Degradation of insulin and insulin-like growth factors by enzyme purified from human erythrocytes. Comparison of degradation products observed with A14- and B26-[125I]monoiodoinsulin.

An insulin-degrading enzyme has been purified from human erythrocytes. This enzyme degraded 125I-labeled insulin-like growth factor I (IGF-I) more slowly than 125I-IGF-II and degraded IGF-II more slowly than 125I-insulin. The time course of 125I-insulin degradation suggested the presence of intermediates, each of which was itself shown to be a substrate for the enzyme. One of these intermediates appeared to be made up entirely of B-chain residues and had HisB10 as its NH2-terminal. The final major radiolabeled degradation product of A14-[125I]monoiodoinsulin was a peptide with TyrA14 at the A-chain NH2 terminal. This peptide could be reduced with dithiothreitol, suggesting that it contained amino acid residues from both A- and B-chains. It was partially precipitated by trichloroacetic acid and anti-insulin antibody but bound poorly to IM-9 lymphocytes. The final major degradation product of B26-[125I]monoiodoinsulin was a peptide whose NH2-terminal was TyrB26 and could not be reduced by dithiothreitol. It was partially precipitated by anti-insulin antibody but was precipitated poorly, if at all, by trichloroacetic acid and bound poorly to IM-9 lymphocytes. The results show that this enzyme degraded insulin by sequential cleavage of peptide bonds on both A- and B-chains. We identified LeuA13-TyrA14, SerB9-HisB10, and PheB25-TyrB26 as three of the bonds that are cleaved.

The fate of insulin in rat hepatocytes. Evidence for the release of an immunologically active fragment.

We have investigated the fate of 125I-insulin after binding by rat hepatocytes. Approximately 30% of the bound radioactivity dissociated from the cells as intact 125I-insulin; approximately 56% dissociated as 125I- and 125I-Tyr. The remaining radioactivity was recovered as peptides that we postulate are intermediate products of insulin metabolism. Experiments were performed in the presence of chloroquine (0.1 mM), an agent known to inhibit the intracellular processing of 125I-insulin. As expected, chloroquine increased the amount of radioactivity recovered as intact 125I-insulin (P less than 0.005) and decreased the amount of 125I- and 125I-Tyr (P less than 0.005). In addition, chloroquine decreased the amount of one of the insulin peptides (P less than 0.005), but increased the amount of the other (P less than 0.01). These data suggest the presence of two pathways of insulin metabolism in rat hepatocytes, one of which is inhibited by chloroquine. We have found a second pathway by which insulin is degraded due to the removal of several amino acids from the carboxy-terminus of the B-chain. The resulting fragment bound poorly to insulin receptors on IM-9 cultured human lymphocytes, and probably has little if any biologic activity. However, this fragment bound well to anti-insulin antibody and constituted about 20% of the immunoreactive radioactivity that dissociated from the hepatocytes.

Inhibition of insulin-stimulated glucose transport by factor extracted from serum of insulin-resistant patient.

We report a 31-yr-old nondiabetic male patient with acanthosis nigricans whose hyperinsulinemia and insulin resistance could not be explained by anti-receptor antibodies or by an intrinsic defect of insulin binding to his cells. An acid-alcohol extract of the patient's serum contained a factor that inhibited insulin-stimulated glucose transport in rat adipocytes. Low levels of the factor could be detected in 9 of 13 unselected patients with non-insulin-dependent diabetes. The factor was heat stable and resistant to treatment with acid, base, and various lytic enzymes. It eluted from a Bio-Gel P-2 column with an apparent molecular weight of 300. The factor also inhibited stimulation of glucose transport in adipocytes by the insulin mimickers hydrogen peroxide and sodium vanadate. In vitro incubation of rat soleus muscles in the presence of the factor resulted in inhibition of insulin-stimulated glucose transport. The factor enhanced 125I-labeled insulin binding in both adipocytes and muscle. A preparation of insulin receptors obtained from muscles incubated with serum factor showed increased binding of 125I-insulin to the alpha-subunit of the insulin receptor. Autophosphorylation of the beta-subunit and phosphorylation of exogenous substrate were increased in the receptor preparation obtained from muscles that had been incubated with serum factor. However, the increase in kinase activity was approximately the same as the increase in binding activity. No difference in kinase activity was observed when assayed under conditions in which 125I-insulin binding activity had been equalized.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of fasting on insulin binding to hepatocytes and liver plasma membranes from rats.

