Glucose and type 2A protein phosphatase regulate the interaction between catalytic and regulatory subunits of AMP-activated protein kinase

We have expressed in yeast the different subunits of AMP-activated protein kinase (AMPK) and, by using the two-hybrid system, we have found a glucose-regulated interaction between alpha 2 catalytic and gamma 1 regulatory subunits. This regulation was not affected by known regulators of the corresponding yeast orthologue, the SNF1 complex, such as Reg1 or Hxk2, but it was affected by deletion of regulatory subunits of yeast type 2A protein phosphatase (PP2A) complex. We have also found that Tpd3 and PR65 alpha, the corresponding yeast and mammalian A subunits of PP2A, interacted with AMPK alpha 2 both in yeast and mammals, respectively. This interaction occurred only through the regulatory domain of this subunit. These results suggested a direct involvement of PP2A complex in regulating the interaction between AMPK alpha 2 and gamma 1 in a glucose-dependent manner.     

Activation of the fatty acid alpha-dioxygenase pathway during bacterial infection of tobacco leaves. Formation of oxylipins protecting against cell death

A pathogen-induced oxygenase showing homology to prostaglandin endoperoxide synthases-1 and -2 was recently characterized by in vitro experiments as a fatty acid alpha-dioxygenase catalyzing formation of unstable 2(R)-hydroperoxy fatty acids. To study the activity of this enzyme under in vivo conditions and to elucidate the fate of enzymatically produced 2-hydroperoxides, leaves of tobacco were analyzed for the presence of alpha-dioxygenase-generated compounds as well as for lipoxygenase (LOX) products and free fatty acids. Low basal levels of 2-hydroxylinolenic acid (0.4 nmol/g leaves fresh weight) and 8,11,14-heptadecatrienoic acid (0.1 nmol/g) could be demonstrated. These levels increased strongly upon infection with the bacterium Pseudomonas syringae pv syringae (548 and 47 nmol/g, respectively). Transgenic tobacco plants overexpressing alpha-dioxygenase were developed, and incompatible infection of such plants led to a dramatic elevation of 2-hydroxylinolenic acid (1778 nmol/g) and 8,11,14-heptadecatrienoic acid (86 nmol/g), whereas the levels of LOX products were strongly decreased. Further analysis of oxylipins in infected leaves revealed the presence of a number of 2-hydroxy fatty acids differing with respect to chain length and degree of unsaturation as well as two new doubly oxygenated oxylipins identified as 2(R),9(S)-dihydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid and 2(R),9(S)-dihydroxy-10(E),12(Z)-octadecadienoic acid. alpha-Dioxygenase-generated 2-hydroxylinolenic acid, and to a lesser extent lipoxygenase-generated 9-hydroxyoctadecatrienoic acid, exerted a tissue-protective effect in bacterially infected tobacco leaves.     

Purification and characterization of an X-prolyl-dipeptidyl peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55 degrees C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K(m) s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 microM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 microM, respectively. Among peptides, beta-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

Structure of the periplasmic domain of Pseudomonas aeruginosa TolA: evidence for an evolutionary relationship with the TonB transporter protein

The crystal structure of the C-terminal domain III of Pseudomonas aeruginosa TolA has been determined at 1.9 A resolution. The fold is similar to that of the corresponding domain of Escherichia coli TolA, despite the limited amino acid sequence identity of the two proteins (20%). A pattern was discerned that conserves the fold of domain III within the wider TolA family and, moreover, reveals a relationship between TolA domain III and the C-terminal domain of the TonB transporter proteins. We propose that the TolA and TonB C-terminal domains have a common evolutionary origin and are related by means of domain swapping, with interesting mechanistic implications. We have also determined the overall shape of the didomain, domains II + III, of P.aeruginosa TolA by solution X-ray scattering. The molecule is monomeric-its elongated, stalk shape can accommodate the crystal structure of domain III at one end, and an elongated helical bundle within the portion corresponding to domain II. Based on these data, a model for the periplasmic domains of P.aeruginosa TolA is presented that may explain the inferred allosteric properties of members of the TolA family. The mechanisms of TolA-mediated entry of bateriophages in P.aeruginosa and E.coli are likely to be similar.     

