Intestinal guanylate cyclase mediates the action of the heat-stable enterotoxin to cause a decrease in intestinal fluid absorption and to increase chloride secretion, ultimately causing diarrhea. An endogenous ligand that acts on this guanylate cyclase has not previously been found. To search for a potential endogenous ligand, we utilized T84 cells, a human colon carcinoma-derived cell line, in culture as a bioassay. This cell line selectively responds to the toxin in a very sensitive manner with an increase in intracellular cyclic GMP. In the present study, we describe the purification and structure of a peptide from rat jejunum that activates this enzyme. This peptide, which we have termed guanylin, is composed of 15 amino acids and has the following amino acid sequence, PNTCEICAYAACTGC, as determined by automated Edman degradation sequence analysis and electrospray mass spectrometry. Analysis of the amino acid sequence of this peptide reveals a high degree of homology with heat-stable enterotoxins. Solid-phase synthesis of this peptide confirmed that it stimulates increases in T84 cyclic GMP levels. Guanylin required oxidation for expression of bioactivity and subsequent reduction of the oxidized peptide eliminated the effect on cyclic GMP, indicating a requirement for cysteine disulfide bond formation. Synthetic guanylin also displaces heat-stable enterotoxin binding to cultured T84 cells. Based on these data, we propose that guanylin is an activator of intestinal guanylate cyclase and that it stimulates this enzyme through the same receptor binding region as the heat-stable enterotoxins.Pathogenic strains of E. coli and other bacteria produce a family of heat-stable enterotoxins (STs) that activate intestinal guanylate cyclase. STs are acidic peptides that contain 18 or 19 amino acids with six cysteines and three disulfide bridges that are required for full expression of bioactivity (6). The increase of intestinal epithelial cyclic GMP elicited by STs is thought to cause a decrease in water and sodium absorption and an increase in chloride secretion (7,8). These changes in intestinal fluid and electrolyte transport then act to cause secretory diarrhea. In developing countries, the diarrhea resulting from STs causes many deaths, particularly in the infant population (9). STs are also considered a major cause of traveler's diarrhea in developed countries (10). They have also been reported to be a leading cause of morbidity and death in domestic animals (11).In the present study, we designed a bioassay to search for a potential endogenous ligand that activates the intestinal guanylate cyclase. This bioassay is based on the demonstration that T84 cells in culture respond to ST in a selective and sensitive manner with graded increases of intracellular cyclic GMP. This bioassay revealed that the intestine as well as the kidney possessed an active material. Purification of this material from the rat intestine was accomplished and the structure was determined to be a 15-amino acid peptide with 4 cysteines t...

Chronic inflammation is postulated to be involved in development of end stage renal disease (ESRD) in diabetes, but which specific circulating inflammatory proteins contribute to this risk remains unknown. To study this we examined 194 circulating inflammatory proteins in subjects from three independent cohorts with Type 1 and Type 2 diabetes. In each cohort we identified an extremely robust K idney R isk I nflammatory S ignature (KRIS) consisting of 17 novel proteins enriched for TNF Receptor Superfamily members that was associated with a 10-year risk of ESRD. All these proteins had a systemic, non-kidney source. Our prospective study findings provide strong evidence that KRIS proteins contribute to the inflammatory process underlying ESRD development in both types of diabetes. These proteins may be used as new therapeutic targets, new prognostic tests for high risk of ESRD and as surrogate outcome measures where changes in KRIS levels during intervention can reflect the tested therapy’s effectiveness.

This paper presents electrospray mass spectrometric analysis of mixtures containing monoglycerides, diglycerides, and triglycerides. Sample compounds were dissolved in concentrations of 1-50 pmol/microL in chloroform:methanol (70:30, v:v), which was modified by the addition of alkall-metal or ammonium salts or by addition of formic acid to favor the addition of a cationic species to the sample molecules. Electrospray mass spectrometric analysis of acylglycerol standards yielded positive-ion current signals for (M + Na)+ or (M + NH4)+ of all the species that were present at low picomole per microliter concentrations with no fragmentation. For equimolar concentrations of these sample compounds, there was a general decrease in ion current response as the analyte polarity decreased. Therefore, acylglycerols that contained unsaturated fatty acid chains were observed to exhibit a response in the mass spectrum greater than those with saturated chains, and ion signals resulting from the molecular adduct ions of monoglycerides were more abundant than those of diglycerides, which were more abundant than those of triglycerides in the mass spectrum. Electrospray mass spectrometric analysis of an unknown lipid material recovered from a mammalian cell culture reactor revealed a mixture of triglycerides containing mostly C14, C16, and C18 fatty acids with varying degrees of unsaturation. The results obtained by electrospray mass spectrometry compared favorably to those obtained by gas chromatography after saponification and methylation of fatty acid components of the triglycerides. MS/MS fragmentation of sodiated acylglycerols required a dissociation energy significantly greater than that required for fragmentation of ammoniated acylglycerols, so MS/MS characterization of acylglycerols was generally performed on the ammoniated compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

