Determination of free carrier protein in protein-polysaccharide conjugate vaccines by micellar electrokinetic chromatography.

Protein-polysaccharide conjugate vaccines offer the prospect of reducing morbidity and mortality due to bacterial pneumonia and meningitis but, because of their size and heterogeneity, are often a challenge to characterize by traditional analytical methods. Vaccines consisting of Streptococcus pneumoniae, or Neisseria meningiditis polysaccharide covalently linked to formaldehyde-inactivated diphtheria toxoid carrier protein were resolved from non-conjugated toxoid by micellar electrokinetic chromatography. Separation was achieved using alkaline sodium borate solutions containing sodium dodecyl sulfate in excess of the critical micellar concentration. No sample pretreatment was required prior to analysis. Diphtheria toxoid peak migration times were highly reproducible. Measurement of absolute toxoid peak area showed poor precision, but good precision was observed when peak area was normalized against an internal standard (myoglobin). Good linearity was observed over useful ranges of both protein content and injection time.

Quantitation of low level unconjugated polysaccharide in tetanus toxoid-conjugate vaccine by HPAEC/PAD following rapid separation by deoxycholate/HCl.

A simple and rapid acid precipitation method has been applied successfully for separating free capsular polysaccharide of Haemophilus influenzae type b (polyribosyl ribitol phosphate, PRP) from PRP tetanus toxoid conjugate (PRP-T) in a final dosage amount of low-level materials. The unconjugated PRP was found to stay in the supernatant without precipitation, while conjugated PRP-T was fully precipitated. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has been applied for analysis of the PRP content in the supernatant after the separation. This method requires minimum sample handling and is specific, sensitive and reproducible making it suitable for release and stability testing of PRP-T in final containers.

Capillary electrophoretic analysis of meningococcal polysaccharide-diphtheria toxoid conjugate vaccines.

Protein-polysaccharide conjugate vaccines are large, complex molecules that present challenges in terms of characterization. Free solution capillary electrophoresis using alkaline (pH 9-10) borate or glycine/NaOH buffers permitted electrophoresis of diphtheria toxoid (Dt) or meningococcal polysaccharide-Dt conjugates as well-formed, relatively broad peaks. Resolution was observed between the conjugate and the Dt peaks for some serotypes; however, overlap between Dt and one or more conjugate peaks was observed with both buffers. Inclusion of SDS in the separation buffer at a level above the critical micelle concentration allowed separation of Dt and Dt-conjugates of meningococcal serotypes A, C, Y and W135. Using borate/SDS, a linear relationship between peak area and Dt concentration was observed between approximately 2 mg/ml and 20 microg/ml. A linear relationship between Dt peak area and injection times from 10 to 50 seconds was also observed. Capillary electrophoresis may therefore be a useful method for quantifying free protein level in meningococcal-Dt conjugate vaccines.

Quantification of free polysaccharide in meningococcal polysaccharide-diphtheria toxoid conjugate vaccines.

A precipitation method using deoxycholate/HCI has been applied successfully to separate unconjugated free polysaccharide from carrier protein-bound material in meningococcal polysaccharide-diphtheria toxoid conjugate vaccines. The method effectively separated free and bound polysaccharide in conjugate vaccines prepared from Neisseria meningitidis serotypes A, C, W135 and Y. Free polysaccharide remained in the supernatant after deoxycholate treatment while protein-bound polysaccharide was fully precipitated. Testing by both colorimetric assay and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has confirmed the selective loss of protein-bound polysaccharide in samples of conjugate vaccine or conjugate vaccine mixed with known amounts of free polysaccharide. This rapid separation method requires minimum sample handling and is specific, reproducible, and allows assessment of free polysaccharide levels in vaccines at final container concentration.

Preliminary study of the relationship between guilt resolution and depression in a gerontological population.

