Genetic and physical analysis of the bacteriophage P22 tail protein

A plasmid based genetic system was developed for the tail protein of the Salmonella typhimurium bacteriophage P22 and used to isolate and characterize tail protein mutants. The tail protein is a trimeric structural protein of the phage and an endorhamnosidase whose activity is essential for infection. The gene for the tail protein has previously been cloned into a plasmid expression vector and sequenced. A plate complementation assay for tail protein produced from the cloned gene was developed and used to isolate 27 tail protein mutants following mutagenesis of the cloned gene. These mutations were mapped into 12 deletion intervals using deletions which were made on plasmids in vitro and crossed onto P22. The base substitutions were determined by DNA sequencing. The majority of mutants had missense or nonsense mutations in the protein coding portion of the gene; however four of the mutants were in the putative transcription terminator. The oligomeric state of tail protein from the 15 missense mutants was investigated using SDS and nondenaturing polyacrylamide gel electrophoresis of cell lysates. Wild-type tail protein retains its trimeric structure in SDS gels at room temperature. Two of the mutant proteins also migrated as trimers in SDS gels, yet one of these had a considerably faster mobility than wild-type trimer. Its migration was the same as wild-type in a nondenaturing gel, so it is thought to be a trimer which is partially denatured by SDS. Four of the mutants produced proteins which migrate at the position of a monomer in an SDS gel but cannot be seen on a nondenaturing gel. These proteins are thought to be either monomers or soluble aggregates which cannot enter the nondenaturing gel. The remainder of mutants produce protein which is degraded. The mutant tail protein which had normal trimeric mobility on SDS and nondenaturing gels was purified. This protein has essentially wild-type ability to attach to phage capsids, but its endorhamnosidase activity is only 4% of wild-type. ^

PROTECTION FOR THE "TURNAROUND WORKER"

This document characterizes the types and magnitude of exposures to toxic substances faced by various turnaround workers in the petroleum industry. The safety and health professional is acquainted with the basic petroleum refinery, refinery equipment and causes for scheduled or emergency turnarounds. Common work procedures during the turnaround are discussed with emphasis on performing the job safely and without adverse consequence to worker health. A listing of commonly encountered substances with a corresponding summary of recognized exposure limits, recommended personal protection, hygiene measures and hazard information is provided to equip the safety and health professional with a ready checklist for worker protection.^ The use of this document was tested and found to improve the average prescription of work procedures and equipment from 38% appropriate (prior to receipt of information) to 84% appropriate (post receipt of information). All participants statistically improved their ability to protect the health and safety of the turnaround worker. ^

Generation of an hTERT-restricted, CMV-augmented oncolytic adenovirus capable of tumor-specific transgene expression: Development, characterization, and optimization

Current shortcomings in cancer therapy require the generation of new, broadly applicable, potent, targeted treatments. Here, an adenovirus is engineered to replicate specifically in cells with active human telomerase promotion using a modified hTERT promoter, fused to a CMV promoter element. The virus was also modified to contain a visible reporter transgene, GFP. The virus, Ad/hTC-GFP-E1 was characterized in vitro and demonstrated tumor specific activity both by dose and over time course experiments in a variety of cell lines. In vivo, Ad/hTC-GFP-E1 was affected at suppressing tumor growth and providing a survival benefit without causing any measurable toxicity. To increase the host range of the vector, the fiber region was modified to contain an RGD-motif. The vector, AdRGD/hTC-GFP-E1, was recharacterized in vitro, revealing heightened levels of infectivity and toxicity however maintaining a therapeutic window between cancer and normal cell toxicity. AdRGD/hTC-GFP-E1 was administered in vivo by limb perfusion and was observed to be tumor specific both in expression and replication. To further enhance the efficacy of viral vectors in lung delivery, asthma medications were investigated for their abilities to enhance transgene delivery and expression. A combination of bronchodilators, mast cell inhibitors, and mucolytic agents was devised which demonstrated fold increases in expression in immunocompetent mouse lungs as single agents and more homogenous, intense levels of expression when done in combination of all agents. To characterize the methods in which some cancers are resistant or may become resistant to oncolytic treatments, several small molecule inhibitors of metabolic pathways were applied in combination with oncolytic infection in vitro. SP600125 and PD 98059, respective JNK and ERK inhibitors, successfully suppressed oncolytic toxicity, however did not affect infectivity or transgene expression of Ad/hTC-GFP-E1. JNK and ERK inhibition did significantly suppress viral replication, however, as analyzed by lysate transfer and titration assays. In contrast, SB 203580, an inhibitor for p38, did not demonstrate any protective effects with infected cells. Flow cytometric analysis indicated a possible correlation with G1 arrest and suppressed viral production, however more compounds must be investigated to clarify this observation. ^

Sleep quality and quantity and associated factors among high school students from south Texas

Background. Insufficient and poor quality sleep among adolescents affects not only the cognitive functioning, but overall health of the individual. Existing research suggests that adolescents from varying ethnic groups exhibit differing sleep patterns. However, little research focuses on sleep patterns and associated factors (i.e. tobacco use, mental health indicators) among Hispanic youth. ^ Methods. The study population (n=2,536) included students in grades 9-12 who attended one of the three public high schools along the Texas-Mexico border in 2003. This was a cross sectional study using secondary data collected via a web-based, confidential, self-administered survey. Separate logistic regression models were estimated to identify factors associated with reduced (<9 hours/night) and poor quality sleep on average during weeknights. ^ Results. Of participants, 49.5% reported reduced sleep while 12.8% reported poor quality sleep. Factors significantly (p<0.05) associated with poor quality sleep were: often feeling stressed or anxious (OR=5.49), being born in Mexico (OR=0.65), using a computer/playing video games 15+ hours per week (OR=2.29), working (OR=1.37), being a current smoker (OR=2.16), and being a current alcohol user (OR=1.64). Factors significantly associated with reduced quantity of sleep were: often feeling stressed or anxious (OR=2.74), often having headaches/stomachaches (OR=1.77), being a current marijuana user (OR=1.70), being a current methamphetamine user (OR=4.92), and being a current alcohol user (OR=1.27). ^ Discussion. Previous research suggests that there are several factors that can influence sleep quality and quantity in adolescents. This paper discusses these factors (i.e. work, smoking, alcohol, etc.) found to be associated with poor sleep quality and reduced sleep quantity in the Hispanic adolescent population. A reduced quantity of sleep (81.20% of the participants) and a poor quality of sleep (12.80% of the participants) were also found in high school students from South Texas. ^