The human malaria parasite, Plasmodium falciparum, de-grades nearly all its host cell hemoglobin during a short segment of its intraerythrocytic development. This massive catabolic process occurs in an acidic organelle, the digestive vacuole. Aspartic and cysteine proteases have been implicated in this pathway. We have isolated three vacuolar proteases that account for most of the globin-degrading activity of the digestive vacuole. One is the previously described aspartic hemoglobinase that initiates hemoglobin degradation. A second aspartic protease is capable of cleaving hemoglobin with an overlapping specificity, but seems to prefer acid-denatured globin. The third is a cysteine protease that does not recognize native hemoglobin but readily cleaves denatured globin. It is synergistic with the aspartic hemoglobinase, both by in vitro assay of hemoglobin degradation, and by isobologram analysis of protease inhibitor-treated parasites in culture. The cysteine protease is highly sensitive to chloroquine-heme complex, suggesting a possible mechanism of 4-aminoquinoline antimalarial action. The data suggest an ordered pathway of hemoglobin catabolism that presents an excellent target for chemotherapy. (J. Clin. Invest.

The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the ␣ and ␤ chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4 positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases.

BackgroundInflammation signaled by Janus kinases (JAKs) promotes progression of diabetic kidney disease (DKD). Baricitinib is an oral, reversible, selective inhibitor of JAK1 and JAK2. This study tested the efficacy of baricitinib versus placebo on albuminuria in adults with Type 2 diabetes at high risk for progressive DKD.MethodsIn this Phase 2, double-blind, dose-ranging study, participants were randomized 1:1:1:1:1 to receive placebo or baricitinib (0.75 mg daily; 0.75 mg twice daily; 1.5 mg daily; or 4 mg daily), for 24 weeks followed by 4–8 weeks of washout.ResultsParticipants (N = 129) were 63±9.1 (mean±standard deviation) years of age, 27.1% (35/129) women and 11.6% (15/129) African-American race. Baseline hemoglobin A1c (HbA1c) was 7.3±1% and estimated glomerular filtration rate was 45.0±12.1 mL/min/1.73 m2 with first morning urine albumin–creatinine ratio (UACR) of 820 (407–1632) (median; interquartile range) mg/g. Baricitinib, 4 mg daily, decreased morning UACR by 41% at Week 24 compared with placebo (ratio to baseline 0.59, 95% confidence interval 0.38–0.93, P = 0.022). UACR was decreased at Weeks 12 and 24 and after 4–8 weeks of washout. Baricitinib 4 mg decreased inflammatory biomarkers over 24 weeks (urine C–X–C motif chemokine 10 and urine C–C motif ligand 2, plasma soluble tumor necrosis factor receptors 1 and 2, intercellular adhesion molecule 1 and serum amyloid A). The only adverse event rate that differed between groups was anemia at 32.0% (8/25) for baricitinib 4 mg daily versus 3.7% (1/27) for placebo.ConclusionsBaricitinib decreased albuminuria in participants with Type 2 diabetes and DKD. Further research is required to determine if baricitinib reduces DKD progression.

To determine the effect of tirzepatide, a dual agonist of glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 receptors, on biomarkers of nonalcoholic steatohepatitis (NASH) and fibrosis in patients with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODSPatients with T2DM received either once weekly tirzepatide (1, 5, 10, or 15 mg), dulaglutide (1.5 mg), or placebo for 26 weeks. Changes from baseline in alanine aminotransferase (ALT), aspartate aminotransferase (AST), keratin-18 (K-18), procollagen III (Pro-C3), and adiponectin were analyzed in a modified intentionto-treat population. RESULTSSignificant (P < 0.05) reductions from baseline in ALT (all groups), AST (all groups except tirzepatide 10 mg), K-18 (tirzepatide 5, 10, 15 mg), and Pro-C3 (tirzepatide 15 mg) were observed at 26 weeks. Decreases with tirzepatide were significant compared with placebo for K-18 (10 mg) and Pro-C3 (15 mg) and with dulaglutide for ALT (10, 15 mg). Adiponectin significantly increased from baseline with tirzepatide compared with placebo (10, 15 mg). CONCLUSIONSIn post hoc analyses, higher tirzepatide doses significantly decreased NASH-related biomarkers and increased adiponectin in patients with T2DM.The prevalence of nonalcoholic fatty liver disease (NAFLD) is ;25% globally and ;60-75% in patients with type 2 diabetes mellitus (T2DM) (1,2). Nonalcoholic steatohepatitis (NASH) (NAFLD with inflammation and hepatocyte injury, with or without fibrosis) can progress to cirrhosis, liver failure, hepatocellular carcinoma, and increased cardiovascular risk (3,4). T2DM increases the risk of NASH twofold (5). Weight loss through lifestyle modification reduces liver fat; weight reductions $10% can induce NASH resolution in most patients (6).Glucagon-like peptide 1 receptor agonists (GLP-1 RAs) promote weight loss and may have efficacy in NASH (7). Tirzepatide, a 39-amino acid synthetic peptide, has agonist activity at both glucose-dependent insulinotropic polypeptide (GIP) and GLP-1