Non-esterified fatty acids are deleterious for human pancreatic islet function at physiological glucose concentration.

AIMS/HYPOTHESIS: Whether excess glucose (glucotoxicity) and excess non-esterified fatty acids (lipotoxicity) act synergistically or separately to alter beta-cell function on Type 2 diabetes remains controversial. We examined the influence of non-esterified fatty acids, with or without concomitant increased glucose concentrations, on human islet function and on the expression of genes involved in lipid metabolism. METHODS: Human islets isolated from non-diabetic and non-obese donors were cultured with 5.5, 16 or 30 mmol/l glucose, and when appropriate with 1 or 2 mmol/l non-esterified fatty acids. After 48 h, glucose-stimulated insulin secretion, insulin content, triglyceride content and expression of different genes were evaluated. RESULTS: Non-esterified fatty acids decreased glucose-stimulated insulin secretion, insulin content and increased triglyceride content of human isolated islets, independently from the deleterious effect of glucose. Increased glucose concentrations also decreased glucose-stimulated insulin secretion and insulin content, but had no influence on triglyceride content. Glucose-stimulated insulin secretion of islets appeared to be significantly correlated with their triglyceride content. Glucose and non-esterified fatty acids modified the gene expression of carnitine palmitoyltransferase-I, acetyl-CoA carboxylase, acyl-CoA oxidase and uncoupling protein 2. CONCLUSION/INTERPRETATION: In our model of isolated human islets, increased glucose and non-esterified fatty acids separately reproduced the two major beta-cell alterations observed in vivo, i.e. loss of glucose-stimulated insulin secretion and reduction in islet insulin content. Our results also suggest that this deleterious effect was, at least in part, mediated by modifications in lipid metabolism gene expression.

Combined effects of inhaled nitric oxide (iNO) and oxidant agents on the production of methemoglobinemia in newborn piglets.

OBJECTIVE: To investigate the effects of the association of inhaled nitric oxide (iNO) and oxidant drugs (acetaminophen, phytomenadione, and EMLA cream) on methemoglobinemia during the neonatal period. DESIGN: Prospective, randomized, experimental study. SETTING: University Experimental Pharmacology laboratory. SUBJECTS: Sixty newborn piglets weighing 1.5-2.0 Kg. INTERVENTIONS: Twelve groups of five piglets were anaesthetized, mechanically ventilated, and studied for 3 hrs. Eight groups received iNO (40 ppm or 80 ppm) alone or in association with a single intravenous dose of acetaminophen (120 mg/kg propacetamol), phytomenadione (5 mg vitamin K1) or EMLA cream (2.5 g) applied to the ventral lower abdomen for 3 hrs. Three other groups received, respectively, acetaminophen, phytomenadione, or EMLA cream without iNO. The last group (control group) received neither drugs nor iNO. MEASUREMENTS AND MAIN RESULTS: Methemoglobinemia was measured before the beginning of each experiment, 30 mins later, and every hour for 3 hrs. There was no significant difference in methemoglobinemia at any time between groups receiving acetaminophen (0.90%+/-0.12%), phytomenadione (0.88%+/-0.11%), or EMLA cream alone (0.97%+/-0.11%) and the control group (0.92%+/-0.12%). At 3 hrs, methemoglobinemia was slightly but significantly increased in group receiving iNO alone (1.04%+/-0.17% at 40 ppm iNO and 1.14%+/-0.16% at 80 ppm iNO; p < .05). Conversely, methemoglobinemia increased as a function of time in groups in which iNO was associated to drug administration and was significantly greater than the control group at 3 hrs (80 ppm iNO + acetaminophen, 2.80%+/-0.47%; 80 ppm iNO + phytomenadione, 2.38%+/-0.45%; 80 ppm iNO + EMLA cream, 2.33%+/-046%; p < .001). CONCLUSIONS: These results demonstrate that if oxidant drugs (acetaminophen, phytomenadione, or EMLA cream) did not increase blood methemoglobinemia in neonatal piglets, their association with iNO caused an increase in methemoglobin. Special care should be taken to monitor methemoglobinemia when iNO is combined to such drugs in newborn infants.

Inhaled nitric oxide neither alters oxidative stress parameters nor induces lung inflammation in premature lambs with moderate hyaline membrane disease.

