Common Degradative Pathways of Morpholine, Thiomorpholine, and Piperidine by Mycobacterium aurum MO1: Evidence from 1H-Nuclear Magnetic Resonance and Ionspray Mass Spectrometry Performed Directly on the Incubation Medium

In order to see if the biodegradative pathways for morpholine and thiomorpholine during degradation by Mycobacterium aurum MO1 could be generalized to other heterocyclic compounds, the degradation of piperidine by this strain was investigated by performing ¹H-nuclear magnetic resonance directly with the incubation medium. Ionspray mass spectrometry, performed without purification of the samples, was also used to confirm the structure of some metabolites during morpholine and thiomorpholine degradation. The results obtained with these two techniques suggested a general pathway for degradation of nitrogen heterocyclic compounds by M. aurum MO1. The first step of the degradative pathway is cleavage of the C—N bond; this leads formation of an intermediary amino acid, which is followed by deamination and oxidation of this amino acid into a diacid. Except in the case of thiodiglycolate obtained from thiomorpholine degradation, the dicarboxylates are completely mineralized by the bacterial cells. A comparison with previously published data showed that this pathway could be a general pathway for degradation by other strains of members of the genus Mycobacterium.     

Spermatogenèsein vitro: une nouvelle voie de recherche

Studies in our laboratory have aimed to settle two culture systems allowing some steps of rodent spermatogenic cell differentiation to occurin vitro. The first system is a coculture of pachytene spermatocytes from adult rats together with Sertoli cells from 20-day-old animals. In the second system, small pieces of spermatogenic tubules are seeded. In that way the cell-cell junctions which are presentin vivo are better maintainedin vitro; in addition every type of germ cell present in the tubule segments at the time of seeding can be studied. Our results have shown that the two meiotic divisions or even most of the meiotic process can occur over a 2 to 4 week-culture period. These results should have applications in basic research, toxicology, clinical studies and biotechnology. However, for these latter, some improvements and verifications are still needed in order to assess the feasibility and the innocuity of these procedures.     

Activity of NMN+, nicotinamide ribose and analogs in alcohol oxidation promoted by horse-liver alcohol dehydrogenase. Improvement of this activity and structural requirements of the pyridine nucleotide part of the NAD+ coenzyme

Kinetic constants of horse-liver alcohol-dehydrogenase-mediated oxidation of alcohol by nicotinamide mononucleotide and nicotinamide ribose were determined and the role of different adenine moieties complementing the reaction mixtures was investigated. Five nicotinamide ribose analogs were synthesized and their activities as NAD+ inhibitors and as cofactors in this dehydrogenase-mediated oxidation of alcohol were assayed. In the light of these results, structural requirements of the pyridine ribofuranosyl part of the NAD+ are discussed.     

Salt-Induced Conformation and Interaction Changes of Nucleosome Core Particles

Small angle x-ray scattering was used to follow changes in the conformation and interactions of nucleosome core particles (NCP) as a function of the monovalent salt concentration C_s. The maximal extension (D_max) of the NCP (145 ± 3-bp DNA) increases from 137 ± 5 Å to 165 ± 5 Å when C_s rises from 10 to 50 mM and remains constant with further increases of C_s up to 200 mM. In view of the very weak increase of the R_g value in the same C_s range, we attribute this D_max variation to tail extension, a proposal confirmed by simulations of the entire I(q) curves, considering an ideal solution of particles with tails either condensed or extended. This tail extension is observed at higher salt values when particles contain longer DNA fragments (165 ± 10 bp). The maximal extension of the tails always coincides with the screening of repulsive interactions between particles. The second virial coefficient becomes smaller than the hard sphere virial coefficient and eventually becomes negative (net attractive interactions) for NCP₁₄₅. Addition of salt simultaneously screens Coulombic repulsive interactions between NCP and Coulombic attractive interactions between tails and DNA inside the NCP. We discuss how the coupling of these two phenomena may be of biological relevance.     

