction. The subject was a 15-year-old female basketball player who sustained a unilateral ACL tear and underwent subsequent ACL reconstruction using a patellar tendon autograft. The subject immediately began participation in a "decelerated" rehabilitation program in which the intensity and rate of progression was decelerated, emphasizing a prolonged period of maximum graft protection. Progress was objectively quantified with a battery of diagnosis-specific tests at scheduled intervals. Results at 52 weeks postoperative revealed normal range of motion, proprioception, balance, knee stability, quadriceps strength, hamstring strength, and subjective assessment values, and only a 4.0% deficit in functional scores. Our results suggest a "decelerated" rehabilitation program may be appropriate for the population with generalized ligamentous hyperelasticity by yielding excellent functional results without compromising the integrity of the graft and, ultimately, knee stability.

Adolescent↗
PubMed2006

Tumor targeting by an aptamer.

UNLABELLED: Aptamers are small oligonucleotides that are selected to bind tightly and specifically to a target molecule. We sought to determine whether aptamers have potential for in vivo delivery of radioisotopes or cytotoxic agents. METHODS: TTA1, an aptamer to the extracellular matrix protein tenascin-C, was prepared in fluorescent and radiolabeled forms. After in vivo administration, uptake and tumor distribution of Rhodamine Red-X-labeled aptamer was studied by fluorescence microscopy. In glioblastoma (U251) and breast cancer (MDA-MB-435) tumor xenografts, biodistribution and imaging studies were performed using TTA1 radiolabeled with (99m)Tc. Tenascin-C levels and tumor uptake were studied in a variety of additional human tumor xenografts. To assess the effect of radiometal chelate on biodistribution, mercapto-acetyl diglycine (MAG(2)) was compared with diethylenetriaminepentaacetic acid and with MAG(2)-3,400-molecular-weight PEG (PEG(3,400)). RESULTS: Intravenous injection of fluorescent aptamer TTA1 produced bright perivascular fluorescence in a xenografted human tumor within 10 min. In the ensuing 3 h, fluorescence diffused throughout the tumor. Labeled with (99m)Tc, TTA1 displayed rapid blood clearance, a half-life of less than 2 min, and rapid tumor penetration: 6% injected dose (%ID)/g at 10 min. Tumor retention was durable, with 2.7 %ID/g at 60 min and a long-lived phase that stabilized at 1 %ID/g. Rapid tumor uptake and blood clearance yielded a tumor-to-blood ratio of 50 within 3 h. Both renal and hepatic clearance pathways were observed. Using the (99m)Tc-labeled aptamer, images of glioblastoma and breast tumors were obtained by planar scintigraphy. Aptamer uptake, seen in several different human tumors, required the presence of the target protein, human tenascin-C. Modification of the MAG(2) radiometal chelator dramatically altered the uptake and clearance patterns. CONCLUSION: TTA1 is taken up by a variety of solid tumors including breast, glioblastoma, lung, and colon. Rapid uptake by tumors and rapid clearance from the blood and other nontarget tissues enables clear tumor imaging. As synthetic molecules, aptamers are readily modified in a site-specific manner. A variety of aptamer conjugates accumulate in tumors, suggesting imaging and potentially therapeutic applications.

Animals↗
PubMed1987

[Expression of human leukocyte interferon type A in Saccharomyces cerevisiae under the control of regulatory elements of the yeast gene URA3].

Expression of a chromosomal gene for human leucocyte interferon A was obtained due to interaction of 5'-nontranslating region of a cloned interferon gene with the regulatory sequences from UNA3 yeast gene. The sequence of 5'-nontranslating region from interferon gene essential for its expression in yeast is localized within 130 b p. from the initiating codon. Due to increasing of plasmid stability and copy number a 60-fold increase. in interferon yield was obtained in yeast transformants reaching the level of 6 X 10(7) u X 1(-1). The data are presented supposing the existence of functional polycistronic mRNA in yeast.

Base Sequence↗
PubMedDate not supplied

Psychometric assessment of the Jalowiec Coping Scale.