Binding of [125I]iodoinsulin to isolated hepatocytes from fed and fasted rats was measured. Hepatocytes from fed rats bound significanlty more [125I]iodoinsulin at insulin concentrations ranging from 0.15--100 nM. Scatchard and average affinity profile data analyses showed no significant difference in the affinities of the binding sites but indicated a decrease in the receptor concentration of the hepatocytes from fasted rats. In contrast to hepatocytes, liver plasma membranes of fasted rats showed increased insulin binding at all insulin concentrations, except the highest level of 100 nM. Analysis of the data indicated a significant increase in the affinity of the receptors but no change in the receptor concentration. Evidence is presented that the differences in the results obtained with hepatocytes and membranes were not due to the differences in the recovery of the plasma membranes. The results suggest that insulin binding by intact hepatocytes does not necessarily reflect the receptor protein content of the cell membrane and that the liver cell modulates its response to insulin by changes in the architecture of the plasma membrane.

Inhibition of insulin degradation by insulin-like growth factors.

The effect of insulin-like growth factors (IGF 1 and IGF 2) on insulin degradation was studied with the use of a preparation of insulin protease from rat skeletal muscle. Insulin, IGF 1 and IGF 2 inhibited 125I-insulin degradation by this enzyme. IGF 2 was the most potent inhibitor and IGF 1 was the least potent. These results are similar to what has been reported previously for the insulin-degrading activity in the serum of a diabetic patient who was resistant to sc and im insulin. Insulin protease also degraded 125I-iodo IGF 1 and 125I-iodo IGF 2. 125I-iodo IGF 2 was degraded more rapidly than was 125I-iodo IGF 1. 125I-iodoinsulin was degraded more rapidly than 125I-iodo IGF 2. With all three peptides, immunoprecipitation was a more sensitive measure of degradation than was trichloroacetic acid precipitation. The results suggest that insulin protease may be responsible for the degradation of insulin-like growth factors as well as of insulin.

Insulin degradation in serum of a patient with apparent insulin resistance.

A case is presented of a thin diabetic male who was resistant to large doses of sc and im insulin but responded to small dose of insulin given iv. His serum contained an enzyme that degraded [125I]insulin in vitro. We postulate that his apparent insulin resistance was due to inactivation of insulin at the injection site. We propose that the sera of patients with insulin resistance be tested for [125I]insulin-degrading activity as a possible means of identifying patients with this syndrome.

Phase II study of gemcitabine, 5-fluorouracil, and leucovorin in patients with pancreatic cancer.

The primary goal of this phase II study was to determine the efficacy of gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) plus 5-fluorouracil in patients with pancreatic cancer. Eligibility criteria included nonresectable locally advanced or metastatic pancreatic adenocarcinoma and measurable disease. Gemcitabine at 1,000 mg/m(2) and leucovorin at 20 mg/m(2) were administered intravenously 30 minutes before 5-fluorouracil 600 mg/m(2), weekly for 3 of every 4 weeks. Twenty nine patients were enrolled. The overall response rate was 21% (95% confidence interval: 8% to 40%), consisting of one complete response and five partial responses; 16 patients (55%) had stable disease. Median survival was 8.4 months (95% confidence interval: 2.6 to 14.2), and actuarial 1-year survival was 36%. Neutropenia (grade 3 only) was reported in 3.4% of patients, but was generally of short duration. No thrombocytopenia or evidence of cumulative myelosuppression was observed. The only significant nonhematologic events were grade 3 diarrhea and alopecia (both 3.4%). Gemcitabine plus 5-fluorouracil is active and well tolerated compared with results reported for each of these single agents. Thus, this combination justifies future comparative clinical trials. Semin Oncol 28 (suppl 10):44-49.

Analysis of expressed sequence tags from Plasmodium falciparum.

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

Effects of insulin and streptozotocin-diabetes on isoproterenol-stimulated cyclic AMP production in myocytes isolated from rat heart.

Isoproterenol (ISO), at concentrations of up to 10(-5) mol/L, caused a dose-dependent stimulation of cyclic AMP (cAMP) production in rat cardiomyocytes. At higher ISO concentrations, relative inhibition was seen. Insulin augmented ISO-stimulated cAMP production and appeared to mitigate the toxic effects of high ISO concentrations. This synergy between insulin and ISO observed in cardiomyocytes was not observed in adipocytes. Streptozotocin (STZ)-diabetes abolished the stimulatory effects of insulin on ISO-induced cAMP production in cardiomyocytes.