Conflict and post-conflict behavior in a small group of chimpanzees

Chimpanzee research plays a central role in the discussions of conflict negotiation. Reconciliation, or the attraction and affiliation of former opponents following conflict, has been proposed as a central element of conflict negotiation in chimpanzees and various other taxa. In an attempt to expand the database of chimpanzee conflict resolution, conflict and post-conflict behavior were recorded for a small group of socially housed chimpanzees at the Chimpanzee and Human Communication Institute, at Central Washington University. Data were collected over six 6-week periods between 1997 and 2000, for a total of 840 hours of observation, resulting in a substantial post-conflict (PC) and matched control (MC) data set. The data demonstrate this group’s tendencies to maintain visual contact and closer proximity after conflicts. Dyadic corrected conciliatory tendencies ranged between 0 – 37.5% and averaged 17.25% across all dyads. Individual corrected conciliatory tendencies ranged between 5.8 and 32%. The results of this study combined with recent publications on captive and free-ranging chimpanzee post-conflict behavior suggest that variation in post-conflict behavior may be important to our understanding of chimpanzee conflict negotiation, and may also have implications for the design and management of captive chimpanzee enclosures and social groups, respectively.     

Sperm coating mechanism from the 1.8 A crystal structure of PDC-109-phosphorylcholine complex

Bovine seminal plasma PDC-109 binds to sperm surface choline lipids and promotes sperm capacitation by stimulating the efflux of cholesterol and phospholipids. The structure of PDC-109 with bound phosphorylcholine was solved using MAD data of a single platinum site. Its two globular (40 x 50 x 20 A(3)) Fn2 domains are linked and clustered by a short polypeptide. The choline binding sites lie at the same face of the molecule. Phosphorylcholine binds to the Fn2 domains through a cation-pi interaction between the quaternary ammonium group and a core tryptophan, plus hydrogen bonding between hydroxyls of exposed tyrosines and the phosphate group. The structure of the PDC-109-oPC complex provides a structural ground for the sperm membrane-coating mechanism underlying PDC-109-induced capacitation.     

Development of specific fluorescent oligonucleotide probes for in situ identification of wine lactic acid bacteria

A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising technique for the rapid identification of LAB, allowing positive identification in a few hours (4-16 h).     

Purification and characterization of a prolyl aminopeptidase from Debaryomyces hansenii

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45 degrees C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg(2+), which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K(m) for proline-7-amido-4-methylcoumarin was 40 micro M. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.     

Determination of risperidone and 9-hydroxyrisperidone in human plasma by liquid chromatography: application to the evaluation of CYP2D6 drug interactions

A high-pressure liquid chromatography with ultra-violet detection method for the simultaneous determination of risperidone and 9-hydroxyrisperidone in plasma after liquid-liquid extraction has been developed. The limit of quantitation was 5 nmol/L, and the inter-day coefficient of variation was less than 8% for both compounds. The mean recoveries of risperidone and 9-hydroxyrisperidone added to plasma were 96.8 and 99.4%, with an intra-day coefficient of variation of under 5 and 6%, respectively. Studies of analytical interference showed that the most commonly co-administered antidepressants and benzodiazepines did not interfere. The method was used for the determination of the plasma concentrations of a schizophrenic patient treated daily with an oral dose of 4.5 mg risperidone. The patient suffered severe extrapyramidal side-effects after adding risperidone to his previous medication of haloperidol and levomepromazine. The risperidone plasma concentration was well above the average (182 nmol/L), which suggests that a pharmacokinetic interaction occurred, presumably due to inhibition of the enzyme CYP2D6.