Professionalism and academic medicine: the Mayo Clinic program in professionalism.

Public trust in the medical profession has declined and with it physician morale and well being. This has undesirable consequences for patients, physicians and for medical education and training at academic medical centers. The recent upsurge of interest in professionalism may be attributed in part to a desire to regain public trust and restore the image and morale of the profession. The importance of professionalism led to a formal program being established at Mayo clinic that informs clinical practice, conducts educational initiatives and supports novel research into the topic. It may serve as a guide and template for the establishment of similar programs promoting professionalism within academic medical centers.

The gypsy insulator can act as a promoter-specific transcriptional stimulator.

Insulators define chromosomal domains such that an enhancer in one domain cannot activate a promoter in a different domain. We show that the Drosophila gypsy insulator behaves as a cis-stimulatory element in the larval fat body. Transcriptional stimulation by the insulator is distance dependent, as expected for a promoter element as opposed to an enhancer. Stimulation of a test alcohol dehydrogenase promoter requires a binding site for a GATA transcription factor, suggesting that the insulator may be facilitating access of this DNA binding protein to the promoter. Short-range stimulation requires both the Suppressor of Hairy-wing protein and the Mod(mdg4)-62.7 protein encoded by the trithorax group gene mod(mdg4). In the absence of interaction with Mod(mdg4)-62.7, the insulator is converted into a short-range transcriptional repressor but retains some cis-stimulatory activity over longer distances. These results indicate that insulator and promoter sequences share important characteristics and are not entirely distinct. We propose that the gypsy insulator can function as a promoter element and may be analogous to promoter-proximal regulatory modules that integrate input from multiple distal enhancer sequences.

High throughput genotyping technologies for pharmacogenomics.

Genetic differences between individuals play a role in determining susceptibility to diseases as well as in drug response. The challenge today is first to discover the range of genetic variability in the human population and then to define the particular gene variants, or alleles, that contribute to clinically important outcomes. Consequently, high throughput, automated methods are being developed that allow rapid scoring of microsatellite alleles and single nucleotide polymorphisms (SNPs). Many detection technologies are being used to accomplish this goal, including electrophoresis, standard fluorescence, fluorescence polarization, fluorescence resonance energy transfer, and mass spectrometry. SNP alleles may be distinguished by any one of several methods, including single nucleotide primer extension, allele-specific hybridization, allele-specific primer extension, oligonucleotide ligation assay, and invasive signal amplification. Newer methods require less sample manipulation, increase sensitivity, allow more flexibility, and decrease reagent costs. Recent developments show promise for continuing these trends by combining amplification and detection steps and providing flexible, miniaturized platforms for genotyping.

Polarity of transcriptional enhancement revealed by an insulator element.

Transcriptional enhancers for genes transcribed by RNA polymerase II may be localized upstream or downstream of the stimulated promoter in their normal chromosomal context. They stimulate transcription in an orientation-independent manner when assayed on circular plasmids. We describe a transient transformation system to evaluate the orientation preference of transcriptional enhancers in Drosophila. To accomplish this, the gypsy insulator element was used to block bidirectional action of an enhancer on circular plasmids. In this system, as in the chromosome, blocking of enhancer activity requires wild-type levels of the su(Hw) protein. We evaluated the orientation preference for the relatively large (4.4 kb) Adh larval enhancer from Drosophila melanogaster, used in conjunction with a luciferase reporter gene under the control of a minimal Adh promoter. An orientation preference was revealed by insertion of a single copy of the insulator between the enhancer and the promoter. This orientation effect was greatly amplified when the promoter was weakened by removing binding sites for critical transcription factors, consistent with a mechanism of insulator action in which the insulator intercepts signals from the enhancer by competing with the promoter. The orientation preference, as much as 100-fold, is a property of the enhancer itself because it is displayed by gene constructions introduced into the chromosome regardless of the presence of the insulator in a distal location. These findings are most easily reconciled with a facilitated tracking mechanism for enhancer function in a native chromosomal environment.

