Comparison of two teaching methods in a chiropractic clinical science course.

This study was done to determine whether the test performances of chiropractic students taught by traditional didactic methods (n = 50) and those undergoing problem-based learning (PBL) (n = 54) would differ. A 25-question multiple-choice test of knowledge synthesis was given to both groups of students. Although the novelty of the PBL approach was a cause of concern among the PBL students, who tended to be uncertain about what was expected of them and to lack confidence in the depth of their knowledge, their performance on the test was equivalent to that of the traditional students. The usefulness of PBL for educating allied health professionals adept at problem solving for future practice needs to be examined further.

Zinc ion mediated amino acid discrimination by threonyl-tRNA synthetase.

Accurate translation of the genetic code depends on the ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids. In order to investigate the basis of amino acid recognition and to understand the role played by the zinc ion present in the active site of threonyl-tRNA synthetase, we have determined the crystal structures of complexes of an active truncated form of the enzyme with a threonyl adenylate analog or threonine. The zinc ion is directly involved in threonine recognition, forming a pentacoordinate intermediate with both the amino group and the side chain hydroxyl. Amino acid activation experiments reveal that the enzyme shows no activation of isosteric valine, and activates serine at a rate 1,000-fold less than that of cognate threonine. This study demonstrates that the zinc ion is neither strictly catalytic nor structural and suggests how the zinc ion ensures that only amino acids that possess a hydroxyl group attached to the beta-position are activated.

tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments. Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM). By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA. Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe). Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA.

The predominant elF4G-specific cleavage activity in poliovirus-infected HeLa cells is distinct from 2A protease.

Human enteroviruses and rhinoviruses rapidly and selectively abolish translation from cellular mRNA upon infection of susceptible cells. Expression of the poliovirus 2A protease (PV 2Apro) is sufficient to cause host translation shutoff through cleavage of elF4G (formerly p220, elF4 gamma) either directly or indirectly through activation of a cellular factor. Evidence exists for both direct and indirect cleavage mechanisms; however, factors presumed to participate in an indirect mechanism have not yet been purified or defined. Here we show that the dominant elF4G cleavage activity in lysates from infected HeLa cells was separable from PV 2Apro by size exclusion chromatography. 2Apro separated into two peak fractions which contained activity which cleaved a peptide substrate derived from the poliovirus polyprotein. These peak 2Apro fractions did not cleave elF4G or an elF4G-derived peptide, as expected, due to the poor efficiency of direct cleavage reactions. Conversely, fractions which contained peak elF4G cleavage activity and only trace amounts of 2Apro efficiently cleaved a peptide substrate derived from the previously mapped elF4G cleavage site and also cleaved a peptide derived from the poliovirus 1D2A region. The dominant elF4G cleavage activity was highly purified through four chromatography steps and found to be devoid of all traces of 2Apro or its precursors. Quantitation of 2Apro from lysates of infected cells showed that during infections in HeLa cells, 2Apro does not reach molar excess over elF4G, as previously shown to be required for direct elF4G cleavage in vitro. Further, infection of HeLa cells in the presence of 2 mM guanidine-HCl, a potent inhibitor of viral RNA replication, suppressed accumulation of 2Apro and its precursor 2ABC below detectable levels but was unable to delay the onset of elF4G proteolysis in vivo. The elF4G cleavage activity was still easily detectable in in vitro assays using fractions from guanidine-treated cells. Thus, the data suggest that poliovirus utilizes two catalytic activities to ensure rapid cleavage of elF4G in vivo. Although it was not directly measurable here, 2Apro likely does cleave a portion of elF4G in cells. However, the data suggest that a cellular factor which can be activated by small quantities of 2Apro constitutes the bulk of the elF4G-specific cleavage activity in infected cells and is responsible for the rapid and efficient elF4G cleavage activity observed in vivo.

Direct cleavage of elF4G by poliovirus 2A protease is inefficient in vitro.

Previously, the purified recombinant 2A proteases (2Apro) of coxsackievirus B4 (CVB4) and human rhinovirus type 2 (HRV2) were shown to cleave synthetic peptides derived from human or rabbit elF4G as well as elF4G protein purified from rabbit reticulocytes. These results were in contrast to previous evidence which supported the view that elF4G cleavage activity in poliovirus-infected HeLa cells required a cellular factor(s) activated by poliovirus (PV) 2Apro. In the present study, recombinant PV 2Apro was shown to cleave either rabbit or human elF4G or their derived peptides in direct cleavage reactions, but cleaved the 4G-derived peptides with 100-fold lower efficiency than with a peptide derived from the poliovirus polyprotein. In these experiments, up to 25-fold molar excess of 2Apro over elF4G protein was required to cause greater than 50% cleavage. CVB4 2Apro was also tested in peptide cleavage assays under the same conditions as PV 2Apro and was found to cleave all elF4G substrates with efficiencies similar to PV 2Apro. Finally, cleavage reactions utilizing recombinant elF4G containing a G486E substitution at the cleavage site for CVB4 and HRV2 proteases resulted in drastically reduced cleavage by PV 2Apro, similar to the reduction previously seen with HRV2 and CVB4 2Apro, confirming that all three viral 2A proteases recognize the same cleavage site on elF4G. These data show that PV 2Apro can directly cleave elF4G in vitro with efficiencies similar to those of CVB 2Apro, but cleavage efficiency of elF4G is approximately 1000-fold lower than cleavage of a peptide derived from the authentic 2A cleavage site on the poliovirus polyprotein.