The purpose of this investigation was to examine whether inhaled nitric oxide (NO) may alter oxidative stress parameters and induce lung inflammation in moderate hyaline membrane disease (HMD). Eighteen moderately premature lambs (130 days gestation, term = 147 days) were randomly assigned to treatment with 20 ppm inhaled NO (n = 8) from the onset of ventilation or used as control (n = 10). Except inhaled NO, treatments were intentionally similar to those applied in clinical situations. The main studied parameters were oxidative stress index measurements on lung parenchyma and in circulating blood, lung parenchyma microscopic examination and bronchoalveolar lavage cell count. We found that 20 ppm of inhaled NO for 5 h did not change significantly either malondialdehyde and total antioxidant status levels in circulating blood, or malondialdehyde, reduced glutathione, glutathione peroxidase and glutathione reductase in lung parenchyma. Amino-imino-propene bond generation, which are lipoperoxidation markers, was similar in both groups. Furthermore, no significant changes in the number of inflammatory cells in lung lavage products and in lung parenchyma microscopic examination could be found. Therefore, these data do not support the hypothesis that short-term NO inhalation increases oxidative stress and lung inflammation in an experimental model of moderate HMD.

Colonic xanthelasma due to glyceride accumulation associated with an adenoma.

The case is reported of a 66-year-old woman who presented with endoscopic and histological features of multiple lipid deposits in the mucosa of the sigmoid colon associated with an adenoma. Associated clinical features were abdominal pain and diarrhea. Colectomy led to the complete resolution of symptoms. Biochemical analysis disclosed the presence of glycerides in the mucosa. The pathogenesis of lipid deposits and the possible link with the formation of an adenoma is discussed.

Squid spermiogenesis: molecular characterization of testis-specific pro-protamines.

Cuttlefish spermiogenesis is characterized by a two-step nuclear protein transition: histones-->spermatid-specific protein (protein T)-->sperm protamine (protein Sp). A similar situation can be observed in another Cephalopod species, the squid Loligo pealeii. The protein T from Loligo consists of two structural variants, T1 and T2 (molecular masses: 10788 and 10791 Da respectively), phosphorylated to different degrees (2-6 phosphate groups). The primary structures of these two variants and of the protamine variant Sp2 were established from sequence analysis and mass spectrometric data of the proteins and their fragments. T1 and T2 are closely related proteins of 79 residues. The complete structural identity of the C-terminal domain (residues 22-79) of protein T2 with the sperm protamine Sp2 (molecular mass 8562 Da, 58 residues) strongly suggests that the testis-specific protein T2 is indeed the precursor of the protamine. The transition between the precursor protein T and protein Sp results from a hydrolytic cleavage similar to that found in many proteins that are synthesized as precursors. The processing mechanism involves the specific cleavage of a Gly-Arg bond in the sequence Met/Leu18-Lys-Gly-Gly-Arg-Arg23. Furthermore, the study provides molecular evidence on the taxonomic relationship between Loligo and Sepia.

Isolation and characterization of a small putative zinc finger protein from cuttlefish epididymal sperm cells.

At the end of spermiogenesis, sperm chromatin stabilization is ensured by protamine dephosphorylation and, in mammals, by the formation during epididymal transit, of intra- and inter-molecular disulfide bridges between protamines. In cuttlefish, the nuclear protein transition histones-->spermatid-specific protein T-->protamine Sp is very similar to that occurring in mammals during spermiogenesis. However, in cuttlefish, the protamine Sp is devoid of cysteine residues. The protein complement of cuttlefish epididymal sperm nuclei has been investigated. A minor basic protein, called protein E, has been isolated. Its primary structure was established from sequence analysis and mass spectrometry data of the protein and its fragments. Protein E contains a motif -Cys-Xaa2-Cys-Xaa23-His-Cys-Xaa2-Cys- which is likely to adopt a zinc finger conformation. Reduced protein E does fix zinc whereas alkylation of cysteine residues abolishes this ability. The sequence of protein E does not correspond to that of any known protein, but presents some similarities with a part of ZFY protein, a putative human transcription factor specifically expressed in germinal cells and which could be involved in spermatogenesis.

Cuttlefish spermatid-specific protein T. Molecular characterization of two variants T1 and T2, putative precursors of sperm protamine variants Sp1 and Sp2.

In cuttlefish, as in selachians and mammals, spermiogenesis is characterized by the double nuclear protein transition histones----intermediate protein (protein T)----protamine (protein Sp). The cuttlefish protein T, which consists of two structural variants phosphorylated at different degrees, is the first invertebrate spermatid-specific protein to be fully characterized and sequenced. The primary structures of these two variants were established from sequence analysis and mass spectrometric data of the proteins and their fragments. T1 and T2 are two highly related proteins of 78 and 77 residues, respectively, which differ only by four conservative substitutions, two inversions Ser in equilibrium with Arg, and the deletion of 1 residue of arginine in variant T2. The asymmetrical distribution of the hydrophobic and basic residues determines two well defined domains: an amino-terminal domain (residues 1-21) devoid of arginine and aromatic residues and containing all the aliphatic hydrophobic residues and a highly basic carboxyl-terminal domain (residues 22-77 or 78) that contains 77% of arginine, all the tyrosine residues, and most of the phosphorylated serine residues present in the protein. The complete structural identity of the basic carboxyl-terminal domain of spermatidal proteins T1 and T2 with the protamine variants Sp1 and Sp2 isolated from cuttlefish spermatozoa strongly suggests that T1 and T2 could be precursors of Sp1 and Sp2, respectively.