Polymorphic markers for the arylsulfatase A gene reveal a greatly expanded meiotic map for the human 22q telomeric region.

Two microsatellite markers, D22S1743 and D22S1744, were developed for the arylsulfatase A (ARSA) region of chromosome 22q. Linkage analysis for 171 families, using nine reference markers covering all of 22q, placed these new markers 2.0 Kosambi cM distal to D22S526, making them more distal than any microsatellite markers currently on the Généthon or Marshfield linkage maps. Recombination between proximal markers D22S270/D22S683 and D22S446/D22S311 exhibited increased rates of female meiotic recombination compared to male recombination (P < 0.01). In contrast, the region encompassing sJCW16, D22S526, D22S1743, and D22S1744 exhibited relatively greater recombination in males (1.1 cM for females and 7.5 cM for males; chi(2); P < 0.005). These four distal markers lie in a region of hyperrecombination having a sex-averaged recombination ratio of between 8.3 (D22S1843/D22S1744) and 12 cM (sJCW16/D22S526) per megabase.

Cis-acting sequences contributing to expression of the Drosophila affinidisjuncta Adh gene in both larvae and adults.

To characterize the cis-acting sequences required for expression of the alcohol dehydrogenase (Adh) gene of the Hawaiian picture-winged fruit fly Drosophila affinidisjuncta, germ-line transformation was used to introduce altered forms of this gene into Drosophila melanogaster. Genes were constructed lacking regions corresponding to known or putative regulatory elements within the D. melanogaster gene as well as to sequences previously shown to be required for expression of the D. affinidisjuncta gene in the larval fat body as assayed by transient transformation. Measurement of alcohol dehydrogenase (EC 1.1.1.1) activity levels produced by the transfected genes indicates that multiple elements in both the 5' and 3' regions of the gene contribute to expression. The dispersed elements appear to function redundantly to ensure high levels of expression. Moreover, in contrast to what has been reported for other Drosophila Adh genes, some of the regulatory elements influence expression in both larvae and adults. In total, these results reveal a regulatory system in which the transcribed region in imbedded in an extended genomic segment rich in cisacting regulatory information.

cis-Acting sequences controlling the adult-specific transcription pattern of the Drosophila affinidisjuncta Adh gene.

The cis-acting sequences required for the adult-specific expression pattern of the alcohol dehydrogenase (Adh) gene of the Hawaiian picture-winged fruit fly, Drosophila affinidisjuncta were analyzed by germline transformation. Normally this gene produces two developmentally regulated transcripts. The upstream (distal) promoter produces a distal transcript, which makes up about 80% of the total in adults, while the downstream (proximal) promoter produces a corresponding proximal transcript, which accounts for the remainder. Previously constructed genes lacking regions corresponding to regulatory elements within the Drosophila melanogaster Adh gene or regions known to be required for full expression of the D. affinidisjuncta Adh gene in larvae were analyzed by introduction into the germline of D. melanogaster followed by RNase-protection analysis of RNA levels. In addition, to test a model of preferential promoter utilization by which transcription at the proximal promoter is inhibited by transcription initiated at the upstream distal promoter, a construction lacking the distal promoter was analyzed. Sequences homologous to the adult enhancer of the Adh gene of D. melanogaster appear to play a similar role in the D. affinidisjuncta gene. In contrast to what has been reported for other Drosophila Adh genes, this and some other regulatory elements are shared by the two promoters of the D. affinidisjuncta gene. Taken together, the results favor a model of stage-specific switching between the two promoters of the D. affinidisjuncta gene that involves competition for limiting components stimulating transcription, rather than interference by read-through from the upstream promoter.

Profiles of the Endocrine Clinic: the Mayo Clinic.

It is evident that clinical endocrinology, as a discipline, is entering a particularly exciting period in its evolution. Knowledge gained from basic and clinical research is being translated at the bedside for the benefit of our patients. The emergence of new drugs and novel treatment strategies has equipped clinical endocrinologists with the tools to more successfully combat many old enemies, such as diabetes and osteoporosis. Realization of full benefit from these exciting new tools requires a practice model in which the clinical endocrinologist's role is preeminent and is coordinated and integrated with those of practitioners drawn from other disciplines. The Mayo Division of Endocrinology, Metabolism, and Nutrition provides one such model of highly integrated care. We believe that as the pace of knowledge regarding basic mechanisms of disease and their treatment quickens, such integrated divisions will prove well suited to deliver the highest quality care to people with endocrine disorders.

