Use of brevibacterium sp 19-derived amidase for the synthesis of hydroxamic fatty acids

Summary Oleylhydroxamic acid (Oleyl HA) was produced by acylation of neutralized hydroxylamine with oleic acid in buffered or in solvent media using the broad-spectrum amidase activity ofBrevibacterium (BB) sp 19 cells.     

Plasma Membrane Profiling Defines an Expanded Class of Cell Surface Proteins Selectively Targeted for Degradation by HCMV US2 in Cooperation with UL141

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2’s impact on HCMV pathogenesis.     

Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Effect of adrenocorticotrophic hormone on the activity of sterol ester hydrolase from rat brain

Adrenocorticotrophic hormone (ACTH) stimulates in vitro the hydrolysis of oleoylcholesterol by a sterol ester hydrolase from rat brain synaptosomes. The stimulatory effect involves an alkaline shift of the pH optimum of catalysis and culminates at pH 6 with a 15 to 20-fold increase in lipolytic rates. The effect requires trace amount of organic solvent in the substrate emulsion and is dependent on the NH₂-terminal sequence extending through the basic amino acid residues at positions 15–18. The hormonal stimulation is decreased when a lipid-depleted preparation is used as enzyme source, and fully restored upon addition of lecithin. The results raise the possibility that ACTH may have a neuro-hormonal role in brain via modulation of local lipolytic processes.     

Nitrilases and amidases: determination of activity by proton magnetic resonance spectrometry.

A rapid and easy method based on proton magnetic resonance spectrometry is proposed for the determination of nitrilase and amidase activities. The technique is applicable to many substrates of different chemical types: aliphatic, α-ethylenic, α-hydroxy, and α-amino nitriles and amides. The rate of hydrolysis of the above substrates may be monitored in a continuous fashion by using proton magnetic resonance. A good correlation has been achieved between the results obtained by proton magnetic resonance and those found by gas-liquid chromatography.     

Interaction of alpha 2-HS-glycoprotein with immobilized triazine dyes

We studied the interaction of alpha 2-HS-glycoprotein with immobilized Cibacron blue F3-GA (Blue A) and Procion red HE-3B (Red A). When whole plasma was applied on the Blue A, alpha 2-HS-glycoprotein remained unbound, together with other plasma proteins. In contrast, when this fraction was applied on the Red A, alpha 2-HS-glycoprotein was shown to bind tightly and was eluted with a linear sodium chloride gradient between 0.5 and 0.8 M. This proved to be a useful two-step technique for the purification of alpha 2-HS-glycoprotein. Further characterization revealed that the protein appeared homogeneous by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis with yields greater than 30%. A small (less than 5%) but significant fraction of alpha 2-HS-glycoprotein with a same molecular weight as the native protein was consistently found in the wash of the Red A column, and may correspond to alpha 2-HS-glycoprotein bound to a yet unidentified ligand.     

Selection and study of a Candida molischiana mutant derepressed for β-glucosidase production

Abstract A mutant strain of Candida molischiana was selected. Analysis of the exocellular activity of Candida molischiana 35M5N grown on different carbon sources revealed that the biosynthesis of β-glucosidase is derepressed in this yeast strain. The strain is not a hyper-producer mutant. There were no observed differences in the endocellular and parietal activities of the wild and mutant strains. However, the mutant strain produced 35-fold more enzyme than the wild-type in the culture medium with glucose as carbon source. When glucose was used as carbon source, the mutant strain produced 90% more exocellular enzyme than when cellobiose was used as the carbon source.     

Genetic studies of the leptin receptor gene in morbidly obese French Caucasian families

Family studies have shown that in some populations up to 75% of the variation of body mass index can be explained by genetic factors. However, in humans, no major obesity gene has been identified to date. In contrast, there are a number of genetically well defined animal models for obesity. In two of those models (ob/ob and db/db), defects in the same pathway are responsible for obesity. Recently, some evidence has been found for the OB gene also being involved in human obesity. In this study we investigated the potential role of the OB receptor (OBR) in the etiology of massive obesity in humans using familial linkage analyses and case-control association studies. The typing of two microsatellite markers (D1S198 and D1S209), flanking the OBR gene, in 256 sib pairs showed no evidence for linkage with obesity. In order to be able to detect small gene effects, association studies with a 3′-UTR insertion/deletion polymorphism were carried out. The results of these analyses remained non-significant (χ² = 3.442, P = 0.18). However, subjects heterozygous for the insertion/deletion polymorphism showed a slight trend towards lower insulin values 30 min after an oral glucose load compared to homozygous individuals (P = 0.02). In summary, our results do not support a major role of the human OBR gene in the development of morbid obesity in our population. Received: 4 December 1996 / Accepted: 25 June 1997     

Molecular Co-occupancy Identifies Transcription Factor Binding Cooperativity In Vivo

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