Kinetics of initiation, propagation, and termination for the [rac-(C(2)H(4)(1-indenyl)(2))ZrMe][MeB(C(6)F(5))(3)]-catalyzed polymerization of 1-hexene.

Metallocene-catalyzed polymerization of 1-alkenes offers fine control of critical polymer attributes such as molecular weight, polydispersity, tacticity, and comonomer incorporation. Enormous effort has been expended on the synthesis and discovery of new catalysts and activators, but elementary aspects of the catalytic processes remain unclear. For example, it is unclear how the catalyst is distributed among active and dormant sites and how this distribution influences the order in monomer for the propagation rates, for which widely varying values are reported. Similarly, although empirical relationships between average molecular weights and monomer have been established for many systems, the underlying mechanisms of chain termination are unclear. Another area of intense interest concerns the role of ion-pairing in controlling the activity and termination mechanisms of metallocene-catalyzed polymerizations. Herein we report the application of quenched-flow kinetics, active site counting, polymer microstructure analysis, and molecular weight distribution analysis to the determination of fundamental rate laws for initiation, propagation, and termination for the polymerization of 1-hexene in toluene solution as catalyzed by the contact ion-pair, [rac-(C(2)H(4)(1-indenyl)(2))ZrMe][MeB(C(6)F(5))(3)] (1) over the temperature range of -10 to 50 degrees C. Highly isotactic (>99% mmmm) poly-1-hexene is produced with no apparent enchained regioerrors. Initiation and propagation processes are first order in the concentrations of 1-hexene and 1 but independent of excess borane or the addition of the contact ion-pair [PhNMe(3)][MeB(C(6)F(5))(3)]. Active site counting and the reaction kinetics provide no evidence of catalyst accumulation in dormant or inactive sites. Initiation is slower than propagation by a factor of 70. The principal termination process is the formation of unsaturates of two types: vinylidene end groups that arise from termination after a 1,2 insertion and vinylene end groups that follow 2,1 insertions. The rate law for the former termination process is independent of the 1-hexene concentration, whereas the latter is first order. Analysis of (13)C-labeled polymer provides support for a mechanism of vinylene end group formation that is not chain transfer to monomer. Deterministic modeling of the molecular weight distributions using the fundamental rate laws and kinetic constants demonstrates the robustness of the kinetic analysis. Comparisons of insertion frequencies with estimated limits on the rates of ion-pair symmetrization obtained by NMR suggest that ion-pair separation prior to insertion is not required, but the analysis requires assumptions that cannot be validated.

Assessing medical students' awareness of and sensitivity to diverse health beliefs using a standardized patient station.

PURPOSE: To assess students' performances on a health-beliefs communication OSCE station to determine whether there were differences in cultural competence based on the students' ethnic backgrounds. METHOD: A total of 71 students completed a health-beliefs communication OSCE station in which they were required to address the health beliefs and cultural concerns of a standardized patient (SP) portraying an African American woman with diabetes. The SPs rated students' performances on a ten-item interview assessment checklist. Scores on the station were standardized within SPs to adjust for differences in their use of the rating scale. A factor analysis was performed to determine conceptual constructs on the interview assessment checklist. Subscale means were computed for each student. T-tests of these subscale scores were conducted to investigate gender and ethnic differences between subgroups of students. The underrepresented minority (URM) students (five African Americans and three Mexican Americans) were compared with all other students, and the white students were compared with all others. To assess the magnitudes of the differences between subgroups, effect sizes (ES(m)) were computed for means comparisons. RESULTS: Factor analysis formed two factors: Disease Beliefs and Management, and Cultural Concerns. Two remaining items loaded on a third factor that had reliability too low to support further analysis. Meaningful differences were found in cultural sensitivity based on students' ethnic backgrounds. The URM students performed better than did all other students in addressing the patient's concerns about altering culturally-based dietary behaviors for diabetes self-care [URM students' mean standardized score (SD) = 0.42 (0.15); all others = -0.01 (0.67); ES(m) = 1.05]. White students performed better than did all other students in assessing the patient's concerns about using insulin to control her blood sugar levels [white students' mean standardized score (SD) = 0.13 (0.40); all others = -0.10 (0.64); ES(m) = 0.4]. CONCLUSION: Cultural competency deficits and differences were measurable using a health-beliefs communications station, and these differences were meaningful enough to warrant faculty discussion and research about how to ensure that students master this competency.

