Involvement of a region of 23S ribosomal RNA of {\it E. coli\/} in decoding of termination codons

Involvement of E. coli 23S ribosomal RNA (rRNA) in decoding of termination codons was first indicated by the characterization of a 23S rRNA mutant that causes UGA-specific nonsense suppression. The work described here was begun to test the hypothesis that more 23S rRNA suppressors of specific nonsense mutations can be isolated and that they would occur non-randomly in the rRNA genes and be clustered in specific, functionally significant regions of rRNA.^ Approximately 2 kilobases of the gene for 23S rRNA were subjected to PCR random mutagenesis and the amplified products screened for suppression of nonsense mutations in trpA. All of the suppressor mutations obtained were located in a thirty-nucleotide part of the GTPase center, a conserved rRNA sequence and structure, and they and others made in that region by site-directed mutagenesis were shown to be UGA-specific in their suppression of termination codon mutations. These results proved the initial hypothesis and demonstrated that a group of nucleotides in this region are involved in decoding of the UGA termination codon. Further, it was shown that limitation of cellular availability or synthesis of L11, a ribosomal protein that binds to the GTPase center rRNA, resulted in suppression of termination codon mutations, suggesting the direct involvement of L11 in termination in vivo.^ Finally, in vivo analysis of certain site-specific mutations made in the GTPase center RNA demonstrated that (a) the G$\cdot$A base pair closing the hexanucleotide hairpin loop was not essential for normal termination, (b) the "U-turn" structure in the 1093 to 1098 hexaloop is critical for normal termination, (c) nucleotides A1095 and A1067, necessary for the binding to ribosomes of thiostrepton, an antibiotic that inhibits polypeptide release factor binding to ribosomes in vitro, are also necessary for normal peptide chain termination in vivo, and (d) involvement of this region of rRNA in termination is determined by some unique subset structure that includes particular nucleotides rather than merely by a general structural feature of the GTPase center.^ This work advances the understanding of peptide chain termination by demonstrating that the GTPase region of 23S rRNA participates in recognition of termination codons, through an associated ribosomal protein and specific conserved nucleotides and structural motifs in its RNA. ^

Source of health insurance and immunization rates: Findings from the 1996 Texas immunization survey

This study was a further investigation of the 1996 Texas Immunization Survey conducted by the Associateship for Disease Control and Prevention of the Texas Department of Health. The 1996 survey was conducted through 4,599 completed telephone interviews of families with a child between the ages of 3–35 months concerning the immunization status of Texas children. The present study determined differences in immunization rates for children aged 3–35 months for the last shot in the immunization series that should be completed before 2 years of age, a total of four shots, both overall and for different health insurance groups. Life tables were used to determine the percentage and distribution over time of completed vaccination rates for each shot. Emphasis was placed on the proportion of children that were immunized at the end of the recommended range of the immunization schedule, and at 2 years of age. Univariate and multivariate analysis was also performed in order to ascertain which risk factors predict whether or not a child will be immunized. RESULTS: Between 80–90% were immunized for the last shot of Hepatitis B; Measles, Mumps, and Rubella; and Polio at 2 years of age. Approximately 2/3 of the sample was immunized for Diphtheria, Pertussis, and Tetanus. Most of the children were immunized by the end of the recommended range of the immunization schedule except for Measles, Mumps, and Rubella. Children of parents with private indemnity insurance were significantly more likely to have received two of the four shots; children of uninsured parents were significantly less likely to have received three of the four shots. In multivariate analysis, maternal education was the only variable that consistently predicted immunization status for the different shots. Results indicate that a substantial gap exists for immunization rates between children with private insurance and uninsured children, despite recent policy changes to provide immunizations free of charge. Health care providers should pay extra attention to the poor and uninsured to make sure that all children receive timely immunizations. ^