Thyroid hormones and illness.

OBJECTIVE: To review illness-associated changes in thyroid hormone levels with respect to the alterations in metabolism, regulation, and transport of thyroid hormones present in such a setting. METHODS: We summarize normal pituitary-thyroid function and categorize the types of changes that have been noted during illness. In addition, we examine studies that have addressed the metabolic status of patients with low thyroid hormone levels and the potential utility of thyroid hormone treatment in severely ill patients. RESULTS: The finding of altered thyroid hormone levels in hospitalized patients confirms the participation of these factors in the adaptive response to illness. Thyroid hormone alterations that occur during illness can be classified into three categories: (1) decreased production of triiodothyronine, (2) decreased serum binding of thyroid hormones, and (3) decreased secretion of thyrotropin. Study of these hormonal changes has provided insight into normal thyroid hormone regulation and metabolism as well as into neuroendocrine adaptations to illness. CONCLUSION: It seems reasonable to conclude that illness-associated changes in thyroid hormone levels are generally beneficial to the patient as a whole; however, the possibility of concomitant regional or individual tissue hypothyroidism exists. Conclusions about the potential utility of pharmacologic administration of triiodothyronine in selected diseases and clinical settings must await additional clinical trials. Finally, the predictable occurrence of altered thyroid hormone levels in hospitalized patients necessitates caution in both the selection and the interpretation of thyroid hormone tests in this patient population.

Human NAD(P)H:quinone oxidoreductase induction in human hepatoma cells after exposure to industrial acrylates, phenolics, and metals.

Induction of the endogenous human NAD(P)H:quinone oxidoreductase (HQOR1) gene in the human hepatoma cell line HepG2 was measured at both the enzyme activity and RNA levels after exposure to a variety of industrial compounds. An RNA probe was designed that was complementary to portions of both the coding region and the 3'-nontranslated region unique to the largest (2.7-kilobase) HQOR1 transcript. Induction by three strong inducers of HQOR1 verified the utility of the antisense RNA probe. Ten industrial chemicals were evaluated as potential inducers, i.e. acrylonitrile, Sb2O3, BaO, CdCl2, CuCl, ethyl acrylate, methyl acrylate, MoO3, phenol, and toluene. Induction at the RNA level was about 2-fold higher than at the enzyme activity level except in the case of acrylonitrile, for which induction at the enzyme activity and RNA levels was similar. There was no preferential induction of the 2.7-kilobase transcript for any chemical tested, including 2,3,7,8-tetrachloro-dibenzo-p-dioxin, which had previously been reported to preferentially induce this transcript. Six of the 10 industrial chemicals, including four previously untested chemicals (phenol, Sb2O3, CuCl, and MoO3), were found to induce the HQOR1 gene. By comparison, previous studies in rodent systems failed to accurately predict the human HQOR1 gene response. Two chemicals previously shown to be inducers in rodent systems (methyl acrylate and CdCl2 failed to induce the HQOR1 gene. These results emphasize the importance of analyzing induction of the endogenous human gene, rather than simply extrapolating from rodent systems or gene fusion experiments.

Molecular organization of the alcohol dehydrogenase loci of Drosophila grimshawi and Drosophila hawaiiensis.

To determine sequences involved in conserved and species-specific aspects of alcohol dehydrogenase (Adh) gene expression in Hawaiian Drosophila, a 3644-base pair (bp) region containing the D. grimshawi gene and the homologous 3335-bp region containing the D. hawaiiensis Adh gene were sequenced. These genes have the two-promoter and exon-intron structure seen for many Drosophila Adh genes. Analysis of putative and known regulatory sequences of the D. grimshawi and D. hawaiiensis genes in comparison to those of D. affinidisjuncta (the only other Hawaiian species for which the promoter organization is known) highlighted elements likely to be involved in conserved aspects of Adh gene expression as well as sequences that may account for species-specific differences in tissue-specific expression. Sequence comparisons, in the context of regulatory roles previously assigned to particular gene fragments, indicated that multiple insertions and deletions in the promoter regions are responsible for differences in tissue-specific regulation displayed by these genes.