The Jalowiec Coping Scale consists of 40 coping behaviors culled from a comprehensive literature review, which are rated on a 1- to 5-point scale to indicate degree of use. Twenty judges classified the items to permit analysis of the coping behaviors according to a problem-oriented/affective-oriented dichotomy; 15 problem and 25 affective items resulted. Overall agreement by the judges was 85%, with greater consensus on problem items. Evaluation of stability using a two-week retest interval (N = 28) yielded significant rhos of .79 for total coping scores, .85 for problem, and .86 for affective. With a one-month interval (N = 30) coefficients were .78, .84, and .83, respectively. Alpha reliability coefficients of .86 (N = 141) and .85 (N = 150) supported instrument homogeneity. Content validity is substantiated by the systematic manner of tool development, by the large number of items used, and by the inclusion of diverse coping behaviors. Factor analysis (N = 141) was used to investigate construct validity. A two-factor solution to evaluate the validity of the dichotomous classification showed that 80% of the problem items loaded on Factor I, but only 56% of the affective items loaded on Factor II. To examine this multidimensional aspect, several other factor solutions were explored. Ultimately, the four-factor solution provided the most intelligible conceptual pattern with the least loss of information. Conceptual composition of these factors is discussed, and several tentative labels for each factor are suggested.

Adaptation, Psychological↗
PubMed1980

A novel purification procedure for Penicillium notatum phospholipase B and evidence for a modification of phospholipase B activity by the action of an endogenous protease.

The purification of Penicillium notatum phospholipase B was greatly improved using phosphatidylserine-AH Sepharose 4B affinity chromatography. This chromatography simplified the procedure, stabilized the enzyme activity and gave six-fold higher yields and two-fold higher specific activity for the enzyme than the previous method (Kawasaki, N. and Saito, K. (1973) Biochim. Biophys. Acta 296, 426-430). During purification, it was found that an endogenous protease attacked phospholipase B and produced a modified type which exhibited extremely reduced phospholipase B (phosphatidylcholine hydrolysis) activity but maintained native lysophospholipase (lysophosphatidylcholine hydrolysis) activity. This protease was completely inhibited by phenylmethylsulfonylfluoride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that native and modified phospholipases B had exactly the same molecular weight (90 000) in the absence of beta-mercaptoethanol. However, the latter gave three bands in the presence of beta-mercaptoethanol, whereas the former only a single band. These results strongly suggested that the endogenous protease cleaved phospholipase B at only a few sites, thereby changing its phospholipase B activity.

Chromatography, Affinity↗
PubMed1995

[Production and catalytic properties of point mutants Phe41--->His and Phe143--->Glu of horseradish peroxidase, expressed in Escherichia coli].

Recombinant horseradish peroxidase and its single-point mutants, F4IH and H143E, have been reactivated from E. coli inclusion bodies with a 25% yield. Both mutations affect heme entrapment as well as enzyme stability and activity. A more than 40-fold decrease in the specific activity towards ABTS is associated with different steps of peroxidase catalysis. F41H replacement results in a drop of both rate constants by two and one orders or magnitude for hydrogen peroxide and ABTS, respectively. The mechanism of iodide oxidation by the F41H mutant fits into a ternary interaction. The F143E replacement mainly affects the steps of ABTS oxidation and product dissociation. It is suggested that the role of replaced phenylalanine residues consists in the formation of a highly hydrophobic pocket allowing for strong non-covalent binding of the heme porphyrin ring.

Amino Acid Sequence↗
PubMed1996

Specimen stability for DNA-based diagnostic testing.