Cuttlefish sperm protamines. 1. Amino acid sequences of two distinct variants.

The amino acid sequences of two cuttlefish protamine variants Sp1 and Sp2 have been established from automated sequence analysis and mass spectrometry data. Sp1 (57 residues) and Sp2 (56 residues) have molecular masses of 8410 and 8253 Da, respectively. They are almost identical proteins which differ only by one residue of arginine and the position of two of the serine residues (14 and 37 in Sp1; 13 and 35 in Sp2). With an arginine content of about 77%, cuttlefish protamine is one of the most basic proteins which have ever been characterized and the first typical protamine sequenced in invertebrates. It is closely similar to sperm basic proteins identified in squids but strongly differs from the protamine-like components isolated from the sperm of bivalve molluscs.

Histone H4 from cuttlefish testis is sequentially acetylated. Comparison with acetylation of calf thymus histone H4.

The differently acetylated subfractions of histone H4 isolated from cuttlefish testis and from calf thymus were separated by ion exchange chromatography on sulfopropyl-Sephadex, using a shallow linear gradient of guanidine hydrochloride in the presence of 6 M urea at pH 3.0. The tetra-, tri-, di-, mono-, and nonacetylated forms of cuttlefish H4 represent 2, 6.4, 18, 32.2, and 41.4% of the whole histone, respectively. The di-, mono-, and nonacetylated forms of calf H4 represent 11.7, 41.3, and 44% of the whole histone, respectively. The acetylation sites were determined in each subfraction by identification of the acetylated peptides. In each acetylated H4 subfraction, the acetylated tryptic peptides were identified by peptide mapping and amino acid analysis with reference to the peptide map of nonacetylated H4. In cuttlefish testis H4, lysine 12 is the main site of acetylation in the monoacetylated subfraction; lysines 5 and 12 are found acetylated in diacetylated H4; lysines 5, 12, and 16 are found acetylated in triacetylated H4. From these results and the stoichiometry of the different H4 subfractions, it can be concluded that lysine 5 is acetylated after lysine 12. In calf thymus, lysine 16 is the only site of acetylation in the monoacetylated subfraction. All the diacetylated forms are acetylated in lysine 16, the second site of acetylation being, in decreasing order, lysine 12, lysine 5, or lysine 8. These observations suggest that acetylation occurs in a sequential manner. Moreover, the sites of acetylation depend upon the biological event in which acetylation is involved.

[Assay of proteins in the urine and cerebrospinal fluid. Choice of a method in centrifugal analysis]. / Dosage de la protéinurie et de la protéinorachie. Choix d'une méthode en analyse centrifuge.

The study consists in adapting two methods allowing estimation of proteins in cerebrospinal fluid and urine on a centrifugal analyzer using Coomassie Blue-SDS and benzethonium chloride. Results obtained with the two reagents are compared with turbidimetric technic using trichloroacetic acid, by statistic methods.

The amino-acid sequence of histone H2A from cuttlefish Sepia officinalis.

The amino acid sequence of cuttlefish testis histone H2A (124 residues) was established from structural data obtained by automated sequencing of large peptides generated by the cleavage of the protein with V8 staphylococcal protease or by limited chymotryptic hydrolysis. Compared to the calf thymus homologous histone, cuttlefish H2A shows 14 substitutions (most of them conservative) and 5 deletions. Extensive evolutionary changes were mainly observed in the basic amino-terminal and carboxy-terminal regions of the molecule, which are the primary DNA-binding sites. Few punctual changes are observed in the central region (residues 18-118), which interacts strongly with histone H2B to form the dimer H2A-H2B.

Determination of amniotic fluid phospholipids by thin-layer chromatography, with use of a hydrogen flame ionization detector.

We describe a new specific method for measuring lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid by thin-layer chromatography, with use of a hydrogen flame ionization detector. After extraction and acetone precipitation to isolate surface-active lecithin, the phospholipids are separated on a thin rod of refractory and chemically stable material, having an outer coating of a bonded, sintered partition medium. Two solvent systems are used to develop the chromatograms, chloroform/methanol/water to separate lecithin and sphingomyelin, tetrahydrofuran/methylal/methanol for phosphatidylglycerol. Then the rod is passed through an hydrogen flame; the resulting ions produced generate a current, which is amplified and fed to a potentiometric recorder. The height of integral curves was proportional to the area under each peak. For quantitation we used an internal standard (lysolecithin in the first system. phosphatidyldimethylethanolamine in the second). The method requires less than 5 mL of amniotic fluid; results are available within 6 h.