EXPRESSION OF THE HUMAN PLACENTAL LACTOGEN GENES

The placenta is the site of synthesis of various peptide and steroid hormones related to pregnancy. Human placental lactogen (hPL) is the predominant peptide hormone secreted by term placenta and its synthesis is tissue-specific and coupled to placenta development. The objective of this work was to study the structure and expression of the hPL.^ Poly(A('+))RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to pre-hPL and a minor band comigrating with mature hPL, represent (TURN)15% of the total radioactively labeled proteins. Analysis of the poly(A('+))RNA showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with {('32)P}-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460 and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A('+))RNA was used to construct a cDNA library, of which approximately 5% of the clones were found to hybridize to hPL DNA sequences. Heteroduplexes constructed between a clone containing a 815 bp hPL cDNA insert and a hPL genomic DNA clone revealed four small intervening sequences which can account for the lengths observed in hnRNA molecules.^ Recombinant plasmid HCS-pBR322 containing a 550 bp insert of a cDNA transcript of human placental lactogen (hPL) mRNA was ('3)H-labeled an hybridized in situ to human chromosome preparations. These experiments allowed assignment of the hPL and growth hormone (hGH) genes, which have over 90% nucleotide homology in their coding sequences, to band q22-24 of chromosome 17. A gene copy number experiment showed that both genes are present in (TURN)3 copies per haploid genome.^ Experiments were designed to determine if all members of the hPL gene cluster, consisting of four non-allelic genes, are transcribed in term placenta. Advantage was taken of differences in restriction endonuclease sites in the coding portions of the different hPL genes, to distinguish the putative cDNAs of the transcriptionally active genes. Two genes were found to be represented in the cDNA library and their cDNA transcripts were isolated and characterized. Three independent methods showed that their corresponding mRNAs are about equally represented in the hPL mRNA population. The two cDNAs code for prehPL proteins which differ at a single amino acid position. However the secreted hPLs have identical amino acid sequences. A tetramer insertion duplication was found in a palindrome area of the 3' untranslated region of one of the hPL mRNAs. ^

Determinants of patients' expectations about total knee arthroplasty outcomes

Knee osteoarthritis (OA) is the most prevalent form of arthritis in the US, affecting approximately 37% of adults. Approximately 300,000 total knee arthroplasty (TKA) procedures take place in the United States each year. Total knee arthroplasty is an elective procedure available to patients as an irreversible treatment after failure of previous medical treatments. Some patients sacrifice quality of life and endure many years of pain before making the decision to undergo total knee replacement. In making their decision, it is therefore imperative for patients to understand the procedure, risks and surgical outcomes to create realistic expectations and increase outcome satisfaction. ^ From 2004-2007, 236 OA patients who underwent TKA participated in the PEAKS (Patient Expectations About Knee Surgery) study, an observational longitudinal cohort study, completed baseline and 6 month follow-up questionnaires after the surgery. We performed a secondary data analysis of the PEAKS study to: (1) determine the specific presurgical patient characteristics associated with patients’ presurgical expectations of time to functional recovery; and (2) determine the association between presurgical expectations of time to functional recovery and postsurgical patient capabilities (6 months after TKA). We utilized the WOMAC to measure knee pain and function, the SF-36 to measure health-related quality of life, and the DASS and MOS-SSS to measure psychosocial quality of life variables. Expectation and capability measures were generated from panel of experts. A list of 10 activities was used for this analysis to measure functional expectations and postoperative functional capabilities. ^ The final cohort consisted of 236 individuals, was predominately White with 154 women and 82 men. The mean age was 65 years. Patients were optimistic about their time to functional recovery. Expectation time of being able to perform the list activities per patient had a median of less than 3 months. Patients who expected to be able to perform the functional activities by 3 months had better knee function, less pain and better overall health-related quality of life. Despite expectation differences, all patients showed significant improvement 6 months after surgery. Participant expectation of time to functional recovery was not an independent predictor of capability to perform functional activities at 6 months. Better presurgical patient characteristics were, however, associated with a higher likelihood of being able to perform all activities at 6 months. ^ This study gave us initial insight on the relationship between presurgical patient characteristics and their expectations of functional recovery after total knee replacement. Future studies clarifying the relationship between patient presurgical characteristics and postsurgical functional capabilities are needed.^