The two small introns of the Drosophila affinidisjuncta Adh gene are required for normal transcription.

All Drosophila alcohol dehydrogenase (Adh) genes sequenced to date contain two small introns within the coding region. These are conserved in location and, to some extent, in sequence between the various species analyzed. To determine if these introns play a role in Adh gene expression, derivatives of the Drosophila affinidisjuncta Adh gene lacking one or both introns were constructed and analyzed by germline and transient transformation of Drosophila melanogaster. Removal of both introns lowered expression, whether measured by enzyme activity or by RNA levels. The decrease was seen in both germline transformed and transiently transformed larvae, with the effect being larger for germline transformants. Similar decreases (averaging 5-fold) were also seen at the embryonic and adult stages for germline transformants. Nuclear run-off transcription with nuclei from germline transformed embryos indicated that the reduction in RNA levels is due to decreased transcription. However, LacZ fusion constructs designed to test for the presence of a classical enhancer in the introns provided no evidence for such a mechanism. Removal of each intron individually resulted in more complex phenotypes. The introns have smaller, additive effects on expression in adults. In larvae, removal of the upstream intron significantly increases RNA levels but modestly decreases enzyme activity. Removal of the downstream intron lowers expression in both germline and transiently transformed larvae, but also increases position effects in germline transformants. Therefore, the small introns are clearly needed for optimal transcription of this Adh gene, but multiple mechanisms are involved.

Nongoitrous (type I) amiodarone-associated thyrotoxicosis: evidence of follicular disruption in vitro and in vivo.

Treatment with the antiarrhythmic agent amiodarone results in alterations in thyroid hormone metabolism, and can induce either hypothyroidism or hyperthyroidism (amiodarone-associated thyrotoxicosis, AAT). AAT occurs in patients both with and without preexisting goiter. In our study of the nongoitrous variety, the effect in vitro of amiodarone treatment and of concurrent treatment with potential inhibitors on thyroid cells (FRTL-5) was assessed by measuring the release of radiolabeled chromium (51Cr). In addition, thyroid histopathology was evaluated in autopsy specimens from six amiodarone-treated patients who had no pretreatment evidence of thyroid disease. Histopathologic examination revealed minimal or no evidence of thyroid follicular damage in specimens from amiodarone-treated euthyroid patients (n = 4). In contrast, moderate to severe follicular damage and disruption were present in glands from patients with AAT (n = 2). Studies in vitro showed amiodarone to be cytotoxic to thyroid cells; this effect was inhibited by treatment with dexamethasone (10(-3) mmol) or perchlorate (2.5 micrograms/mL). In summary, we demonstrate evidence in vitro and in vivo of amiodarone-induced thyroid follicular damage and disruption in specimens from patients with nongoitrous AAT and in cultured normal thyroid cells. In addition, we demonstrate inhibition of this effect following treatment in vitro with dexamethasone or perchlorate. Our findings support the concept that nongoitrous (type I) AAT results from direct drug toxicity with disruption of thyroid follicles and subsequent release of preformed thyroid hormone.

A transcriptional role for conserved footprinting sequences within the larval promoter of a Drosophila alcohol dehydrogenase gene.

