Jeffrey P. Rabek

Evidence for posttranscriptional regulation of C/EBPalpha and C/EBPbeta isoform expression during the lipopolysaccharide-mediated acute-phase response.

The mRNAs of the CCAAT/enhancer-binding trans-activator proteins (C/EBPalpha and C/EBPbeta) serve as templates for the differential translation of several isoforms which have specific transcriptional regulatory functions. By using an oligonucleotide corresponding to the C/EBP binding site of the mouse alpha1-acid glycoprotein promoter, we detected multiple forms of C/EBPalpha and C/EBP++ beta proteins in the mouse liver that have DNA-binding activity. By using specific antisera, we detected C/EBPalphas with molecular masses of 42, 38, 30, and 20 kDa that have DNA-binding activity. The pool levels of the 42- and 30-kDa isoforms were high in control nuclear extracts and decreased significantly after lipopolysaccharide (LPS) treatment. The binding activity and protein levels of the 20-kDa isoform are low in controls and increase dramatically after LPS treatment. C/EBPbeta isoforms with molecular masses of 35, 20, and 16 kDa were also detected. The 35-kDa pool level did not change whereas the 20-kDa isoform was strongly induced in response to LPS. Western (immunoblot) and Southwestern (DNA-protein) analyses show that p42 C/EBPalpha forms specific complexes with the alpha1-acid glycoprotein oligonucleotide in control nuclear extract and that p20 C/EBP beta forms complexes in LPS-treated liver. Our studies suggest that synthesis of specific C/EBPalpha and C/EBPbeta isoforms occurred in the normal liver in vivo and that LPS mediated a differential initiation and inhibition of translation at specific AUG sites within each mRNA. The qualitative and quantitative changes in C/EBPalpha and C/EBPbeta isoform pool levels suggest that LPS or an LPS-stimulated factor can regulate the selection of AUG start sites for both activation and repression of translation. This regulation appears to involve an LPS-mediated down-regulation of initiation at the first AUG codon of the 42-kDa C/EBPalpha and dramatic translational up-regulation at the fifth AUG codon of the 20-kDa C/EBPalpha and the third AUG codon of the 20-kDa C/EBPbeta. These regulatory events suggest the existence of proteins that may act as translational trans-acting factors.

Carbonylation of ER chaperone proteins in aged mouse liver

Progressive accumulation of oxidative damage to macromolecules in aged tissues is thought to contribute to the decline in tissue function characteristic of the aged phenotype. Mitochondria are a major intracellular source of reactive oxygen species (ROS); however, other organelles are also endogenous sources of oxyradicals and oxidants, which can damage macromolecules. We, therefore, sought to examine the relationship between aging and oxidative damage to ER resident proteins, which exist in a strongly oxidizing environment necessary for disulfide bond formation. In these studies, we have fractionated young and aged liver homogenates, resolved the proteins by 2D gel electrophoresis, assayed for oxidative damage as indicated by protein carbonylation, and identified BiP/Grp78, protein disulfide isomerase (PDI), and calreticulin as exhibiting an age-associated increase in oxidative damage. Increased carbonylation of these key proteins in aged liver suggests an age-associated impairment in protein folding, disulfide crosslinking, and glycosylation in the aged mouse liver.

Regulation of LPS-mediated induction of C/EBPδ gene expression in livers of young and aged mice

Abstract The C/EBP family of transcription factors plays a major role in the regulation of families of stress response genes, in particular, the acute phase response genes. We have examined expression of the C/EBPδ gene during the bacterial lipopolysaccharide mediated induction of the acute phase response in livers of young (4 months) and aged (24–28 months) male C57Bl/6 mice by Northern, Western, and Southwestern analyses. C/EBP δ mRNA is present at a low constitutive level, is induced by lipopolysaccharide, and reaches the same induced level in young and aged mice. Aged mice, however, show a higher constitutive, uninduced mRNA pool level and a delay in recovery to uninduced levels after lipopolysaccharide treatment. C/EBP δ mRNA is observable 30 min after lipopolysaccharide in total RNA, cytoplasmic and polysomal fractions. Specific full length 28-kDa nascent peptides are detectable in polysomes 90 min after lipopolysaccharide. mRNA and nascent peptides cosediment with large polysomes and C/EBP δ mRNA is shifted to larger polysomes in lipopolysaccharide treated aged mice, consistent with an increased rate of initiation. Specific DNA-binding activity of C/EBP δ protein in nuclear extracts was examined by electromobility shift and antibody supershift assay. The levels of C/EBP δ binding-activity, are consistent with the changes in mRNA levels in young lipopolysaccharide treated livers. These studies support our hypothesis that aged mice exhibit a state of chronic inflammation or stress in the absence of a stressor.

Effects of comprehensive educational reforms on academic success in a diverse student body

Medical Education 2010: 44: 1232–1240

Comprehensive changes in the learning environment: subsequent step 1 scores of academically at-risk students.

