Signal peptides and signal peptidase in Drosophila melanogaster.

Translation of poly(A)-containing RNA from the female fat body of Drosophila melanogaster in a rabbit reticulocyte cell-free system results in the synthesis of previtellogenin polypeptides (PVs) having higher apparent molecular weights (46,000 and 45,000) than the forms seen after an in vivo pulse labeling. However, when this RNA is translated in the presence of EDTA-stripped microsomal membranes from the dog pancreas, vitellogenin precursors are produced that, upon SDS-polyacrylamide gel electrophoresis, comigrate with the in vivo forms (apparent molecular weights, 45,000 and 44,000). These processed forms are sequestered within the microsomal lumen, as evidenced by their insensitivity to trypsin digestion. Neither processing nor sequestration occur posttranslationally. In addition, a microsomal membrane fraction derived from Drosophila embryos is able to cotranslationally process the PVs as well as a murine pre-light chain IgG. These observations support a signal-mediated mode of secretion in Drosophila, and suggest that signal sequence recognition and signal peptidase activities are conserved even between mammalian and insect systems.

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.

Expression of tumor-associated aldehyde dehydrogenase gene in rat hepatoma cell lines.

Significant changes in aldehyde dehydrogenase (ALDH) activity occur during rat hepatocarcinogenesis in vivo. To compare the structure and expression of the tumor aldehyde dehydrogenase gene in rat hepatoma cell lines and normal rat liver, several rat hepatoma cell lines, including HTC, H4-II-EC3, JM2, McA-RH7777, and four lines established in this laboratory have been examined for T-ALDH gene expression using a tumor ALDH complementary DNA. Northern blot analysis of polyadenylate-containing RNA from log-phase cells and normal rat liver with T-ALDH complementary DNA indicates production of a single major 1.7-kilobase transcript in the high activity lines HTC, JM2, RLT-2M, RLT-3C, RLT-9F, and intermediate activity line RLT-5G. There is a direct correlation between expression of T-ALDH enzyme activity and the amount of 1.7-kilobase transcript. S1 nuclease protection experiments confirm that there is only one major T-ALDH transcript in the high activity lines. Thus, cell line differences in T-ALDH activity are reflected in the level of a single T-ALDH transcript. Southern analysis was used to identify the T-ALDH gene in genomic DNA. The results indicate that no significant amplification or rearrangement of the T-ALDH gene has occurred in these hepatoma cells. DNA methylation has been proposed to play an important role in gene expression. Genomic DNA from HTC, JM2, McA-RH7777, H4-II-EC3, RLT-2M, RLT-9F, RLT-3C, RLT-5G, rat embryo and normal rat liver were digested with MspI and HpaII to examine methylation patterns. A digestion pattern consistent with hypomethylation was detected only in DNA from the high T-ALDH activity cell lines HTC, JM2, RLT-2M, and RLT-9F. This suggests that constitutive expression of T-ALDH in the hepatoma cells is related to changes in DNA methylation patterns.

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.

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.

Tissue-specific expression phenotypes of Hawaiian Drosophila Adh genes in Drosophila melanogaster transformants.

Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.

Developmentally regulated RNA transcripts coding for alcohol dehydrogenase in Drosophila affinidisjuncta.

The organization of the gene coding for alcohol dehydrogenase (Adh) in Drosophila affinidisjuncta has been determined by physically mapping Adh RNA transcripts to cloned genomic DNA. Two distinct transcript types accumulate with developmental specificity. Because only a single genomic Adh locus is detected in D. affinidisjuncta, and since all Adh transcripts appear to be identical except at their termini, the two Adh RNA types are products of the same gene. One type of transcript, abundant in adults, contains a small 5' terminal exon that is completely lacking in the other type of transcript, which accumulates in larvae. This 5' end difference suggests that the D. affinidisjuncta Adh gene, like the homologous gene from the distantly related species D. melanogaster, is expressed from two promoters. According to the transcription map, these D. affinidisjuncta promoters are separated by approximately 560 base pairs of genomic DNA sequence. D. affinidisjuncta Adh transcripts also resemble D. melanogaster Adh transcripts in both their overall organization and their developmental distribution. Multiple 3' ends are responsible for the size heterogeneity of both types of D. affinidisjuncta Adh RNA, and some of these also appear with stage specificity.

Outcome of anesthesia and surgery in hypothyroid patients.

We retrospectively examined the outcome of anesthesia and surgery in 59 hypothyroid patients and in 59 paired euthyroid matched controls. Hypothyroid patients had more preoperative risk factors but did not differ as a group from controls with regard to duration of surgery or anesthesia, lowest temperature and BP recorded during surgery, need for vasopressors, time to extubation, fluid and electrolyte imbalances, incidence of arrhythmias, pulmonary and myocardial infarction, sepsis, need for postoperative respiratory assistance, bleeding complications, or time to hospital dismissal. Analysis of subsets of hypothyroidism (thyroxine level, less than 1.0, less than 3.0, and greater than or equal to 3.0 micrograms/dL) also failed to disclose any significant differences compared with matched controls. Among patients with mild or moderate hypothyroidism, we found no evidence to justify deferring needed surgery until the hypothyroidism has been corrected.

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.

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.

Diagnosis of pancreatic islet hyperplasia causing hypoglycemia in a patient with portacaval anastomosis.

