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Biomedical subjects

B J Goldstein

Publications and source records attributed to B J Goldstein.

At least 91 records · Page 5Linked to original sources
PubMed1989

Analysis of mRNA heterogeneity by ribonuclease H mapping: application to the insulin receptor.

The major species of human insulin receptor mRNA (5.9, 7.5, 8.5 and 10.2 kb) and those in rat tissues (7.4 and 9.6 kb) are each much larger than the 4.2 kb required to encode the insulin receptor precursor. To evaluate the structural basis for this mRNA size heterogeneity, we performed a ribonuclease H mapping technique. A small insulin receptor cDNA insert was annealed to human and rat poly(A) RNA, followed by site-specific enzymatic cleavage with ribonuclease H. Subsequent Northern blot analysis with cDNA probes specific to the 5' end of the cDNA revealed a single fragment from each of the human and rat insulin receptor mRNA species. The size of this fragment indicated that each mRNA contains approximately 0.4 kb of 5' untranslated mRNA. In contrast, a 3' region probe demonstrated multiple mRNA fragments after cleavage. The sizes of these fragments indicated that the human insulin receptor mRNA species contain from 1.5 to 5.4 kb, and the rat insulin receptor mRNAs either 2.8 or 5.3 kb, of 3' untranslated RNA. Thus, the presence of varied, but extensive, 3' untranslated sequences in insulin receptor mRNA transcripts accounts for their size heterogeneity and may affect mRNA stability and/or translation efficiency.

Animals↗
PubMed1989

Identification of persistent defects in insulin receptor structure and function capillary endothelial cells from diabetic rats.

Insulin actions and receptors were studied in capillary endothelial cells cultured from diabetic BB rats and their nondiabetic colony mates. The endothelial cells from diabetic rats of 2 mo duration had persistent biological and biochemical defects in culture. Compared with normal rats, endothelial cells from diabetic rats grew 44% more slowly. Binding studies of insulin and insulin-like growth factor I (IGF-I) showed that cells from diabetic rats had 50% decrease of insulin receptor binding (nondiabetic: 4.6 +/- 0.7; diabetic: 2.6 +/- 0.4% per milligram protein, P less than 0.01), which was caused by a 50% decrease in the number of binding sites per milligram protein, whereas IGF-I binding was not changed. Insulin stimulation of 2-deoxy-glucose uptake and alpha-aminoisobutyric acid uptake were also severely impaired with a 80-90% decrease in maximal stimulation, in parallel with a 62% decrease in insulin-stimulated autophosphorylation (P less than 0.05). 125I-insulin cross-linking revealed an 140-kD alpha subunit of the insulin receptor similar to that in cells from nondiabetic rats, although bands at greater than 200 kD were also detected. The molecular weight of the insulin receptor beta subunit (by SDS-PAGE) was smaller in cells from diabetic than from normal rats (88-90 vs. 95 kD). Neuraminadase treatment of the partially purified insulin receptors decreased the molecular weight of the insulin receptors from nondiabetic rats to a greater degree than its diabetic counterpart. In contrast, Northern blot analysis of insulin receptor mRNAs using human cDNA probes revealed two species of 9.4 and 7.2 kb with no difference in mRNA abundance between cells from diabetic and nondiabetic rats. We conclude that the exposure of capillary endothelial cells to a diabetic milieu in vivo can cause specific and persistent changes in the insulin receptor and insulin action.

Animals↗
PubMed1989

Insulin receptor messenger ribonucleic acid sequence alterations detected by ribonuclease cleavage in patients with syndromes of insulin resistance.