An individual interview process was used to investigate the relationship between proneness to depression in a gerontological sample and their evaluation of critical life incidents. High and low depressed individuals were asked to recall prideful and guiltful incidents from their past, and discrepancy scores were obtained between their past and present evaluations of those events. Limited evidence supported the hypothesis that individuals with higher proneness to depression showed a greater negative discrepancy in evaluations of critical life incidents. Subjects did not differ in the content of their recalled incidents as a function of guilt type or guilt referent.

A kinematic comparison of ergometer and on-water rowing.

The purpose of this investigation was to compare on-water rowing with ergometer rowing by identifying the important kinematic variables, using vector loop analysis. Two hypotheses were investigated that provided evidence that rowing ergometers are valid simulators of actual rowing and that the trunk segment is the major contributor to the total linear oar velocity. Thirty subjects were filmed rowing on-water and on an ergometer. The film was digitized for X and Y coordinates of the body joint locations for a cycle of rowing from catch to finish. A model of rowing was designed such that the rowing movement could be defined as comprising two closed vector loops. The digitized data were fitted to the vector loop model to derive kinematic variables from the drive phase of rowing. Five kinematic variables that described the contributions of five body segments to the total linear oar velocity were analyzed statistically to find differences in the patterns of on-water and ergometer rowing. The results indicated that the kinematics of the upper arm and forearm segments were significantly different in the two types of rowing. These differences were of minor importance because of the small contributions made by the arms at the catch. The differences at the finish were a result of the lifting of the oar from the water not exhibited in ergometer rowing. The vector loop analysis provided the major finding that the trunk segment contributed significantly more to the drive phase portion of the rowing cycle for both on-water and ergometer rowing although the legs initiated the drive phase.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of a brief relaxation treatment for dental anxiety on measures of state and trait anxiety.

Used a brief deep muscle relaxation procedure to reduce patient (N = 39) anxiety during a dental appointment. State anxiety decreased significantly for a relaxed (treated) group from the waiting room period to the actual contact with the dentist. This reduction in state anxiety was maintained for the duration of the dental visit. There were no changes in trait anxiety. Implications for the reduction of state and trait anxiety in an in vivo situation were discussed.

Inhibition of Bacillus subtilis growth and sporulation by threonine.

A 1-mg/ml amount of threonine (8.4 mM) inhibited growth and sporulation of Bacillus subtilis 168. Inhibition of sporulation was efficiently reversed by valine and less efficiently by pyruvate, arginine, glutamine, and isoleucine. Inhibition of vegetative growth was reversed by asparate and glutamate as well as by valine, arginine, or glutamine. Cells in minimal growth medium were inhibited only transiently by very high concentrations of threonine, whereas inhibition of sporulation was permanent. Addition of threonine prevented the normal increase in alkaline phosphatase and reduced the production of extracellular protease by about 50%, suggesting that threonine blocked the sporulation process relatively early. 2-Ketobutyrate was able to mimic the effect of threonine on sporulation. Sporulation in a strain selected for resistance to azaleucine was partially resistant. Seventy-five percent of the mutants selected for the ability to grow vegetatively in the presence of high threonine concentrations were found to be simultaneously isoleucine auxotrophs. In at least one of these mutants, the threonine resistance phenotpye could not be dissociated from the isoleucine requirement by transformation. This mutation was closely linked to a known ilvA mutation (recombination index, 0.16). This strain also had reduced intracellular threonine deaminase activity. These results suggest that threonine inhibits B. subtilis by causing valine starvation.

The use of paraprofessionals in university mental health settings; a national survey.

A national survey of 218 counseling centers was conducted to determine the extent and nature of paraprofessional use. Responses from 119 centers indicated that only 38% employ paraprofessionals. Those programs that do exist tended to be at larger, public institutions. Paraprofessionals were generally salaried, and they functioned in multiple roles. The selection procedure most frequently employed was professional staff interviews with an emphasis on "interpersonal characteristics" as the selection criterion. Training and evaluation procedures were usually conducted on an ongoing rather than on a "one-shot" basis. Reasons for not employing paraprofessionals varied considerably from center to center.