Resistance to subcutaneous and intramuscular insulin associated with deficiency of insulin-like growth factor (IGF) 2.

A diabetic patient is described whose serum was deficient in IGF 2. The patient responded appropriately to intravenous insulin but was resistant to subcutaneous and intramuscular insulin. His serum degraded insulin in vitro. This degradation was inhibited by IGF 2 and to a lesser extent by IGF 1 and insulin. We propose that this patient inactivated insulin at the injection site because of an insulin protease in his tissues that would normally be inhibited by serum IGF 2.

Insulin binding and glucose transport activity in cardiomyocytes of a diabetic rat.

We studied insulin binding and glucose transport in isolated adult cardiomyocytes from rats with 2-wk streptozotocin-induced diabetes. At 37 degrees C, cells from diabetic rats bound less 125I-insulin and exhibited lower rates of 3-O-methylglucose transport than cells from control rats. In contrast, the amount of 125I-insulin bound to myocytes at 4 degrees C was the same in both groups. Preincubation of cells from both groups with 10-10,000 ng/ml insulin significantly increased their basal rates of glucose transport by approximately 40%. However, the augmented rates in diabetics were still approximately 36% lower than the corresponding insulin-stimulated rates in the controls. When the glucose transport data were expressed as percent maximal insulin effect and plotted as a function of the amount of insulin bound, the curves obtained from both diabetic and nondiabetic controls were superimposable. These data demonstrate that 1) heart cells from diabetic rats bind less insulin than from control rats under conditions in which they exhibit impaired glucose transport rates, 2) there is no apparent difference in total receptor number between the two groups, but internalization of intact insulin appears to be diminished in diabetes, 3) coupling exists between insulin binding and glucose transport in both groups, and 4) these impaired processes are completely reversed by insulin treatment in vivo but not in vitro.

Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed.

Insulin metabolism in rat hepatocytes. Evidence for generation of an insulin fragment missing a portion of the B chain involved in receptor binding.

Insulin metabolism by isolated rat hepatocytes was studied, utilizing A14 [125I]monoiodoinsulin, [3H]PheB1 semisynthetic insulin, and [3H]insulins synthesized by rat islets. Degradation was assessed by gel filtration, polyacrylamide gel electrophoresis, precipitation by anti-insulin antibody, and binding to specific insulin receptors on IM-9 human lymphocytes. When incubations were performed at 15 degrees C or less, insulin bound to hepatocytes remained intact for up to 2 h. At 37 degrees C we detected the generation of an insulin fragment with an apparent molecular weight of approximately 5000 whose electrophoretic mobility was greater than that of insulin. The fragment bound well to anti-insulin antibodies but poorly to insulin receptors. Information about the structure of the fragment was obtained by comparing the metabolism of [3H]PheB1 semisynthetic insulin with that of [3H]PheB1,24,25 insulin. The data suggest that the fragment contains PheB1 but is missing the PheB24 and PheB25. Treatment of the fragment with trypsin and carboxypeptidase B did not affect its electrophoretic mobility indicating that the fragment is also missing ArgB22. Incubation in the presence of 0.1 mM chloroquine led to accumulation of both intact insulin and the insulin fragment, suggesting that both are degraded by lysosomes. The results of this study suggest the presence of two pathways for insulin degradation in liver: a chloroquine-insensitive pathway by which a portion of the B chain consisting of at least 10 amino acids is removed and a chloroquine-sensitive pathway by which both insulin and the fragment are degraded.

125I-insulin metabolism by rat liver cells: preferential binding and degradation of insulin labeled at TyrA14.

The binding and degradation of highly purified A14-[125I]monoiodoinsulin and A19-[125I]monoiodoinsulin were compared in isolated rat hepatocytes. A14-125I-insulin was bound more rapidly and was degraded more rapidly than was A19-125I-insulin. The difference in the rate of degradation between the two preparations was apparent when either tricholoroacetic acid precipitation or binding to specific insulin receptors on cultured human lymphocytes was used for assay. These results provide evidence that binding to its receptor is the first step in insulin degradation by liver cells.

A transcriptional activator, FleQ, regulates mucin adhesion and flagellar gene expression in Pseudomonas aeruginosa in a cascade manner.

