Acid phosphatase and adenosine triphosphatase activities in the cell wall of baker's yeast.

In order to establish whether a specific adenosine triphosphatase is present in yeast cell wall, hydrolysis rates for p-nitrophenylphosphate (acid phosphatase activity) and for ATP (ATPase activity) were compared under various conditions. Rate determinations were made with both, intact cells and with preparations containing secreted enzymes from protoplasts. Acid phosphatase and ATPase activities had the same pH profile and were susceptible in the same way to the repression by orthophosphate and to the inhibition by 2-deoxyglucose. The Lineweaver-Burk plot shows biphasic kinetic behaviour for the hydrolysis of either p-nitrophenylphosphate or ATP. This suggests the existence of two enzymes with different affinities for the substrates, or one enzyme with at least two active sites. The two activities differ in thermostability and only one activity could be completely abolished by heat treatment. The thermostable enzyme activity had K-m values of 0.475 mM for p-nitrophenylphosphate, and 0.040 mM for ATP. ATP behaved as a partially competitive inhibitor of p-nitrophenylphosphate hydrolysis. Substrate competition studies showed that only a non-specific acid phosphatase is responsible for the hydrolysis of ATP.

Purification of protoplast-secreted acid phosphatase from baker's yeast. Effect on adenosine triphosphatase activity.

In order to examine acid phosphatase (EC 3.1.3.2) and ATPase (EC 3.6.1.3) activities of baker's yeast (pH optimum 3.5) a protoplast-secreted enzyme preparation was purified and some physical and chemical properties were studied. Three protein fractions containing ATPase and acid phosphatase activities, in the same ratio as the initial preparation, were separated by ion-exchange chromatography. The first fraction which had the highest protein content yielded a homogeneous preparation after Sepharose 4B chromatography and was used in further studies. An attempt to estimate molecular weight of this protein was made. Attempts to separate acid phosphatase and ATPase activities by ion-exchange chromatography, gel filtration, isoelectric focusing and sucrose density gradient centrifugation have been unsuccessful. Both activities behaved the same way to heat and urea denaturation. These results suggest that the two activities are associated with the same protein molecule.

Role of glycosylation in secretion of yeast acid phosphatase.

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.

Assessment of contemporary social phobia verbal report instruments.

The Social Phobia Scale (SPS), the Social Interaction Anxiety Scale (SIAS) and the Social Phobia and Anxiety Inventory (SPAI) were compared to each other and evaluated in patients with social phobia. We examined the relationship of these three contemporary social phobia verbal report instruments with each other, as well as with behavioral and self-report cognitive criteria. As expected, the three social phobia scales were significantly intercorrelated, although they differed in their relationship to the behavioral and cognitive measures. Specifically, the SPS had a significant negative relationship with time spent in a speech behavioral assessment test. The higher the anxiety scores were on the SPS, the less time patients spent giving an impromptu speech in front of a small audience. The SIAS was consistently related to negative and positive self-reported thoughts in speech and conversation behavioral assessment tests. All instruments differentiated patients with speech phobia from those having both generalized social phobia and avoidant personality disorder; on the SPAI and the SIAS, however, distinguished the former group from individuals with generalized social phobia but not without avoidant personality disorder. All three social phobia instruments were sensitive to treatment changes. Results are discussed in terms of the relative utility of each of these measures' total sores and any and their subscales.

[Artefact reducing in diagnosis of lung embolism using spiral CT with saline bolus]. / Artefaktreduzierung bei der Lungenemboliediagnostik mittels Spiral-CT unter Verwendung eines Kochsalzbolus.

PURPOSE: To improve the diagnostic efficacy of bolus-enhanced spiral CT (SCT) in the detection of pulmonary embolism using a saline push immediately after bolus injection of the contrast medium. PATIENTS AND METHODS: The study included 90 patients with suspected acute or chronic pulmonary embolism. The CT scan was performed in a caudocephaled direction. In Group I (n = 60) we applied a bolus contrast injection (120 ml, 3 ml/s, 300 mg J/ml), after a median delay of 25 s. Group II (n = 30) had the same contrast injection which was immediately followed by an additional saline push (60 ml, 2 ml/s). Streak artifacts originating from high contrast concentrations in the superior vena cava were rated on a 4-point scale for different locations: right pulmonary artery, pars basalis, truncus anterior, and the segmental upper lobe arteries. RESULTS: The incidence of artifacts in group I was nearly twice as high as in group II. The difference was significant (p < 0.05) for the upper and anterior superior lobe artery, the right pulmonary artery and the pars basalis. CONCLUSION: The presented protocol significantly reduces artifacts mainly by a washout of contrast medium in the superior vena cava.