The use of molecular diagnostic testing is increasing in the clinical setting; therefore, data regarding DNA stability in clinical specimens are essential for correct test performance and interpretation. This study was designed to determine DNA stability in peripheral blood and solid tissue under different storage conditions. DNA quality and yield were assayed by spectrophotometric absorbance, gel electrophoresis, and suitability for Southern hybridization and polymerase chain reaction (PCR), the most widely employed clinical DNA analyses. A second goal of the study was to evaluate DNA stability during storage at 4 degrees C for 1 month to 3 years. The data show that freezing or refrigeration of separated leukocytes is preferable for short- to intermediate-term storage and freezing is preferable for solid tissue. DNA degradation varying from slight to severe is seen inconsistently with such specimens, probably due to sampling of unevenly frozen-tissue areas. Depending on the degree of DNA degradation, analysis may still be possible by PCR and in some cases even by Southern hybridization. Once isolated, DNA was stable at 4 degrees C for at least 3 years. These results suggest a more flexible approach to specimen requirements for molecular pathology, as some samples that would routinely be rejected gave interpretable results.

Blood Cells↗
PubMed2006

Use of EAF dust as heterogeneous catalyst in Fenton oxidation of PCP contaminated wastewaters.

In this study, chemical oxidation tests using H2O2 were performed on a solution contaminated with 100 mg l(-1) of pentachlorophenol (PCP). The effectiveness of electric arc furnace dust and hematite as heterogeneous catalysts was evaluated. Reactions were conducted at pH 2 for 24 h. Either H2O2 stabilized with KH2PO4 or un-stabilized H2O2 was used. Total organic carbon (T.O.C.) removal and chloride release from PCP molecule were monitored. Results showed that the maximum removal yields for electric arc furnace (EAF) dust (49.2% T.O.C., 56.7% Cl) were achieved when H2O2:PCP ratio was 10:1 and Fe:H2O2 = 1:5 for unstabilized H2O2 and when H2O2:PCP = 10:1 and Fe:H2O2 = 1:1 for stabilized H2O2 (48% T.O.C., 60.6% Cl). The maximum yield using hematite (45.2% T.O.C., 55.2% Cl) was obtained when H2O2:PCP ratio was 10:1 and Fe:H2O2 was 1:2. When EAF dust was used and Fe:H2O2 > 1:5, Cl release was higher than the one expected from T.O.C. removal.

Catalysis↗
PubMed1999

Selection for improved protein stability by phage display.

A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, phage display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the display phages at elevated temperatures was compared to exposure of the phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in phage display.

Amino Acid Sequence↗
PubMed2006

Quality assessment of confocal microscopy slide-based systems: instability.

BACKGROUND: All slide-based fluorescence cytometry detections systems basically include an excitation light source, intermediate optics, and a detection device (CCD or PMT). Occasionally, this equipment becomes unstable, generating unreliable and inferior data. METHODS: A number of tests have been devised to evaluate equipment performance and instability. The following four instability tests are described: galvanometer scanning, stage drift, correct wavelength spectral detection, and long-term laser power. RESULTS: Quality assurance tests revealed that a confocal microscope can become unstable in the following parameters, yielding inaccurate data: laser power, PMTs functionality, spectrophotometer accuracy, galvanometer scanning and laser stability, and stage drift. Long-term laser power stability has been observed to vary greatly. CONCLUSIONS: Confocal systems can become unstable in the following parameters: long-term laser power, galvanometer scanning, spectrophotometer accuracy, and stage stability. Instability in any of these parameters will affect image quality. Laser power fluctuations result from either a defective Acousto-optic tunable filter or improper heat dissipation. Spectrophotometer instability will generate unreliable spectra data, extra light reflections, and poor image quality. Galvanometer scanning instability yields poor image quality while microscope stage drift results in a sample going out of the plane of focus. With minor modifications, these tests may be applicable to other slide-based systems.

Animals↗
PubMed2004

Cold-shock induced high-yield protein production in Escherichia coli.

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.

Amino Acid Sequence↗
PubMed1993

Insulin structure and stability.