Previous work has demonstrated that fleR, the gene for a transcriptional activator belonging to the NtrC subfamily of response regulators, is involved in the regulation of mucin adhesion and flagellar expression by Pseudomonas aeruginosa. This report describes the identification and characterization of fleQ, the gene for another transcriptional regulator which also regulates mucin adhesion and motility in this organism. The complete nucleotide sequence of the fleQ gene was determined on both DNA strands, and an open reading frame (ORF) consisting of 1,493 nucleotides was identified. This ORF coded for a gene product of predicted molecular weight, as confirmed by the overexpression of the fleQ gene as a fusion protein under an inducible promoter. The fleQ gene is flanked by a flagellar operon, fliDSorf126, at the 5' end and the fleSR operon on the 3' end. FleQ also had striking homology to a number of proteins belonging to the NtrC subfamily of response regulators, which work in concert with the alternate sigma factor RpoN (sigma54) to activate transcription. However, FleQ lacks the residues corresponding to Asp-54 and Lys-104 of the NtrC protein which are conserved in most of the members belonging to this subfamily of regulators. In addition, unlike some of the other transcriptional activators of this group, FleQ does not appear to have a cognate sensor kinase. A chromosomal insertional mutation in the fleQ gene abolished mucin adhesion and motility of P. aeruginosa PAK and PAK-NP. Both of these functions were regained by providing the complete fleQ gene on a multicopy plasmid. The location of fleQ immediately upstream of the fleSR operon, which is also necessary for the same process, suggested that these regulators may interact in some way. We therefore examined the regulation of the fleSR operon by fleQ and vice versa. Promoter fusion experiments showed that the fleSR operon was regulated by RpoN and FleQ. On the other hand, the fleQ promoter was independent of RpoN and FleR. FleQ, thus, adds another level of regulation to motility and adhesion in P. aeruginosa, above that of fleSR. We therefore propose the existence of a regulatory cascade which consists of at least two transcriptional regulators, FleQ and FleR, in the control of motility and adhesion in P. aeruginosa.

Cloning and phenotypic characterization of fleS and fleR, new response regulators of Pseudomonas aeruginosa which regulate motility and adhesion to mucin.

This work has identified two genes (designated fleS and fleR) in Pseudomonas aeruginosa which are highly homologous to members of the subclass of two-component systems involved in transcriptional regulation of a diverse array of genes from sigma 54 promoters. The genes are located upstream from fliE, a flagellar gene of P. aeruginosa, and they are arranged in a putative fleSR operon. FleS has a predicted molecular mass of 43.87 kDa and shows strong homology to histidine kinases which in other two-component systems have been shown to be sensor proteins. FleR has a predicted molecular mass of 51.26 kDa and is homologous to other regulatory proteins that bind to specific upstream activating elements to enhance transcription of genes with sigma 54 promoters. The fleSR system is believed to control both flagellar synthesis and adhesion to mucin. Several lines of evidence are presented. (i) A nonpiliated mutant of P. aeruginosa PAK containing a gentamicin cassette in fleR is nonmotile and nonadhesive. (ii) The fleR mutant regained motility and adhesion when complemented with a wild-type copy of fleR. (iii) A Western blot (immunoblot) of the fleR mutant showed no synthesis of flagellin, and electron microscopy of the fleR mutant confirmed the lack of flagella. Previous work has shown that flagellar mutants with mutations in fliA (sigma 28) or fliC (the structural gene for flagellin) retain adhesion; therefore, these new observations suggest that FleSR regulates both the expression of flagella and the nonpilus adhesin(s) for mucin or that one of the flagellar proteins (other than flagellin) may be responsible for adhesion to mucins.

Cloning and characterization of Pseudomonas aeruginosa fliF, necessary for flagellar assembly and bacterial adherence to mucin.