Protein Misfolding as an Underlying Molecular Defect in Mucopolysaccharidosis III Type C

Mucopolysaccharidosis type IIIC or Sanfilippo syndrome type C (MPS IIIC, MIM #252930) is an autosomal recessive disorder caused by deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT, EC 2.3.1.78), which catalyses transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by α-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death causing neurodegeneration and is accompanied by mild visceral and skeletal abnormalities, including coarse facies and joint stiffness. Surprisingly, the majority of MPS IIIC patients carrying missense mutations are as severely affected as those with splicing errors, frame shifts or nonsense mutations resulting in the complete absence of HGSNAT protein.In order to understand the effects of the missense mutations in HGSNAT on its enzymatic activity and biogenesis, we have expressed 21 mutant proteins in cultured human fibroblasts and COS-7 cells and studied their folding, targeting and activity. We found that 17 of the 21 missense mutations in HGSNAT caused misfolding of the enzyme, which is abnormally glycosylated and not targeted to the lysosome, but retained in the endoplasmic reticulum. The other 4 mutants represented rare polymorphisms which had no effect on the activity, processing and targeting of the enzyme. Treatment of patient cells with a competitive HGSNAT inhibitor, glucosamine, partially rescued several of the expressed mutants.Altogether our data provide an explanation for the severity of MPS IIIC and suggest that search for pharmaceutical chaperones can in the future result in therapeutic options for this disease.     

A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.     

Impacts of environmental changes on the hydrology and sedimentary processes at the confluence of St. Lawrence tributaries: potential effects on fluvial ecosystems

This study examines the effects of changes in St. Lawrence water levels and in hydrological regimes of two tributaries of the St. Lawrence, the Richelieu and Saint-François Rivers, on the sedimentary dynamics of their confluence in an historical context and in response to projected climate changes. Analysis of historical data indicates that alteration to St. Lawrence mean water levels and spring flood characteristics (duration, timing, and magnitude) have already impacted flow and sediment dynamics at tributary confluences. The major progradation of more than 1.5 km of the sedimentary front of the Saint-François delta into the shallow water of the fluvial Lake Saint-Pierre between 1859 and 2001 is associated with the combined effect of water level reduction in the lake, as a consequence of dredging of the navigation channel, and discharge regulation. High tributary discharges during periods of low water levels in the St. Lawrence have also contributed to accelerated delta front progression. Increases in temperature and changes in the precipitation regime projected for the current century were used into the HSAMI model to obtain hydrological simulations for the tributaries. Results indicate that the greatest hydrological changes will be observed during the winter and spring seasons, a time that is critical for many ecosystems. The mean spring discharge is projected to decrease by 17% for the Richelieu and 40% for the Saint-François by the end of the century. During this season, the frequency of discharge higher than the sediment transport threshold will decrease by 63% for the Saint-François and by 17% for Richelieu when compared to the reference period. However, during winter, the frequency of these high discharges will be four times higher for both rivers. These changes are expected to occur simultaneously with a decrease of the St. Lawrence water levels, a situation that will have significant impacts on sedimentary processes. A hydro-sedimentological modeling approach based on SEDROUT was used to investigate the morphological response of the river beds to the projected changes on the hydrology of St. Lawrence tributaries and to three base level scenarios. Results show that the combined hydrology and base level effects will lead to an increase in sediment supply compared to the current state, and to the extension of accumulation zones. While the effects will vary among the tributaries according to their particular characteristics, the projected increase in sediment supply will modify the extent of freshwater wetlands at the mouth of the St. Lawrence tributaries with feedback effects on local flow and sediment distributions.     

Oral ingestion of insulin liposomes: Effects of the administration route

We prepared an insulin liposome suspension by hot dispersion (50 °C) of a lipid mixture comprising dipalmitoyl phosphatidylcholine (DPPC) and cholesterol (7:2 molar ratio) in an 80 UI/ml acid bovine insulin solution, followed by two minutes of cold sonification (4 °C). Free insulin was removed by ultracentrifugation and the washed insulin liposomes were resuspended in a 1% aqueous saline solution (pH 3). Administration of these liposomes in the buccal cavity of normal rats caused clear hypoglycemia (?37% of the initial glycemia after one hour and ?44% after $\text{2}\text{1}\text{2}$ hours), but the solution was inactive when introduced by a strictly intragastric route. Hypoglycemic effects were also obtained when a mixture containing a liposome suspension devoid of insulin and 10 UI/100 g b.w. of free insulin was given by the buccal route (?56% of initial glycemia one hour later and ?55% after $\text{2}\text{1}\text{2}$ hours). These results show that the route of liposomal insulin administration strongly influences its biological effects.