[A novel hirudin coating of vascular endoprostheses: experimental results]. / Eine neuartige Hirudinbeschichtung vaskulärer Endoprothesen: Experimentelle Ergebnisse.

PURPOSE: Does hirudin coating improve the patency of iliac artery endoprostheses in comparison to non-hirudin-coated endoprostheses? MATERIALS AND METHODS: Nitinol stents and stentgrafts covered with polytetrafluoroethylene (PTFE) were coated with the polymer polyamino-p-xylylene-co-poly-p-xylylene using chemical vapor deposition (CVD) technique. Hirudin was covalently bound to the surface of the endoprostheses via the amino-group. External factors (mounting of the prosthesis, sterilization, storage time and temperature, release) affecting the hirudin activity were evaluated in vitro. Five types of prostheses were compared in vivo: (1) plain and (2) CVD- and hirudin-coated stents; (3) plain, (4) CVD-coated, and (5) CVD- and hirudin-coated PTFE-stentgrafts. In 20 sheep, 16 protheses of each type were inserted in arteries pretreated with a Fogarty maneuver. The animals were followed for either 1 (n = 10) or 6 (n = 10) months. Immediately after implantation and after 1, 3, and 6 months, intravascular ultrasound (IVUS) and angiography were performed. The vascular specimens were analyzed histologically. RESULTS: Within 10 weeks, the hirudin activity of coated stents dropped 60 % due to external factors; the activity of coated PTFE stentgrafts dropped 20 %. After 1, 3, and 6 months, IVUS and histology revealed a significantly reduced patency of the hirudin-coated stentgrafts compared to the other prostheses. Only IVUS showed a significantly reduced patency of hirudin coated stents after 1 and 3 months compared to plain and CVD-coated PTFE-stentgrafts. The reduced patency was caused by neointimal hyperplasia. CONCLUSIONS: In an experimental setting, hirudin coating did not improve the patency of vascular endoprostheses.

Multimodal comparisons of social phobia subtypes and avoidant personality disorder.

The purpose of the present study was to further clarify the behavioral, physiological, and verbal response of patients with circumscribed social (speech) phobia, generalized social phobia without avoidant personality disorder, and generalized social phobia with avoidant personality disorder. Patients completed a battery of verbal report instruments and participated in two behavioral assessment tests. Measures of avoidance/escape behavior, cardiac response, level of behavioral skill, state anxiety, and positive and negative self-statements during performance were collected. Significant differences across response domains were found between the circumscribed social phobia and the generalized groups. Most of the distinctions were between individuals with circumscribed social phobia and those with both generalized social phobia and avoidant personality disorder, with the former group having less overall psychopathology. In addition, there was substantial overlap of problems between generalized social phobia individuals with and without avoidant personality disorder. Implications for the conceptualization of social phobia are discussed in terms of the differences among social phobia subtypes.

Vertical extrusion using a removable orthodontic appliance.

Vertical extrusion is a useful adjunct to periodontal-restorative procedures, particularly in the anterior segment of the dentition in which esthetic appearance is a primary concern. In the case presented, a multidisciplinary approach was used to treat a tooth successfully with extensive subgingival destruction. Orthodontic and periodontic treatment, as part of an integrated treatment plan, resulted in preservation of esthetic appearance and establishment of sound tooth structure to allow for biologic width and restorative margins.

What makes age diverse teams effective? Results from a six-year research program.

Based on a new model of productivity in age diverse tams, findings from a six-year research program are reported in which data from more than 745 natural teams with 8,848 employees in three different fields (car production, administrative work, financial services) were collected. Moreover, central assumptions of this model were tested with a representative survey of the German workforce (N = 2,000). Results support both significant advantages and disadvantages for age-mixed teams. Based on the findings, the following preconditions for the effectiveness of age diverse teams are identified: high task complexity, low salience and high appreciation of age diversity, a positive team climate, low age-discrimination, ergonomic design of work places, and the use of age differentiated leadership. Based on these insights, we developed a new training for supervisors, which addresses the aforementioned aspects and seeks to improve team performance and health of team members. It was found that the training reduces age stereotypes, team conflicts and enhances innovation. Thus, we can conclude that effective interventions for a successful integration of elderly employees in work groups are available and that combinations of measures that address ergonomic design issues, team composition and leadership are to be strongly recommended for practice.

Role of the carbohydrate part of yeast acid phosphatase.

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.

Study of the carbohydrate part of yeast acid phosphatase.