Pseudomonas aeruginosa adheres to the mucosal surfaces of the lungs. This process appears to be mediated by nonpilus adhesins which bind to mucin. To find this nonpilus adhesin(s), mutagenesis of a nonpiliated mutant of P. aeruginosa with transposon Tn5G, followed by a screen for mucin adhesion, was used to isolate a series of mutants unable to adhere to mucin. All of these mutants were also found to be defective in motility. One such mutant, PAK-RR20, is characterized here. The site of the transposon insertion in PAK-RR20 was localized to a gene which is homologous to the fliF gene of other organisms and was flanked by other motility-related genes, fliE and fliG. Both adhesion and motility defects in PAK-RR20 were complemented by providing the fliF gene in trans. Since complementation could have been due to the presence of an internal promoter in the fliF gene or in the Tn5G transposon, which allowed the transcription of the downstream genes, another chromosomal mutant of the fliF gene was constructed by insertional inactivation with an antibiotic resistance cassette. This mutant was also nonmotile and nonadhesive. However, the two defects in this new mutant could not be complemented by the fliF gene in trans, consistent with the interpretation that there is no internal fliF promoter but possibly a functional promoter in the Tn5G transposon. The complete nucleotide sequences of the fliE and fliF genes and a partial nucleotide sequence of the fliG gene of P. aeruginosa were determined. Control of the promoter upstream of the fliE gene was analyzed by construction of a fliE-lacZ fusion and the introduction of this construct into strains of P. aeruginosa with mutations in several regulatory genes. Beta-Galactosidase expression measurements indicated that the fliE promoter does not utilize RpoF (sigma(28)) or RpoN (sigma(54)) sigma factors. The characterization of this gene as being responsible for the loss of adhesion indicates that basal body structures are probably important for localization of the adhesin.

The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible for mucin adhesion.

Mucin-specific adhesion of Pseudomonas aeruginosa plays an important role in the initial colonization of this organism in the airways of cystic fibrosis patients. We report here that the flagellar cap protein, FliD, participates in this adhesion process. A polar chromosomal insertional mutation in the P. aeruginosa fliD gene made this organism nonadhesive to mucin in an in vitro mucin adhesion assay. The adhesive phenotype was restored by providing the fliD gene alone on a multicopy plasmid, suggesting involvement of this gene in mucin adhesion of P. aeruginosa. Further supporting this observation, the in vitro competition experiments demonstrated that purified FliD protein inhibited the mucin adhesion of nonpiliated P. aeruginosa PAK-NP, while the same concentrations of PilA and FlaG proteins of P. aeruginosa were ineffective in this function. The regulation of the fliD gene was studied and was found to be unique in that the transcription of the fliD gene was independent of the flagellar sigma factor sigma28. Consistent with this finding, no sigma28 binding sequence could be identified in the fliD promoter region. The results of the beta-galactosidase assays suggest that the fliD gene in P. aeruginosa is regulated by the newly described transcriptional regulator FleQ and the alternate sigma factor sigma54 (RpoN).

A membrane abnormality in lymphocytes from diabetic subjects.

The fluorescence properties of the hydrophobic probe 1,6-diphenyl1-1,3,5-hexatriene incorporated in the lymphocytes of 30 diabetic patients and 21 normal control subjects were studied. The mean value of the probe polarization was 0.314 for the control group and 0.294 for the patient group. The difference was significant at p < 0.001. The decreased polarization was correlated with the level of plasma glucose in the patients (p < 0.01). Nanosecond fluorescence results obtained from the lymphocytes of 7 patients and 5 controls indicated that there was no significant difference in the probe lifetimes between the two groups of subjects and suggest that the decreased polarization of the probe in the patient group resulted from a more fluid lipid environment of cell membranes.

Gene sequence tags from Plasmodium falciparum genomic DNA fragments prepared by the "genease" activity of mung bean nuclease.

A genes-first approach to genome sequencing is described which efficiently generates gene sequence tags from genomic DNA. Mung bean nuclease (EC 3.1.30.1) cleaves the genomic DNA of many organisms before and after genes and within some introns. Analysis of gene sequence tags prepared from mung bean nuclease-digested Plasmodium falciparum DNA demonstrates that this method has several advantages over the popular cDNA expressed sequence tag approach. To date, 673 sequence tags containing over 215 kb of sequence have been generated from 400 clones. Sixty clones (15%) have significant similarity to sequences in the protein and translated nucleic acid data bases. These represent 51 unique genes, of which only 5 encode previously known P. falciparum proteins. The identified proteins include those expressed in erythrocytic, exoerythrocytic, and gametocytic stages of the parasite. Thirty percent of clones identified appear to carry complete coding regions. The spacer DNA separating genes is rarely cloned. These gene sequence tags will form a useful data base from which to initiate projects to develop new therapeutics, vaccines, and strategies to control human malaria.