The difficulty of sustaining curricular reforms: a study of "drift" at one school.

In 1997, five years after a major curricular reform at the University of Michigan Medical School, the authors revisited the Goals for Medical Education (written by faculty to guide the reform process) to identify factors that had facilitated or hindered their achievement. By reviewing responses to identical questionnaires circulated to faculty in 1993 and again in 1997, they learned that considerably more lectures were being used to deliver curricular content in the first-year curriculum than the faculty thought was ideal, and that less social science, humanities, and ethics material was being presented in the first year than the faculty thought was ideal. The authors also learned that consensus between faculty basic scientists and faculty clinicians about the content that would make up an ideal first-year curriculum had diverged since adoption of the new curriculum. Movement toward decreasing the amounts of social sciences, humanities, and ethics in the first year of medical school was particularly pronounced among the basic scientists, who felt this material was being taught prematurely and at the expense of essential basic science content. In contrast, by 1997 much closer agreement had developed between the two groups regarding time they would allocate for lectures; this agreement unfortunately reflected a stagnation in the adoption of active learning methods. Movement toward increasing the amount of time for lectures in the first-year curriculum was particularly pronounced among the clinicians, who reported feeling more and more pressured to bring in clinical revenues. Based on faculty comments and the school's experience with centralized governance and centralized funding, the authors propose a direct linkage between institutional funding to departments and the teaching effort of faculty in the departments, and sufficient, centralized funding to relieve pressure on faculty and to foster educational creativity. They maintain that this may be the most effective way to guarantee ongoing innovation, support interdisciplinary teaching, and subsequently move the curriculum and teachers completely away from content that is isolated within traditional department structures. At the same time they acknowledge that changing faculty attitudes presents a challenge.

Upper respiratory tract infections in adolescents.

While upper respiratory tract infections (URTIs) cause much infectious morbidity in infants and young children, adolescents are not immune to infections. Adolescents experience two to four episodes of viral nasopharyngitis annually. In addition to group A streptococcus (GAS), pharyngitis may occur with other streptococci, Arcanobacterium haemolyticum, Epstein-Barr virus, Neisseria gonorrhoeae, and other pathogens. Uvulits, typically in association with GAS, occurs occasionally. Peritonsillar abscess is the most common deep neck space infection seen in adolescents, but retropharyngeal and parapharyngeal abscesses also occur, causing major morbidity. Adolescents experience fewer cases of otitis media than younger children. Rhinosinusitis occurs commonly in adolescents, occasionally leading to chronic sinusitis and serious sequelae such as osteomyelitis. This article reviews the major URTIs likely to be encountered by physicians caring for adolescents. For each entity there is a brief description of the epidemiology, morbidity, microbiology, clinical and laboratory features, treatment, and prevention.

The influence of task formats on the accuracy of medical students' self-assessments.

PURPOSE: Accurate self-assessment is an essential skill for the self-directed learning activities and appropriate patient referral decisions of practicing physicians. However, many questions about the characteristics of self-assessment remain unanswered. One is whether self-assessment is a generalizable skill or dependent on the characteristics of the task. This study examines the self-assessment skills of medical students across two task formats: performance-based and cognitive-based. METHOD: In 1997 and 1998, fourth-year medical students at the University of Michigan assessed their own performances on ten stations of a clinical examination. The examination used two formats: performance tasks (the examination or history taking of standardized patients) and cognitive tasks (interpreting vignettes or test results and then answering paper-and-pencil questions). Three measures of self-assessment accuracy were used: a bias index (average difference between the students' estimates of their performances and their actual scores), a deviation index (average absolute difference between estimate and actual score), and an actual score-estimate-of-performance correlation (the correlation between the estimate and actual scores). RESULTS: The student bias and deviation indices were similar on the cognitive and the performance tasks. The correlations also indicated similarity between the two types of tasks. CONCLUSION: The results indicate that the format of the task does not influence students' abilities to self-assess their performances, and that students' self-assessment abilities are consistent over a range of skills and tasks. The authors also emphasize the importance of sampling tasks while conducting self-assessment research.