An evaluation of a behavioral change intervention for smokeless tobacco use

During the last three decades considerable attention has been placed on the reduction of tobacco use due to cigarette smoking. During this time, studies have been funded and programs have been developed that focus on both prevention and cessation of cigarette smoking. This intense focus has led to a significant decline in cigarette smoking. But now, use of another form of tobacco--smokeless tobacco--is gaining in popularity.^ In 1989, the National Cancer Institute funded a research study at The University of Texas M. D. Anderson Cancer Center, called Working Well, to develop, implement, and evaluate worksite health promotion programs aimed at reducing cancer risks. As part of this program, a behavioral intervention for smokeless tobacco use was developed. This dissertation evaluates the impact of that behavioral change intervention for smokeless tobacco use.^ Data collected during the Working Well program were analyzed to determine the effect of the intervention. The primary outcomes analyzed were smokeless tobacco cessation, stages of change movement, and prevalence. The secondary outcomes analyzed included the prediction of smokeless tobacco use, stage movement, and cessation. Primary outcome analyses were conducted using the worksite as the unit of analysis, while the secondary analyses were conducted using the individual as the unit of analysis.^ Approximately 20% of the male population used smokeless tobacco. Results of intervention analyses indicate that the Working Well program produced no intervention effect on any of the primary outcomes. At the final observation, the experimental worksites achieved a quit rate of 27%, while the control worksites achieved a quit rate of 26% (P = 0.78). Stage movement for the experimental worksites was 49%, while the control worksites experienced stage movement of 43% (P = 0.20). The results of the analyses on smokeless tobacco prevalence followed the same pattern. Predictors of smokeless tobacco use, cessation, and stage movement were also identified.^ Based on the results found in this study, smokeless tobacco should remain a research priority. Future research should focus on smokeless tobacco use, including the identification of the determinants of smokeless tobacco use and the development of measures and effective intervention strategies. ^

Rabies control in the developing world: Results of a cross-sectional study in Bohol, Philippines

Rabies remains a significant problem in much of the developed world, where canine rabies is not well controlled, and the bite of an infected dog is the most common means of transmission. The Philippines continues to report several hundred cases of human rabies every year, and many more cases go undetected. In recent years, the province of Bohol has been targeted by the Philippine government and the World Health Organization for a rabies eradication program. ^ The primary objective of this dissertation research was to describe factors associated with dog vaccination coverage and knowledge, attitudes, and practices regarding rabies among households in Bohol, Philippines. Utilizing a cross-sectional cluster survey design, we sampled 460 households and 541 dogs residing within dog-owning households. ^ Multivariate linear regression was used to examine potential associations between knowledge, attitudes, and practices (KAPs) and variables of interest. Forty-six percent of households knew that rabies was spread through the bite of an infected dog. The mean knowledge score was 8.36 (SD: ± 3.4; range: 1–24). We found that having known someone with rabies was significantly associated with an almost one point increase in the knowledge score (β = 0.88; p = 0.02). The mean attitudes score was 5.65 (SD: ± 0.63; range: 2–6), and the mean practices score was 7.07 (SD: ± 1.7; range: 2–9). Both the attitudes score and the practices score were positively and significantly associated with only the knowledge score and no other covariates. ^ Multivariate logistic regression was used to examine associations between dog vaccination coverage and variables of interest. Approximately 71% of owned dogs in Bohol were reported as vaccinated at some time during their lives. We found that a dog's age was significantly associated with vaccination, and the odds of vaccination increased in a linear fashion with age. We also found that dogs had approximately twice the odds of being vaccinated if they were confined both day and night to the household premises or if the owner was employed; however, these results were only marginally significant (p = 0.07) in the multivariate model. ^ Finally, a systematic review was conducted on canine rabies vaccination and dog population demographics in the developing world. We found few studies on this topic, especially in countries where the burden of rabies is greatest. Overall, dog ownership is high. Dogs are quite young and do not live very long due to disease and accidents. The biggest deterrent to vaccination is the rapid dog population turnover. ^ It is our hope that this work will be used to improve dog rabies vaccination programs around the world and save lives, both human and canine.^

High-sensitivity rod photoreceptor input to the blue-yellow color opponent pathway in macaque retina.

Small bistratified cells (SBCs) in the primate retina carry a major blue-yellow opponent signal to the brain. We found that SBCs also carry signals from rod photoreceptors, with the same sign as S cone input. SBCs exhibited robust responses under low scotopic conditions. Physiological and anatomical experiments indicated that this rod input arose from the AII amacrine cell-mediated rod pathway. Rod and cone signals were both present in SBCs at mesopic light levels. These findings have three implications. First, more retinal circuits may multiplex rod and cone signals than were previously thought to, efficiently exploiting the limited number of optic nerve fibers. Second, signals from AII amacrine cells may diverge to most or all of the approximately 20 retinal ganglion cell types in the peripheral primate retina. Third, rod input to SBCs may be the substrate for behavioral biases toward perception of blue at mesopic light levels.