A patient with biopsy-proved biliary cirrhosis and previous gastrojejunostomy and portacaval anastomosis experienced episodes of severe hypoglycemia. She was found to have hyperinsulinemia and hyperglucagonemia. An oral glucose tolerance test showed postgastrectomy hypoglycemia. Results of the intravenous tolbutamide test were diagnostic for insulinoma, but results of the intravenous glucagon test and prolonged fast (96 hours) were not. Failure, on two occasions, to suppress C-peptide normally during insulin-induced hypoglycemia led to a diagnosis of pancreatogenous hyperinsulinemia. The pancreas showed a 10-fold increase in islet volume, with intensely positive staining with anti-insulin and anti-glucagon antiserums in addition to anti-somatostatin and anti-pancreatic polypeptide antiserums. Incidental findings at pancreatic exploration were a mesothelioma, which did not stain with anti-insulin antiserum, and, at autopsy one year later, a hepatoma.

Pathology of the thyroid in amiodarone-associated thyrotoxicosis.

Among 83 consecutive patients operated on for thyrotoxicosis at the Mayo Clinic between January 1, 1980 and May 1, 1986, four had a combination of pathologic findings that were similar and unexpected: involutional changes; degenerative and destructive follicular lesions; and zones of fibrosis. These four, but none of the remaining 79, were being treated with amiodarone for cardiac tachyarrhythmias; this drug is known to be taken up by the thyroid gland. Characteristically, small groups of involuted follicles exhibited varying degrees of damage ranging from degenerative changes in a few lining cells to total follicular destruction. Damaged follicular cells were swollen and featured granular or vacuolated cytoplasm. This type of cytoplasmic alteration has been reported to occur in pneumocytes and hepatocytes damaged by amiodarone. Therefore, the drug probably is the cause of the thyroid cell damage, and follicular disruption (with consequent release of iodothyronines into the circulation) is likely to be an important contributor to the associated thyrotoxicosis.

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.

Series on pharmacology in practice. 5. Thyroid hormones.

Thyroid hromones are among the most commonly prescribed medications. This article attempts a critical comparison of available preparations in the light of more recent information regarding thyroid physiology and, in particular, peripheral thyroid hormone economy. Indications and complications of therapy are reviewed. Present knowledge of thyroid physiology indicates that the use of L-thyroxine has advantages over other preparations in the treatment of hypothyroidism.

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.

Heterophilic serum antibodies: a cause for falsely elevated serum thyrotropin levels.

We report two cases of euthyroid patients with inappropriately elevated serum levels of thyrotropin (TSH) in whom investigation led to the detection of heterophilic antibodies. In addition, we compare the performance of two TSH immunometric assay systems in this setting. Even when monoclonal assay systems are used, TSH results must be critically interpreted when they are at variance with clinical and other biochemical findings.

Effect of blood glucose control on peripheral nerve function in diabetic patients.

A prospective, stratified, randomized 3-year clinical trial was conducted on the effect of rigorous versus conventional glucose control on peripheral nerve function in 33 insulin-treated diabetic patients with a duration of diabetes of less than 2 years. The goals for conventional glucose control were the mean of fasting and 80-minute postprandial plasma glucose of 150 mg/dl for non-insulin-dependent diabetes and 200 mg/dl for insulin-dependent diabetes. The goal of rigorous glucose control was an approximation of nondiabetic glucose control. No significant difference in glucose control or peripheral nerve function was observed between the rigorously and the conventionally controlled groups. Eight patients in the conventional-control group spontaneously achieved glucose control in the range that was the objective for the rigorous-control group, and five patients in the rigorous-control group never achieved the desired glucose control. In the remaining 20 patients, with similar baseline glucose control and peripheral nerve function characteristics, observed over a median of 2 years, improved blood glucose control (P less than 0.01) was not associated with any significant improvement in peripheral nerve function. Nevertheless, a significant (P less than 0.05) correlation was found between the degree of abnormal nerve function at entry into the study and change in nerve function during the study. If control of hyperglycemia benefits peripheral nerve function of diabetic patients, its demonstration may require a closer approximation of normoglycemia, a larger difference in glucose control between the two study groups, a longer duration of treatment, and the use of patients with more advanced peripheral nerve function abnormalities than those in this study.

Invasive Hürthle cell carcinoma of the thyroid: natural history and management.

Debate surrounds the natural history and appropriate management of Hürthle cell neoplasms of the thyroid. Some of the uncertainty stems from difficulty in the differentiation of benign from malignant lesions. We report the presentation, management, and outcome of patients with invasive Hürthle cell carcinoma who were examined at this institution between 1946 and 1971. We believe that our review allows us to make reasonable recommendations concerning the management of patients with this type of carcinoma.

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.

Amiodarone-associated thyrotoxicosis (AAT): experience with surgical management.

Amiodarone (Cordarone), a benzofuran derivative containing two atoms of iodine per molecule (37% of molecular weight), has recently been released in the United States for the management of refractory cardiac arrhythmias. While it was still under investigation, 529 patients were managed with this drug at the Mayo Clinic between 1981 and 1986; eight of them (1.5%) had thyrotoxicosis 12 to 37 months (median, 28 months) after initiation of treatment. Low 24-hour iodine-131 uptake (less than 4%) and recognized refractoriness to thionamide treatment precluded the use of radioablation or antithyroid drugs. Cessation of the amiodarone treatment was considered undesirable from a cardiac standpoint. Near-total thyroidectomies were undertaken in six patients, and rapid correction of hyperthyroidism followed. There were no intraoperative or postoperative arrhythmias. The median postoperative hospital stay was 4.3 days (range, 2 to 13 days). Subsequently, all patients required thyroid hormone replacement. Near-total thyroidectomy has proved a safe and effective treatment for amiodarone-associated thyrotoxicosis in patients who required drug continuation.