Insulin is composed of 51 amino acids in two peptide chains (A and B) linked by two disulfide bonds. The three-dimensional structure of the insulin molecule (insulin monomer), essentially the same in solution and in solid phase, exists in two main conformations. These differ in the extent of helix in the B chain which is governed by the presence of phenol or its derivatives. In acid and neutral solutions, in concentrations relevant for pharmaceutical formulation, the insulin monomer assembles to dimers and at neutral pH, in the presence of zinc ions, further to hexamers. Many crystalline modifications of insulin have been identified but only those with the hexamer as the basic unit are utilized in preparations for therapy. The insulin hexamer forms a relatively stable unit but some flexibility remains within the individual molecules. The intrinsic flexibility at the ends of the B chain plays an important role in governing the physical and chemical stability of insulin. A variety of chemical changes of the primary structure (yielding insulin derivatives), and physical modifications of the secondary to quaternary structures (resulting in "denaturation," aggregation, and precipitation) are known to affect insulin and insulin preparations during storage and use (Fig. 8). The tendency of insulin to undergo structural transformation resulting in aggregation and formation of insoluble insulin fibrils has been one of the most intriguing and widely studied phenomena in relation to insulin stability. Although the exact mechanism of fibril formation is still obscure, it is now clear that the initial step is an exposure of certain hydrophobic residues, normally buried in the three-dimensional structure, to the surface of the insulin monomer. This requires displacement of the COOH-terminal B-chain residues from their normal position which can only be accomplished via monomerization of the insulin. Therefore, most methods stabilizing insulin against fibrillation share the property of being able to counteract associated insulin from being disassembled. Chemical deterioration of insulin during storage of pharmaceutical preparations is mainly due to two categories of chemical reactions, hydrolysis and intermolecular transformation reactions leading to insulin HMWT products. The predominant hydrolysis reaction is deamidation of Asn residues which in acid solution takes place at residue A21, in neutral medium at residue B3. An amazing hydrolytic cleavage of the backbone A chain, presumably autocatalyzed by an adjacent insulin molecule, has been identified in insulin preparations containing rhombohedral crystals in combination with free zinc ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1983

Purification and characterization of sheep brain cold-stable microtubules.

The isolation of cold-stable microtubules in high yields, described previously only from rodents, was extended to the brain of higher animals. Under optimal conditions, yields of 30 mg of cold-stable microtubles per 100 g of sheep brain could be obtained routinely. Material purified by two polymerization cycles displayed the same stability to cold temperature or to millimolar concentrations of calcium and the same lability to calmodulin and to ATP as did the purified material obtained from the rat [Job, D., Rauch, C.T., Fischer, E.H. & Margolis, R.L. (1982) Biochemistry 21, 509]. Furthermore, DE-52 chromatography of this material yielded a fraction that restored cold stability when added to cold-labile microtubules. Known to bind to calmodulin and to enhance microtubule assembly, tau proteins had no cold-stabilizing activity. Protein profiles of the cold-stabilizing fraction from sheep and rat brain were similar to one another but showed no protein bands corresponding to the tau proteins.

Animals↗
PubMed2001

Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions.

The importance of the stabilization of the quaternary structure of multimeric enzymes has been illustrated using a model reaction with great industrial relevance: the enzymatic synthesis of ampicillin from 6-amino penicillanic acid (6APA) and phenylglycine methyl ester (PGM) catalyzed by the tetrameric enzyme alpha-amino acid ester hydrolase from Acetobacter turbidans. The stabilization of the multimeric structure of the enzyme was achieved by multi-subunit immobilization of the enzyme followed by its further solid-phase chemical intersubunit cross-linking with polyfunctional macromolecules (dextran-aldehyde). This stabilized derivative has permitted the study of the reaction under conditions where nonstabilized enzyme molecules tended to dissociate (e.g., absence of phosphate ions). Synthetic yields improved from around 65%, under conditions where the nonstabilized derivative was stable, to around 85% in conditions where only the stabilized derivative could be utilized (40% methanol and absence of phosphate ions). When using high concentrations of PGM, a significant worsening of the reaction performance was detected with a significant decrease in the yields (below 55%, using 50 mM 6APA and PGM). This problem has been sorted out by using a fed-batch reaction system. By addition of PGM continuously to the reaction mixture (to maintain the concentration between 0.5 and 3 mM), 95% of 6-APA could be transformed to antibiotic (47.5 mM) by only using a 20% excess of acylating ester.

Acetobacter↗