Background During the past 10 years at our institution, a number of changes have been instituted in the learning environment, including instructional techniques, assessment methods, academic support, and explicit board preparation. Method The authors studied the Step 1 performance of students with MCAT scores of 20 to 25 in our former and current curricula. Effect sizes were calculated for score improvement using adjusted means from ANCOVA with covariates of MCAT and age. Results The overall effect size was 0.48, with larger effects seen for underrepresented minority students overall (d = 0.64) and African American students especially (d = 0.77), representing medium to large effects. Overall failure rates decreased by two thirds. Conclusions Comprehensive changes in the learning environment were followed by substantial improvement in Step 1 performance among academically at-risk students.

Analysis of the mechanism of glucocorticoid-mediated down regulation of the mouse α-fetoprotein gene

Regulation of alpha-fetoprotein gene expression by dexamethasone was examined in vivo and in vitro using primary mouse fetal liver cell cultures. Dexamethasone accelerates the developmental down regulation of AFP mRNA pools. However, treatment of primary fetal liver cells in culture does not reduce the AFP mRNA pool and may stabilize both AFP and albumin gene expression. These results indicate that in vivo the effect of dexamethasone may require interaction with another tissue or cell type. The mechanism of the dexamethasone mediated inhibition of AFP was examined by DNase I footprinting and transient expression assays. Two protein-binding regions of the proximal promoter (III and IV) show significant homology to the GRE consensus sequence. DNase I footprinting shows that only region IV can bind purified GR and competition with GRE oligonucleotides indicate that, using adult liver nuclear proteins, no GR is bound in either region. Nuclear protein from adrenalectomized mice show the same protection as controls. These results indicate that GR may not bind to the AFP proximal promoter in the adult. AFP promoter-CAT expression vectors were used to further examine the effect of dexamethasone on AFP expression. AFP promoter-CAT constructs were inhibited by 10(-6) M dexamethasone; while linking of an AFP enhancer to the promoter abolished the effect. We conclude that the in vitro effects on transiently expressed AFP directed expression vectors may be a function of vector structure and/or characteristics of the cells used whereas the in vivo effect may reflect normal regulatory mechanisms.

Effects of mercuric chloride on the regulation of expression of the acute phase response components α1-acid glycoprotein and C/EBP transcription factors

We have previously shown that in response to treatment with HgCl(2), the adult mouse liver exhibits both transcriptional and translational regulation of the acute phase response genes. In this study we asked whether the heavy metal treatment affects the regulation of the C/EBP transcription factors which play a key role in regulation of the acute phase response gene. Our studies have shown that the AGP gene is transcriptionally activated while transcription of the CCAAT/enhancer-binding trans-activating protein (C/EBP)alpha gene is slightly down-regulated and that of the C/EBPbeta gene does not respond. Both the C/EBPalpha and C/EBPbeta mRNAs produce multiple isoforms possibly by alternative translation initiation (ATI) of multiple internal AUG initiation sites. The C/EBPbeta mRNA appears to be stabilized. Although similar regulatory processes occur in response HgCl(2) vs. LPS, our data suggest that the translational processes (ATI) are differentially affected. In addition, a major difference lies in the fact that the C/EBPbeta gene is not transcriptionally activated by HgCl(2). Our data show decreased binding activity and pool levels of the C/EBPalpha isoform (p42(C/EBPalpha)) and increased binding activity and pool levels of C/EBPbeta isoform (p35(C/EBPbeta)) in response to HgCl(2). We propose that this isoform may be involved in the regulation of AGP gene expression in response to heavy metals and that there is a significant difference between the HgCl(2)-mediated and LPS-mediated inflammatory response.

Evolution of Student Assessment Following Implementation of an Integrated Medical Curriculum: Contribution to Improved Educational Outcomes

This paper describes the stimuli and rationale for and the nature of assessment changes implemented after embarking on a new student-centered, problem-based learning curriculum. In 1998, the University of Texas Medical Branch implemented the Integrated Medical Curriculum (IMC), a problem-based curriculum with sequential, interdepartmental, clinically relevant basic science courses and a concurrent Practice of Medicine course. The IMC’s aim was to improve educational quality by emphasizing knowledge application over rote memorization, student-directed learning, problem-solving skills development, basic science and clinical concepts integration, early acquisition of clinical skills, and professional teamwork. During the initial years of the IMC, students’ learning outcomes did not improve over those of the former curriculum because, in part, we did not initially modify student assessment (a driving force for student study habits) to align with the new curricular goals and philosophies. We subsequently took several steps to modify our assessment practices in order to improve student learning and understanding. These steps included (1) increasing the number and quality of higher -order exam questions, (2) sequestering examinations, (3) increasing the number of practice exam items in each course, (4) and emphasizing in-depth discussion in problem-based learning sessions by introducing small-group quiz exercises. Following the assessment modifications, our students’ USMLE Step 1 scores and our pass rate for first-time test takers rose from being consistently below the national Step 1 average to above the national average. This improvement has been sustained for the past seven years. We believe that implementing those assessment modifications within the framework of the newly devised student-centered, problem-based curriculum in an interdepartmental environment resulted in a critical mass of “local” changes. The changes, initiated and supported by students, faculty and administration, catalyzed, produced and sustained more “global” changes that improved students’ learning, thus better preparing them for the USMLE Step 1 examination.