Galactose transfer in the membranes of human milk fat globules (author's transl)]

Galactosyltransferase which catalyzes the transfer from UDP-galactose to either endogeneous glycoproteins, free N-acetylglucosamine or N-acetylglucosaminyl residues in the carbohydrate portion of glycoproteins, or to glucose when alpha-lactalbumin is added, occurs in human milk fat globule membranes. Various treatments (washing of membranes, freezing and thawing) did not affect this activity. In the presence of Triton X-100, the enzyme shows appreciable latency, This detergent was then used to solubilize the enzyme and to study its main characteristics. A competition and a heat stability experiment show that only one enzyme acts on two substrates (free N-acetylglucosamine or desialyzed and degalactosylated fetuin). UDP-galactose hydrolase activities were very low compared to those of the bovine milk fat globule membranes. Other characteristic enzymes of Golgi vesicles were found in human milk fat globules membranes. It is of interest to find out whether this is the result of contamination with cytoplasmic particles or whether it reflects the participation of Golgi vesicles in human milk fat globule secretion.     

Transfert du galactose dans les membranes des globules lipidiques du lait humain

Galactose transfer in the membranes of human milk fat globulesGalactosyltransferase which catalyzes the transfer from UDP-galactose to either endogeneous glycoproteins, free N-actylglucosamine or N-acetylglucosaminyl residues in the carbohydrate portion of glycoproteins, or to glucose when α-lactalbumin is added, occurs in human milk fat globule membranes. Various treatments (washing of membranes, freezing and thawing) did not affect this activity. In the presence of Triton X-100, the enzyme shows appreciable latency. This detergent was then used to solubilize the enzyme and to study its main characteristics. A competition and a heat stability experiment show that only one enzyme acts on two substrates (free N-acetylglucosamine or desialyzed and degalactosylated fetuin). UDP-galactose hydrolase activities were very low compared to those of the bovine milk fat globule membranes. Other characteristic enzymes of Golgi vesicles were found in human milk fat globules membranes. It is of interest to find out whether this is the result of contamination with cytoplasmic particles or whether it reflects the participation of Golgi vesicles in human milk fat globule secretion.     

AMP-activated protein kinase alpha2 activity is not essential for contraction- and hyperosmolarity-induced glucose transport in skeletal muscle

To examine the role of AMP-activated protein kinase (AMPK) in muscle glucose transport, we generated muscle-specific transgenic mice (TG) carrying cDNAs of inactive alpha2 (alpha2i TG) and alpha1 (alpha1i TG) catalytic subunits. Extensor digitorum longus (EDL) muscles from wild type and TG mice were isolated and subjected to a series of in vitro incubation experiments. In alpha2i TG mice basal alpha2 activity was barely detectable, whereas basal alpha1 activity was only partially reduced. Known AMPK stimuli including 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), rotenone (a Complex I inhibitor), dinitrophenol (a mitochondrial uncoupler), muscle contraction, and sorbitol (producing hyperosmolar shock) did not increase AMPK alpha2 activity in alpha2i TG mice, whereas alpha1 activation was attenuated by only 30-50%. Glucose transport was measured in vitro using isolated EDL muscles from alpha2i TG mice. AICAR- and rotenone-stimulated glucose transport was fully inhibited in alpha2i TG mice; however, the lack of AMPK alpha2 activity had no effect on contraction- or sorbitol-induced glucose transport. Similar to these observations in vitro, contraction-stimulated glucose transport, assessed in vivo by 2-deoxy-d-[(3)H]glucose incorporation into EDL, tibialis anterior, and gastrocnemius muscles, was normal in alpha2i TG mice. Thus, AMPK alpha2 activation is essential for some, but not all, insulin-independent glucose transport. Muscle contraction- and hyperosmolarity-induced glucose transport may be regulated by a redundant mechanism in which AMPK alpha2 is one of multiple signaling pathways.     