A comparative study of human muscle and brain creatine kinases expressed in Escherichia coli.

We report the expression of the human muscle (CK-MM) and brain (CK-BB) creatine kinases in Escherichia coli. The proteins have been purified to apparent homogeneity and several of their physical and kinetic properties investigated. In the process, we have conclusively verified the correct DNA sequence of the genes encoding the respective isozymes, and determined the correct primary structure and mass of the gene products. Alignment of the primary sequences of these two enzymes shows 81% sequence identity with each other, and no obvious gross structural differences. However, Western blot analyses demonstrated the general lack of antigenic cross-reactivity between these isozymes. Preliminary kinetic analyses show the K(m) and k(cat) values for the creatine and MgATP substrates are similar to values reported for other isozymes from various tissues and organisms. The human muscle and brain CKs do not, however, exhibit the synergism of substrate binding that is observed, for example, in rabbit muscle creatine kinase.

Teaching residents to teach. An instructional program for training pediatric residents to precept third-year medical students in the ambulatory clinic.

OBJECTIVE: To provide second- and third-year pediatric residents with practical teaching skills for precepting third-year medical students in the outpatient clinic. DESIGN: Educational intervention with 3-month follow-up of participants. SETTING: University teaching hospital. PARTICIPANTS: Second- and third-year pediatric residents. INTERVENTION: A curriculum for a half-day workshop to provide residents with 6 key clinical teaching skills. Residents participated in the workshop and then were observed by trained faculty as they precepted third-year medical students in the pediatric clinic. MAIN OUTCOME MEASURE: Direct observation of resident-student precepting encounters, noting the presence or absence of their use of clinical teaching skills taught in the workshop. RESULTS: Twenty-one of 23 pediatric residents participated in the workshop. Observation of 56 resident teaching encounters before and after the workshop showed that the residents improved their clinical teaching skills. Residents valued the workshop, and many suggested it should also be considered for faculty development. CONCLUSIONS: Residents can be taught clinical teaching skills in a half-day workshop. These skills also are applicable in various clinical venues. With the increasing interest in using community-based primary care physicians for student and resident education, this curriculum is well suited for training practicing clinicians to teach in their own practice sites.

HLA-A typing: comparison between serology, the amplification refractory mutation system with polymerase chain reaction and sequencing.

In this study we typed HLA-A polymorphisms by a new sequence-based typing (SBT) method, which involved one PCR reaction and four sequencing reactions covering exon 2 and exon 3. This method allowed complete identification of all known HLA-A alleles and revealed the presence of a new allele, named HLA-A*2608. We also introduced sequencing of exon 4 for some samples in order to discriminate the allelic pairs that are identical in exon 2 and 3, thus improving SBT resolution. Finally, we compared the results obtained by SBT with data obtained by serological typing and the amplification refractory mutation system (ARMS-PCR). Together, our results suggest that the SBT here described provides an optimal HLA-A typing technique that may be useful in selecting donor-recipient pairs in bone marrow transplantation between unrelated individuals.

A generic sequencing based typing approach for the identification of HLA-A diversity.

Sequencing Based Typing (SBT) is a generic approach for the identification of HLA-A polymorphism. This approach includes the high resolution typing of the HLA-A broad reacting groups, HLA-A subtypes and will identify new alleles directly. The SBT approach described here uses a locus specific amplification of DNA from exon 1 to exon 5. The resulting 2,022 bp PCR product serves as a template for the subsequent sequencing reactions. Amplification is followed by direct sequencing of exons 2, 3 and 4 in both orientations with fluorescently labeled primers to define all polymorphic positions leading to a high resolution typing result. In this study the sequence of exons 2 and 3 of a panel of 49 cell lines was determined. In addition, the exon 4 region of 35 cell lines was also sequenced to evaluate the exon 4 polymorphism. The HLA-A type of most of the cells could be identified by sequencing only exons 2 and 3. However, the sequence of exon 4 was required to discriminate A*0201 from A*0209 and A*0207 from A*0215N. In this panel, an identical new "HLA-A*0103" was identified in two Caucasian samples.