We used a ribonuclease cleavage assay to screen for insulin receptor mRNA sequence alterations in 12 patients with syndromes of severe insulin resistance. Uniformly labeled [32P]antisense RNA probes complementary to insulin receptor mRNA were prepared by an SP6 or T7 RNA polymerase transcription reaction. Four probes ranging in size from 670-1470 bases were used to examine the entire 4.2-kilobase receptor protein-coding region. Patient RNA samples were hybridized to individual probes in solution, and mismatched sequences were detected by susceptibility to cleavage by a mixture of RNAses A and T1. The method was validated with insulin receptor mRNAs from cells transfected with cDNA constructs bearing known point and deletion mutations. Alterations in the insulin receptor mRNA sequence of two patients were detected. A patient with the type A syndrome of severe insulin resistance (A2-Boston) had a mutation in the insulin receptor beta-subunit mRNA sequence that localized to the region coding for amino acid residues 1174-1211 near the tyrosine kinase domain. The second alteration was a sequence polymorphism in the insulin receptor alpha-subunit mRNA in a patient with lipoatropic diabetes (LA-2) that localized to a region within amino acids 268-272. Direct sequence analysis revealed that the ribonuclease cleavage sites in patients A2-Boston and LA-2 were due to distinct single base changes in the insulin receptor gene and mRNA. Additional insulin receptor mRNA sequence polymorphisms were also identified as mismatches between the labeled RNA probes used and mRNA from several cultured human cell types. This study demonstrates that ribonuclease cleavage can rapidly detect and localize insulin receptor mRNA sequence mutations and polymorphic variations as small as single base changes. Further analysis of insulin receptor mRNA sequence alterations identified in this way may elucidate a possible genetic basis for functional insulin receptor defects in patients with severe insulin resistance and can also reveal some insulin receptor sequence polymorphisms that occur in the population at large.

Acanthosis Nigricans↗
PubMed1988

Initial processing of the insulin receptor precursor in vivo and in vitro.

To evaluate the synthesis and initial processing of the insulin receptor precursor, we compared cell-free translation of rat liver poly(A)+ RNA in a reticulocyte lysate system with metabolically labeled rat hepatoma (Fao) cells. In in vitro translation assays, the primary L-[35S]cysteine-labeled products of rat liver mRNA specifically immunoprecipitable with insulin receptor antiserum were two closely migrating polypeptides with a Mr range of 160,000-164,000 (n = 7). This is similar to the size predicted by the insulin receptor cDNA sequence. When heterologous microsomal membranes were included in the cell-free system to process newly synthesized proteins co-translationally, the receptor precursors migrated as larger species of 180 +/- 2 kDa (n = 3). For comparison, when Fao cells were treated with tunicamycin to block core N-glycosylation and pulse-labeled with L-[35S]methionine, two closely migrating precursors were labeled that co-migrated with the unprocessed in vitro translation products (approximately 160 kDa). Pulse labeling of Fao cells in the absence of tunicamycin revealed receptor precursor species of 188 and 198 kDa that rapidly disappeared (t1/2 = 54 min) as the receptor subunits were observed. Thus, the initial products of insulin receptor mRNA translation are two approximately 162-kDa polypeptides that are rapidly processed in intact cells and can only be observed by in vitro studies or by using inhibitors of core glycosylation. Insulin proreceptor species can also be partially glycosylated during cell-free translation by added microsomal membranes. This is the first description of cell-free translation of the insulin proreceptor in a system that will allow detailed characterization of the earliest steps in insulin receptor biogenesis.

Animals↗
PubMed1988

The insulin receptor gene and its expression in insulin-resistant mice.

Defects of insulin receptor binding and tyrosine kinase activity have been described in genetically diabetic (db/db) and obese (ob/ob) mice. To determine if these changes were related to an abnormality in insulin receptor mRNA expression or structure of the receptor gene, we quantitated receptor mRNA from db/db and ob/ob homozygous, heterozygous (db/x, ob/x) and unaffected [db(x/x), ob(x/x)] mice and also analyzed restriction fragment length patterns of genomic DNA. Northern blot analysis of insulin receptor mRNA in livers from each of the genotypes revealed two major species of 7.5 and 9.5 kilobases. In contrast to known decreased receptor number in various tissues of ob/ob and db/db mice, quantitation of liver insulin receptor mRNA revealed that both homozygous affected strains had 2-fold or more increased levels of both major mRNA species compared to unaffected control groups. (P less than 0.05). Restriction fragment length analysis revealed no major insertion or deletion mutations in either the db/db or ob/ob insulin receptor gene. From the number and size of the fragments generated by this analysis, the minimal size of the mouse insulin receptor gene was calculated to be 97 kilobases, and the minimal number of exons was 16. These data indicate that the insulin receptor gene in ob/ob and db/db mice exhibits no major structural abnormality. Decreases in insulin receptor binding and/or kinase activity in affected mice appear to be due to a defect at the posttranscriptional level and occur despite increased levels of receptor mRNA.