CLINICAL TRIAL ENROLLMENT IN A MULTIDISCIPLINARY PROSTATE CANCER

Purpose: Clinical oncology trials are hampered by low accrual rates. Less than 5% of adult cancer patients are treated on a clinical trial. We aimed to evaluate clinical trial enrollment in our Multidisciplinary Prostate Cancer Clinic and to assess if a clinical trial initiative, introduced in 2006, increased our trial enrollment.Methods: Prostate cancer patients with non-metastatic disease who were seen in the clinic from 2004 to 2008 were included in the analysis. Men were categorized by whether they were seen before or after the clinical trial enrollment initiative started in 2006. The initiative included posting trial details in the clinic, educating patients about appropriate clinical trial options during the treatment recommendation discussion, and providing patients with documentation of trials offered to them. Univariate and multivariate (MVA) logistic regression analysis evaluated the impact of patient characteristics and the clinical trial initiative on clinical trial enrollment.Results: The majority of the 1,370 men were white (83%), and lived within the surrounding counties or state (69.4%). Median age was 64.2 years. Seventy-three point five percent enrolled in at least one trial and 28.5% enrolled in more than one trial. Sixty-seven percent enrolled in laboratory studies, 18% quality of life studies, 13% novel studies, and 3.7% procedural studies. On MVA, men seen in later years (p < 0.0001) were more likely to enroll in trials. The proportion of men enrolling increased from 38.9% to 84.3% (p<0.0001) after the clinical trial initiative. On MVA, older men (p < 0.0001) were less likely to enroll in clinical trials. There was a trend toward men in the high-risk group being more likely to participate in clinical trials (p = 0.056). There was a second trend for men of Hispanic, Asian, Native American and Indian decent being less likely to participate in clinical trials (p = 0.054).Conclusion: Clinical trial enrollment in the multidisciplinary clinic increased after introduction of a clinical trial initiative. Older men were less likely to enroll in trials. We speculate we achieved high enrollment rates because 1) specific trials are discussed at time of treatment recommendations, 2) we provide a letter documenting offered trials and 3) we introduce patients to the research team at the same clinic visit if they are interested in trial participation.

c-{\it mos\/} RNA expression in somatic cells and its physiological significance in cell cycle progression

The c-mos proto-oncogene, which is expressed at relatively high levels in male and female germ cells, plays a key role in oocyte meiotic maturation. The c-mos gene product in oocytes (p39$\sp{\rm c-mos}$) is necessary and sufficient to initiate meiosis. p39$\sp{\rm c-mos}$ is also an essential component of the cytostatic factor, which is responsible for arresting vertebrate oocytes at the second meiotic metaphase by stabilizing the maturation promoting factor (MPF). MPF is a universal regulator of both meiosis and mitosis. Much less is understood about c-mos expression and function in somatic cells. In addition to gonadal tissues, c-Mos has been detected in some somatic tissues and non-germ cell lines including NIH 3T3 cells as a protein termed p43$\sp{\rm c-mos}$. Since c-mos RNA transcripts were not previously detected in this cell line by Northern blot or S1 protection analyses, a search was made for c-mos RNA in NIH 3T3 cells. c-mos transcripts were detected using the highly sensitive RNA-PCR method and RNase protection assays. Furthermore, cell cycle analyses indicated that expression of c-mos RNA is tightly controlled in a cell cycle dependent manner with highest levels of transcripts (approximately 5 copies/cell) during the G2 phase.^ In order to determine the physiological significance of c-mos RNA expression in somatic cells, antisense mos was placed under the control of an inducible promoter and introduced into either NIH 3T3 cells or C2 cells. It was found that a basal level of expression of antisense mos resulted in interference with mitotic progression and growth arrest. Several nuclear abnormalities were observed, especially the appearance of binucleated and multinucleated cells as well as the extrusion of microvesicles containing cellular material. These results indicate that antisense mos expression results in a block in cytokinesis. In summary, these results establish that c-mos expression is not restricted to germ cells, but instead indicate that c-mos RNA expression occurs during the G2 stage of the cell cycle. Furthermore, these studies demonstrate that the c-mos proto-oncogene plays an important role in cell cycle progression. As in meiosis, c-mos may have a similar but not identical function in regulating cell cycle events in somatic cells, particularly in controlling mitotic progression via activation/stabilization of MPF. ^

Food advertisements during children's television programming in 2007: Comparison with ads in 1994 and the 2005 dietary recommendations