All Drosophila alcohol dehydrogenase (Adh) genes that are expressed in larvae display strong transcription in the larval fat body. To identify and characterize elements needed for Adh promoter function, footprinting analysis of the Drosophila affinidisjuncta Adh gene was performed with stage-specific nuclear proteins from embryos and larvae. Multiple sites upstream of the larval promoter were protected from deoxyribonuclease digestion by both embryonic and larval extracts. Comparison with foot-printing results for Adh genes from other Drosophila species revealed only one nuclease-protected region that is conserved in both sequence and position. Clustered point mutations in this sequence were analyzed by footprinting analysis, transient transformation and in vitro transcription. Two separate sequences in this footprinting region exerted positive effects on transcription from the Adh proximal promoter in the larval fat body. The effects of these sequences on gene expression were synergistic. One of these sequences, TGATAA, bound in vitro to Drosophila melanogaster box A binding factor protein, as shown by gel mobility shift assays. This is the first direct demonstration of specific protein-DNA interactions influencing transcription of a Drosophila Adh gene in the larval fat body.

Redundant cis-acting elements control expression of the Drosophila affinidisjuncta Adh gene in the larval fat body.

The alcohol dehydrogenase (Adh) gene in the Hawaiian species of fruit fly, Drosophila affinidisjuncta, like the Adh genes from all Drosophila species analyzed, is expressed at high levels in the larval fat body via a larval-specific promoter. To identify the cis-acting elements involved in this highly conserved aspect of Adh gene expression, deleted D. affinidisjuncta genes were introduced into D. melanogaster by somatic transformation. Unlike previously described methods, this transformation system allows analysis of Adh gene expression specifically in the larval fat body. The arrangement of sequences influencing expression of the proximal promoter of this gene in the larval fat body differs markedly from that described for the Adh gene from the distant relative, D. melanogaster. Multiple redundant elements dispersed 5' and 3' to the gene, only some of which map to regions carrying evolutionarily conserved sequences, affect expression in the fat body. D. affinidisjuncta employs a novel mode of Adh gene regulation in which the proximal promoter is influenced by sequences having roles in expression of the distal promoter. This gene is also unique in that far upstream sequences can compensate for loss of sequences within 200 bp of the proximal RNA start site. Furthermore, expression is influenced in an unusual, context-dependent manner by a naturally-occurring 3' duplication of the proximal promoter--a feature found only in Hawaiian species.

Similar tissue-specific expression of the Adh genes from different Drosophila species is mediated by distinct arrangements of cis-acting sequences.

In Drosophila melanogaster transformants, the alcohol dehydrogenase (Adh) genes from D. affinidisjuncta and D. grimshawi show similar levels of expression except in the adult midgut where the D. affinidisjuncta gene is expressed about 10- to 20-fold more strongly. To study the arrangement of cis-acting sequences responsible for this regulatory difference, homologous restriction sites were used to create a series of chimeric genes that switched fragments from the 5' and 3' flanking regions of these two genes. Chimeric genes were introduced into the germ-line of D. melanogaster, and Adh gene expression was analyzed by measuring RNA levels. Various gene fragments in the promoter region and elsewhere influence expression in the adult midgut and in whole larvae and adults. Comparison of these results with earlier studies involving chimeras between the D. affinidisjuncta and D. hawaiiensis genes indicates that expression in the adult midgut is influenced by multiple regulatory sequences and that distinct arrangements of regulatory sequences can result in similar levels of expression both in the adult midgut and in the whole organism.

Multiple cis-acting sequences contribute to evolved regulatory variation for Drosophila Adh genes.

Drosophila affinidisjuncta and Drosophila hawaiiensis are closely related species that display distinct tissue-specific expression patterns for their homologous alcohol dehydrogenase genes (Adh genes). In Drosophila melanogaster transformants, both genes are expressed at high levels in the larval and adult fat bodies, but the D. affinidisjuncta gene is expressed 10-50-fold more strongly in the larval and adult midguts and Malpighian tubules. The present study reports the mapping of cis-acting sequences contributing to the regulatory differences between these two genes in transformants. Chimeric genes were constructed and introduced into the germ line of D. melanogaster. Stage- and tissue-specific expression patterns were determined by measuring steady-state RNA levels in larvae and adults. Three portions of the promoter region make distinct contributions to the tissue-specific regulatory differences between the native genes. Sequences immediately upstream of the distal promoter have a strong effect in the adult Malpighian tubules, while sequences between the two promoters are relatively important in the larval Malpighian tubules. A third gene segment, immediately upstream of the proximal promoter, influences levels of the proximal Adh transcript in all tissues and developmental stages examined, and largely accounts for the regulatory difference in the larval and adult midguts. However, these as well as other sequences make smaller contributions to various aspects of the tissue-specific regulatory differences. In addition, some chimeric genes display aberrant RNA levels for the whole organism, suggesting close physical association between sequences involved in tissue-specific regulatory differences and those important for Adh expression in the larval and adult fat bodies.