Sequence verification of human creatine kinase (43 kDa) isozymes by high-resolution tandem mass spectrometry.

Amino acid sequencing by recombinant DNA technology, although dramatically useful, is subject to base reading errors, is indirect, and is insensitive to posttranslational processing. Mass spectrometry techniques can provide molecular weight data from even relatively large proteins for such cDNA sequences and can serve as a check of an enzyme's purity and sequence integrity. Multiply-charged ions from electrospray ionization can be dissociated to yield structural information by tandem mass spectrometry, providing a second method for gaining additional confidence in primary sequence confirmation. Here, accurate (+/- 1 Da) molecular weight and molecular ion dissociation information for human muscle and brain creatine kinases has been obtained by electrospray ionization coupled with Fourier-transform mass spectrometry to help distinguish which of several published amino acid sequences for both enzymes are correct. The results herein are consistent with one published sequence for each isozyme, and the heterogeneity indicated by isoelectric focusing due to 1-Da deamidation changes. This approach appears generally useful for detailed sequence verification of recombinant proteins.

A novel activity of OmpT. Proteolysis under extreme denaturing conditions.

A novel property of the bacterial outer membrane protein T, OmpT, has been discovered. It is active under extreme denaturing conditions. This finding emerged during characterization of a protease associated with the degradation of recombinant proteins expressed as inclusion bodies in Escherichia coli. These inclusion body proteins are stable to proteolytic degradation until they are solubilized by denaturation. The protease that degrades them under denaturing conditions was identified as OmpT on the basis of substrate specificity, inhibitor profile, and confirmation that its N-terminal sequence is identical with that of OmpT. A previously unknown property of this enzyme, OmpT's preference for denatured substrates, may provide a clue to its physiological function. To facilitate further characterization of this proteolytic activity, we have optimized a system to extract and assay OmpT under denaturing conditions using a soluble substrate, rabbit muscle creatine kinase.

Isolation and structure elucidation of two new calpain inhibitors from Streptomyces griseus.

Two new peptides, a diketopiperazine of N-methyltyrosine (1) and a tetrapeptide containing N-methyltyrosine (2), were isolated from an actinomycete strain Streptomyces griseus. These compounds inhibit the enzyme calpain in the micromolar range and were characterized on the basis of spectroscopic analysis, amino acid analysis and sequencing. The structure of the tetrapeptide N-methyltyrosyl-N-methyltyrosyl-leucyl-alanine (2), was also confirmed by total synthesis.

Arteriolar dilation mediated by capsaicin and calcitonin gene-related peptide in rats.

In addition to altering vascular tone by stimulating primary afferent nerves and acting through reflex pathways, capsaicin acts locally. We examined effects of topically applied capsaicin on arteriolar diameter in striated muscle and tested the hypothesis that capsaicin can alter microvascular tone by releasing substance P (SP) or calcitonin gene-related peptide (CGRP). In anesthetized rats, the right cremaster muscle was exposed and suspended in a tissue bath filled with a physiological salt solution. Diameters of third-order arterioles were displayed and measured using in vivo video microscopy. In 17 of 20 rats, addition of capsaicin (3 x 10(-7) M) to the bath dilated arterioles (85 +/- 14% above control). Failure of a second administration of capsaicin to produce a sustained dilation in 6 of 7 arterioles that had previously dilated to capsaicin is consistent with the hypothesis that this agent causes depletion of an endogenous vasodilator. Pretreatment with an SP inhibitor did not alter capsaicin-induced dilation. CGRP (1 x 10(-10) to 2 x 10(-8) M) caused dilation similar to that caused by capsaicin. Pretreatment with a CGRP inhibitor to the bath prevented capsaicin-induced dilation, but not constriction. These results suggest that capsaicin can dilate microvessels by releasing CGRP, which can modulate tone.