It has been found that the carbohydrate part of acid phosphatase from yeast Saccharomyces cerevisiae consists of 16 N-glycosidically linked carbohydrate chains containing from 14 to about 150 mannose units. The presence of very small amounts of O-glycosidically linked chains was indicated. Acetolysis studies pointed to a high similarity in the structure of acid phosphatase and mannan carbohydrate chains. A new method is described for cross-linking of acid phosphatase specifically via carbohydrate chains. The possibility to cross-link the enzyme subunits intramolecularly is in accordance with the suggestion that carbohydrate chains play a role in subunit associations.

Purification, carbohydrate composition and kinetic properties of the constitutive yeast acid phosphatase.

Constitutive acid phosphatase was purified from yeast cells grown in a medium supplied with 100 mM phosphate. Specific activity of the pure enzyme was 63.5 mumol/min X mg. The enzyme contains 42.5% of protein, 55% of mannose and 2.5% of N-acetylglucosamine. The carbohydrate chains are N-glycosidically linked to the protein. The pure enzyme shows non-linearity in the Lineweaver-Burk plot, thus indicating the presence of two enzyme forms with Km values of about 0.65 mM and 8.5 mM. The pH optimum of the enzyme is at pH 3.3. The enzyme is much more sensitive to heat denaturation than the repressible acid phosphatase.

Physicochemical and kinetic properties of acid phosphatase from Saccharomyces cerevisiae.

Acid phosphatase from yeast Saccharomyces cerevisiae was purified, and its physicochemical and kinetic properties were investigated. The sedimentation coefficient has been determined to be s0(20,w) = 13.6 S. The diffusion constant has been found to be 3.9 X 10(-7) cm2s-1, and the calculated partial specific volume was v = 0.663 cm3/g. From these data, a molecular weight of 252,000 was calculated. Electrophoresis on gel slabs, with a linear concentration gradient of polyacrylamide (4-30%), showed size heterogeneity of the native enzyme preparation and indicated an apparent molecular weight in the range of 170,000 to 360,000. In the presence of sodium dodecyl sulfate, the molecular weight was in the range of 82,000 to 165,000, indicating dimeric structure of the native enzyme, which was confirmed by cross-linking experiments. Isoelectric focusing demonstrated charge heterogeneity of enzyme preparation. From CD spectrum it was calculated that the enzyme contains about 29% of alpha-helical structure. Excitation at 278 nm gave an emission fluorescence spectrum with a maximum at 340 nm. Amino acid analysis revealed a high content of aspartic acid, serine, and threonine. Glycine is found as the NH2-terminal amino acid. Initial velocity dependence on substrate concentration, as well as on pH, and thermostability studies indicated the presence of at least two enzyme forms in the preparation.

Influence of glycosylation on the oligomeric structure of yeast acid phosphatase.

Secreted yeast acid phosphatase is found to be an octamer under physiological conditions rather than a dimer, as previously believed. The octameric form of the enzyme dissociates rapidly into dimers at pH below 3 and above 5, or by treatment with guanidine hydrochloride or urea, without further dissociation of dimers. Crosslinking experiments revealed that the dissociation of the octamer occurs through very unstable hexamers and tetramers, showing that the octamer is built of dimeric units. Dissociation to dimer was in all cases accompanied with a loss of most of the enzyme activity. The underglycosylated acid phosphatase, with less than eight carbohydrate chains per subunit, secreted from cells treated with moderate tunicamycin concentrations, contained besides octamers a high proportion of the dimers. With decreasing levels of enzyme glycosylation, the proportion of dimers increases and the amount of octamers correspondingly decreases. Furthermore, underglycosylated octamers were found to be significantly less stable than the fully glycosylated ones. This showed that carbohydrate chains play a significant role in the octamer formation in vivo, and in stabilization of the enzyme octameric form.

New chromogen for assay of glucose in serum.

We describe a colorimetric assay for glucose determination in human serum, with use of the chromogen 2-amino-4-hydroxybenzenesulfonic acid (AHBS), glucose oxidase, and peroxidase. With this assay, glucose concentrations less than or equal to 27.8 mmol/L can be measured in serum, with a sample/reagent volume ratio as low as 0.0025. The chromogen itself is easily soluble in water and does not require other components for the color change, making the reagent composition less complex. A single working reagent is used, and the reaction is completed within 10 min at 37 degrees C. The absorbance of the yellow reaction product is measured at 415 nm, and a blank sample measurement is not needed. The average analytical recovery of glucose in different human sera was 97.6%, with no significant interference of reducing compounds in serum. The results of the recommended procedure correlated well with those of the phenol/4-aminoantipyrine method of Trinder.