Childhood overweight and obesity are two major public health problems that are of economic and medical concern in the world today (Lobstein, Baur, & Uauy, 2004). Overweight conditions in childhood are important because they are widely prevalent, serious, and carry lifetime consequences for health and well being (Lobstein et al., 2004). Several studies have shown an association between television viewing and obesity in all age groups (Caroli, Argentieri, Cardone, & Masi, 2004; Harper, 2006; Vandewater & Huang, 2006; Wiecha et al., 2006). One mechanism that potentially links television viewing to childhood obesity is food advertising (Story, 2003). ^ The purpose of this study was to examine the types of foods advertised on children's television programming and to determine if there have been any changes in the number and types of commercials over the last 13 years. In addition, the food content of the advertisements was compared to the 2005 Dietary Guidelines to determine if the foods targeted were consistent with the current recommendations. Finally, each television network was analyzed individually to determine any differences between advertising on cable and regular programming. ^ A descriptive analysis was conducted on the most commonly advertised commercials during children's television programming on Saturday morning from 7 a.m. to 10:30 a.m. A total of 10 major television networks were viewed on three different Saturday mornings during June and July 2007. Commercial advertising accounted for approximately 19% of children's total viewing time. Of the 3,185 commercials, 28.5% were for foods, 67.7% were for non-food items, and 3.8% were PSAs. On average, there were 30 commercial advertisements and PSAs per hour, of which approximately nine were for food. ^ Of the 907 food advertisements, 72.0% were for foods classified in the fats, oils, and sugar group. The next largest group (17.3%) was for restaurant food of which 15.3% were for unhealthy/fast food restaurant fare. The most frequently advertised food product on Saturday morning television was regular cereal, accounting for 43.9% of all food advertisements. ^ Cable and regular programming stations varied slightly in the amount, length, and category of commercials. Cable television had about 50% less commercials and PSAs (1098) than regular programming (2087), but only had approximately 150 minutes less total commercial and PSA time; therefore, cable, in general, had longer commercials than regular programming. Overall, cable programming had more advertisements encouraging increased physical activity and positive nutrition behavior with less commercials focusing on the fats, oils, and sugar groups, compared to regular programming. ^ During the last 13 years, food advertisements have not improved, despite the recent IOM report on marketing foods to children (Institute of Medicine-Committee on Food Marketing and the Diets of Children and Youth, 2005), although the frequency of food advertisements has improved slightly. Children are now viewing an average of one food advertisement every 7 minutes, compared to one food advertisement every 5 minutes in 1994 (Kotz & Story, 1994). Therefore, manufacturers are putting a greater emphasis on advertising other products to children. Despite the recent attention to the issue of marketing unhealthy foods to children through television advertisements, not much progress has been noted since 1994. Further advocacy and regulatory issues concerning the content of advertisements during Saturday morning TV need to be explored. ^

NOVEL THERAPEUTIC TARGETS IDENTIFIED BY HIGH-THROUGHPUT TECHNOLOGIES FOR TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancers (TNBC) are characterized by the lack of or reduced expression of the estrogen and progesterone receptors, and normal expression of the human epidermal growth factor receptor 2. The lack of a well-characterized target for treatment leaves only systemic chemotherapy as the mainstay of treatment. Approximately 60-70% of patients are chemosensitive, while the remaining majority does not respond. Targeted therapies that take advantage of the unique molecular perturbations found in triple-negative breast cancer are needed. The genes that are frequently amplified or overexpressed represent potential therapeutic targets for triple-negative breast cancer. The purpose of this study was to identify and validate novel therapeutic targets for triple-negative breast cancers. 681 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in 2 data sets. For two genes, 3 of the 4 siRNAs showed preferential growth inhibition in TNBC cells. These two genes were the low density lipoprotein receptor-related protein 8 (LRP8) and very low-density lipoprotein receptor (VLDLR). Exposure to their cognate ligands, reelin and apolipoprotein E isoform 4 (ApoE4), stimulated the growth of TNBC cells in vitro. Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. High-throughput protein and metabolic arrays revealed that ApoE4 stimulation rescued TNBC cells from serum-starvation induced up-regulation of genes involved in lipid biosynthesis, increased protein expression of oncogenes involved in the MAPK/ERK and DNA repair pathways, and reduced the serum-starvation induction of biochemicals involved in oxidative stress response and glycolytic metabolism. shLRP8 MDA-MB-231 xenografts had reduced tumor volume, in comparison to parental and shCON xenografts. These results indicate that LRP8-APOE signaling confers survival advantages to TNBC tumors under reduced nutrient conditions and during cellular environmental stress. We revealed that the LRP8-APOE receptor-ligand system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro and helps to sustain the growth of MDA-MD-231 xenografts. We propose that inhibitors of LRP8-APOE signaling may be clinically useful therapeutic agents for triple-negative breast cancer.