Functional role of AMP-activated protein kinase in the heart during exercise

AMP-activated protein kinase (AMPK) plays a critical role in maintaining energy homeostasis and cardiac function during ischemia in the heart. However, the functional role of AMPK in the heart during exercise is unknown. We examined whether acute exercise increases AMPK activity in mouse hearts and determined the significance of these increases by studying transgenic (TG) mice expressing a cardiac-specific dominant-negative (inactivating) AMPKalpha2 subunit. Exercise increased cardiac AMPKalpha2 activity in the wild type mice but not in TG. We found that inactivation of AMPK did not result in abnormal ATP and glycogen consumption during exercise, cardiac function assessed by heart rhythm telemetry and stress echocardiography, or in maximal exercise capacity.     

Viability and virulence of experimentally stressed nonculturable Salmonella typhimurium.

Maintenance of pathogenicity of viable but nonculturable Salmonella typhimurium cells experimentally stressed with UV-C and seawater, was investigated relative to the viability level of the cellular population. Pathogenicity, tested in a mouse model, was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by respiratory activity and cytoplasmic membrane and genomic integrities.     

The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas

Vibrio splendidus, strain LGP32, is an oyster pathogen associated with the summer mortalities affecting the production of Crassostrea gigas oysters worldwide. Vibrio splendidus LGP32 was shown to resist to up to 10 µM Cg‐Def defensin and Cg‐BPI bactericidal permeability increasing protein, two antimicrobial peptides/proteins (AMPs) involved in C. gigas immunity. The resistance to both oyster Cg‐Def and Cg‐BPI and standard AMPs (polymyxin B, protegrin, human BPI) was dependent on the ompU gene. Indeed, upon ompU inactivation, minimal bactericidal concentrations decreased by up to fourfold. AMP resistance was restored upon ectopic expression of ompU. The susceptibility of bacterial membranes to AMP‐induced damages was independent of the ompU‐mediated AMP resistance. Besides its role in AMP resistance, ompU proved to be essential for the adherence of V. splendidus LGP32 to fibronectin. Interestingly, in vivo, ompU was identified as a major determinant of V. splendidus pathogenicity in oyster experimental infections. Indeed, the V. splendidus‐induced oyster mortalities dropped from 56% to 11% upon ompU mutation (Kaplan–Meier survival curves, P < 0.01). Moreover, in co‐infection assays, the ompU mutant was out competed by the wild‐type strain with competitive indexes in the range of 0.1–0.2. From this study, ompU is required for virulence of V. splendidus. Contributing to AMP resistance, conferring adhesive properties to V. splendidus, and being essential for in vivo fitness, the OmpU porin appears as an essential effector of the C. gigas/V. splendidus interaction.     

Muscle-specific overexpression of wild type and R225Q mutant AMP-activated protein kinase gamma3-subunit differentially regulates glycogen accumulation

AMP-activated protein kinase (AMPK) is a heterotrimeric complex that works as an energy sensor to integrate nutritional and hormonal signals. The naturally occurring R225Q mutation in the gamma3-subunit in pigs is associated with abnormally high glycogen content in skeletal muscle. Because skeletal muscle accounts for most of the body's glucose uptake, and gamma3 is specifically expressed in skeletal muscle, it is important to understand the underlying mechanism of this mutation in regulating glucose and glycogen metabolism. Using skeletal muscle-specific transgenic mice overexpressing wild type gamma3 (WTgamma3) and R225Q mutant gamma3 (MUTgamma3), we show that both WTgamma3 and MUTgamma3 mice have 1.5- to 2-fold increases in muscle glycogen content. In WTgamma3 mice, increased glycogen content was associated with elevated total glycogen synthase activity and reduced glycogen phosphorylase activity, whereas alterations in activities of these enzymes could not explain elevated glycogen in MUTgamma3 mice. Basal, 5-aminoimidazole-AICAR- and phenformin-stimulated AMPKalpha2 isoform-specific activities were decreased only in MUTgamma3 mice. Basal rates of 2-DG glucose uptake were decreased in both WTgamma3 and MUTgamma3 mice. However, AICAR- and phenformin-stimulated 2-DG glucose uptake were blunted only in MUTgamma3 mice. In conclusion, expression of either wild type or mutant gamma3-subunit of AMPK results in increased glycogen concentrations in muscle, but the mechanisms underlying this alteration appear to be different. Furthermore, mutation of the gamma3-subunit is associated with decreases in AMPKalpha2 isoform-specific activity and impairment in AICAR- and phenformin-stimulated skeletal muscle glucose uptake.