Animals↗
PubMed1987

Variation in insulin receptor messenger ribonucleic acid expression in human and rodent tissues.

The expression of insulin receptor mRNA was studied in human and rodent tissues by Northern analysis. Human EBV-transformed lymphocytes contained four receptor mRNA species of sufficient length to encode the entire proreceptor: 9.5, 7.9, 7.1, and 5.7 kb. In human fibroblasts, the same four species were observed; however, the 7.9 and 5.7 kb mRNAs were markedly decreased. In mouse liver, rat hepatoma cells, and normal rat brain, kidney, liver, and muscle only two mRNA species (7.4 and 9.6 kb) were detected. Each of these human and rodent mRNAs hybridized equally well with cDNA sequences encoding the binding and kinase domains of the insulin receptor. Several smaller polyadenylated mRNAs (approximately 1.8 to 3.3 kb) were also identified in human cell lines that appeared to separately encode either alpha- or beta-subunit sequences of the receptor. In rats, liver had the highest content of insulin receptor mRNA, followed by kidney, brain, and muscle. The relative amount of the two mRNA species also varied among the rat tissues. The ratio of the 9.6-7.4 kb species was 2.7 in brain but only 1.0 to 1.6 in the other tissues (P less than 0.025). Dexamethasone treatment increased the content of the two insulin receptor mRNAs in rat liver by 2-fold. The half-life of both mRNA species was 70 min in rat hepatoma cells. These findings indicate that insulin receptor gene expression is complex and regulated with differential expression of insulin receptor mRNA and/or alterations in mRNA processing among various tissues.

Animals↗
PubMed1986

Comparative efficacy of estazolam, flurazepam, and placebo in outpatients with insomnia.

The efficacy and safety of estazolam, an investigational triazolobenzodiazepine, and flurazepam were compared in 65 insomniac outpatients. Patients completed sleep questionnaires each morning. Global evaluations demonstrated that both treatments were significantly superior to placebo. However, estazolam was preferred over flurazepam in a global rating that reflected how well rested and refreshed the subjects felt on arising. Improvement in complaints of difficulty in going to sleep showed only a trend toward significance favoring estazolam and flurazepam over placebo. Residual daytime drowsiness and fatigue accounted for approximately 70% of all side effects with both active treatments. Significantly more side effects occurred with flurazepam than with estazolam. Flurazepam-treated patients had a significantly more severe rating of adverse reactions than did placebo-treated patients.

Adult↗
PubMed1985

Hypnotic efficacy of a modified triazolodiazepine, brotizolam.

Fifty-nine outpatients with insomnia were treated in a 3-week study to assess the safety and efficacy of a new triazolothienodiazepine with a short t1/2, brotizolam. Therapy was initiated with 0.25 mg at night, but by the end of the study all subjects who were receiving brotizolam were taking 0.50 mg. Results indicate brotizolam efficacy as measured by daily subject and weekly physician questionnaires in essentially all sleep parameters, including global subject and physician evaluation of treatment efficacy, sleep latency and sleep maintenance, and quality of sleep. Tolerance to the hypnotic action of brotizolam was not observed after 21 days of daily dosing.

Azepines↗
PubMed1985

A double-blind placebo-controlled study of fluvoxamine and imipramine in depression.

Outpatients with major affective disorder, unipolar depressed type (N=101), were treated in a 4-week placebo-controlled double-blind study to compare the efficacy and safety of fluvoxamine, a new serotonin reuptake inhibitor antidepressant, with imipramine and placebo. Therapy was initiated at 50 mg/day; thereafter, dosage ranged between 100 and 300 mg/day for both drugs. Results indicate statistically significant efficacy, measured by both patient and physician rating scales, for both active drugs over placebo. Fluvoxamine showed some evidence of earlier onset of action. Anticholinergic side effects were more common in the imipramine-treated patients, while fluvoxamine produced more gastrointestinal distress and insomnia.

Adult↗