Complexity in evolved regulatory variation for alcohol dehydrogenase genes in Hawaiian Drosophila.

The alcohol dehydrogenase gene (Adh gene) of Drosophila affinidisjuncta is expressed at a higher level in the larval midgut and Malpighian tubules than the homologous gene from Drosophila hawaiiensis. This study analyzed the cis-acting sequences responsible for these regulatory differences in larval tissues of Drosophila melanogaster transformants. A series of 10 chimeric and deleted Adh genes was introduced into the germ line of D. melanogaster, and tissue-specific expression levels were quantified by gel electrophoresis of tissue extracts. Sequences in the upstream region of the two genes had the strongest influence on enzyme production in the midgut and Malpighian tubules. Other sequence elements also showed effects, some of which were tissue specific. Most gene fragments displayed context-dependent effects, thus supporting the proposed model of polygenic regulation of Adh gene expression.

Acquired conditions: an improvement to hospital discharge abstracts.

Selected secondary diagnoses (e.g. pulmonary embolism) may provide an efficient and inexpensive source of data for quality assurance (QA) monitoring if their absence at admission were known. In June 1990 we modified our hospital abstracting methods to classify each diagnosis into categories: (1) present on admission, (2) acquired during hospitalization, or (3) uncertain. Our experience has confirmed the identification and elimination from QA reports of the majority of pre-existing secondary diagnoses. Examples of secondary diagnosis codes acquired or uncertain were acute myocardial infarction 48%, pneumonias 25%, pulmonary emoboli 54% and cerebral vascular accident/hemorrhage 35%. Abstracting time has increased less than 2 min per discharge. A reabstraction study showed 87% agreement (kappa = 0.733, p less than 0.001) between initial collection and blinded reabstraction. The separation of secondary diagnoses into preexisting or acquired can: (1) be reliably undertaken by discharge abstracters; (2) be efficient in adding minimal time; and (3) enhance the validity and usefulness of data and increase physician acceptance.

Follicular thyroid cancer treated at the Mayo Clinic, 1946 through 1970: initial manifestations, pathologic findings, therapy, and outcome.

We retrospectively analyzed the outcome of all patients who received their primary treatment for follicular thyroid cancer at the Mayo Clinic between 1946 and 1970. The diagnosis was confirmed by reexamination of preserved tissue specimens. The 57 female and 43 male patients (mean age, 53 years) underwent follow-up for a maximum of 32 years (mean, 17.4 years). All patients were treated surgically, and total removal of primary tumor was thought to have been accomplished in all but three. Only 2 of the 88 patients without distant metastatic lesions at the time of initial diagnosis underwent ablation of the thyroid remnant. At the conclusion of the study, 52 patients had died, thyroid cancer being the cause of death in 19. On the basis of univariate survival analysis, age more than 50 years, tumor size that exceeded 3.9 cm, higher tumor grade, presence of marked vascular invasion, adjacent tissue invasion, and distant metastatic involvement at the time of initial diagnosis were associated with increased cancer mortality. Multivariate analysis (by Cox proportional hazards model), however, identified only age greater than 50 years, marked vascular invasion, and metastatic disease at the time of diagnosis to be independent predictors of follicular thyroid cancer-related mortality. Patients with two or more of these predictors were classified as being high risk. These patients had 5- and 20-year survival rates of 47% and 8%, respectively; the corresponding survival data for the low-risk group were 99% at 5 years and 86% at 20 years. The identification of these risk groups may facilitate a more rational approach to treatment of follicular thyroid cancer.