Defective RNA replication by poliovirus mutants deficient in 2A protease cleavage activity.

2A protease (2Apro) catalyzes the initial cleavage of the poliovirus polyprotein which separates the P1 structural protein precursor from the P2-P3 nonstructural protein precursor. In addition, 2Apro indirectly induces cleavage of the p220 component of eukaryotic initiation factor 4F, which is thought to contribute to the specific inhibition of host cell protein synthesis observed in virus-infected HeLa cells. However, it is unclear whether the trans function of 2Apro which induces host cell shutoff is essential or merely facilitates efficient poliovirus replication. In this study, three point mutations in 2Apro (D38E, Y88L, and Y89L [S. F. Yu and R. E. Lloyd, Virology 182:615-625, 1991]) which cause specific loss of trans but not cis cleavage function were independently introduced into the full-length poliovirus cDNA. In addition, mutations which caused only partial loss of both cis and trans cleavage activities (Y88S) or resulted in a wild-type phenotype (Y88F) were individually introduced. When each of these mutant poliovirus cDNAs was transcribed and translated in vitro, normal proteolytic processing of the viral polyprotein was observed, and p220 was not cleaved in those reactions containing proteases defective in trans function, as expected. Surprisingly, Northern (RNA) blot analysis and reverse transcriptase-PCRs performed after transfection of COS-7 or HeLa cells with these viral RNAs revealed that Y88S and Y88L RNAs replicated at only very low levels. RNA replication could not be detected at all in cells transfected with D38E and Y89L RNAs. Taken together, the results suggest a correlation between the function of 2Apro and productive poliovirus RNA replication in vivo that may be independent of the ability to cause p220 cleavage.

Intrinsic risk factors for exercise-related injuries among male and female army trainees.

Physical training-related injuries are common among army recruits and other vigorously active populations, but little is known about their causation. To identify intrinsic risk factors, we prospectively measured 391 army trainees. For 8 weeks of basic training, 124 men and 186 women (79.3%) were studied. They answered questionnaires on past activities and sports participation, and were measured for height, weight, and body fat percentage; 71% of the subjects took an initial army physical training test. Women had a significantly higher incidence of time-loss injuries than men, 44.6% compared with 29.0%. During training, more time-loss injuries occurred among the 50% of the men who were slower on the mile run, 29.0% versus 0.0%. Slower women were likewise at greater risk than faster ones, 38.2% versus 18.5%. Men with histories of inactivity and with higher body mass index were at greater injury risk than other men, as were the shortest women. We conclude that female gender and low aerobic fitness measured by run times are risk factors for training injuries in army trainees, and that other factors such as prior activity levels and stature may affect men and women differently.

Persistent infection of human erythroblastoid cells by poliovirus.

Human erythroblastoid K562 cells have been recently described as a relatively nonpermissive host for poliovirus replication. During investigations of virus-induced cytopathic effects in this cell line, we discovered that poliovirus easily established persistently infected cultures in K562-Mu cells. In these cultures, most cells remained viable, with overall viability continuously maintained between 67 and 92% over 3 months. Infected K562 cells continued to grow, usually without any major periods of crisis in the culture or large diminutions in cell growth rate. K562-Mu cells produced a slower onset of virus production than observed in HeLa cells, and virus titers in culture supernates rapidly stabilized at levels between 10(5) and 10(6) PFU/ml. In infectious center or limiting dilution assays, only about 10% of K562 cells produced infectious virus after 2 days. However, when assays were extended to 3 to 5 days, most K562 cells in the culture scored as infectious centers, suggesting productive infection of all cells in the culture with delayed kinetics of virus production. Cultures of infected K562 cells could not be cured of virus by prolonged incubation with high-titer neutralizing antibody. Pulse-label SDS-PAGE analysis of infected cultures detected moderate levels of virus protein synthesis which peaked at 9-12 hr postinfection; however, little or no shutoff of host protein synthesis was observed at any time point during infection. Immunoblot analysis with antisera to the p220 subunit of eIF-4F demonstrated extensive but incomplete cleavage of p220 in infected K562 cells at times which correlated with peak viral protein synthesis. Taken together, the results demonstrate a persistent infection in which host cell shutoff does not occur despite significant viral protein synthesis and extensive early degradation of p220.