Lysophospholipase and lysophosphatidylcholine:Lysophosphatidylcholine transacylase from rat lung: Evidence for a single enzyme and some aspects of its specificity

An enzyme preparation was isolated from rat lung cytosol with the capability to transfer the fatty acyl chain from 1-acyl-sn-glycero-3-phosphocholine to water and to another molecule of 1-acyl-sn-glycero-3-phosphocholine. The evidence presented to indicate that a single protein confers both activities includes: (a) both normal and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis showed a single protein band, and (b) heat treatment and preincubation with increasing amounts of diisopropylfluorophosphate resulted in concomitant loss of fatty acid and phosphatidylcholine formation. The enzyme converted 1-[9,10-³H₂]stearoyl-sn-glycero-3-phospho[¹⁴C-methyl]choline into phosphatidylcholine with an isotopic ³H/¹⁴C ratio twice that of the substrate, even when an excess of unlabeled fatty acid was present. The acyl group from palmitoyl-propanediol (1,3)-phosphocholine and palmitoyl-propanediol (1,3)-phosphoethanolamine could be transferred to lysophosphatidylcholine acceptor to yield phosphatidylcholine. Neither acylglycerols and cholesterol nor glycero-3-phosphate and glycero-3-phosphocholine served as acyl acceptors. Lysophosphatidylethanolamine and lysophosphatidyglycerol were converted also into the corresponding diacylphospholipids. Palmitoyllysophosphatidylcholine is preferentially converted into phosphatidylcholine when compared with stearoyllysophosphatidylcholine. The possible involvement of the enzyme in the synthesis of dipalmitoylphosphatidylcholine for the production of lung surfactant is discussed.     

Characterization of novel components of the baculovirus per os infectivity factor complex

Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.     

Animal culture: chimpanzee conformity?

Culture-like phenomena in wild animals have received much attention, but how good is the evidence and how similar are they to human culture? New data on chimpanzees suggest their culture may even have an element of conformity.     

Order of bovine DRB3, DYA,and PRL determined by sperm typing

The order and recombination fractions () between the bovine major histocompatibility complex DRB3, DYA, and prolactin (PRL) genes were determined by typing of 254 sperm from a triply heterozygous bull. A recently developed method, primer extension preamplification (PEP), was used to amplify the bovine sperm genome prior to amplification of specific loci by the polymerase chain reaction (PCR). At least 28 copies of the DRB3, PRL, or DYA gene were obtained from 50 cycles of PEP. For sperm typing, alleles of each locus were discriminated by restriction endonuclease cleavage of PCR products and polyacrylamide gel electrophoresis of the restriction fragments. The most likely gene order is PRL-DRB3-DYA, with =0.025 (±0.012) and =0.150 (±0.024), respectively. The odds are 128:1 in favor of this order in comparison with the second most likely order DRB3-PRL-DYA. Our results demonstrate the power of sperm typing in concert with PEP for multilocus gene mapping.     

The additional value of TGFβ1 and IL-7 to predict the course of prostate cancer progression

Background

Given the fact that prostate cancer incidence will increase in the coming years, new prognostic biomarkers are needed with regard to the biological aggressiveness of the prostate cancer diagnosed. Since cytokines have been associated with the biology of cancer and its prognosis, we determined whether transforming growth factor beta 1 (TGFβ1), interleukin-7 (IL-7) receptor and IL-7 levels add additional prognostic information with regard to prostate cancer-specific survival.

Materials and methods

Retrospective survival analysis of forty-four prostate cancer patients, that underwent radical prostatectomy, was performed (1989–2001). Age, Gleason score and pre-treatment PSA levels were collected. IL-7, IL-7 receptor and TGFβ1 levels in prostate cancer tissue were determined by quantitative real-time RT-PCR and their additional prognostic value analyzed with regard to prostate cancer survival. Hazard ratios and their confidence intervals were estimated, and Akaike’s information criterion was calculated for model comparison.

Results

The predictive ability of a model for prostate cancer survival more than doubled when TGFβ1 and IL-7 were added to a model containing only the Gleason score and pre-treatment PSA (AIC: 18.1 and AIC: 6.5, respectively).

Conclusion

IL-7 and TGFβ1 are promising markers to indicate those at risk for poor prostate cancer survival. This additional information may be of interest with regard to the biological aggressiveness of the diagnosed prostate cancer, especially for those patients screened for prostate cancer and their considered therapy.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration

Leukocyte transendothelial migration (TEM) has been modeled as a multistep process beginning with rolling adhesion, followed by firm adhesion, and ending with either transcellular or paracellular passage of the leukocyte across the endothelial monolayer. In the case of paracellular TEM, endothelial cell (EC) junctions are transiently disassembled to allow passage of leukocytes. Numerous lines of evidence demonstrate that tyrosine phosphorylation of adherens junction proteins, such as vascular endothelial cadherin (VE-cadherin) and beta-catenin, correlates with the disassembly of junctions. However, the role of tyrosine phosphorylation in the regulation of junctions during leukocyte TEM is not completely understood. Using human leukocytes and EC, we show that ICAM-1 engagement leads to activation of two tyrosine kinases, Src and Pyk2. Using phospho-specific Abs, we show that engagement of ICAM-1 induces phosphorylation of VE-cadherin on tyrosines 658 and 731, which correspond to the p120-catenin and beta-catenin binding sites, respectively. These phosphorylation events require the activity of both Src and Pyk2. We find that inhibition of endothelial Src with PP2 or SU6656 blocks neutrophil transmigration (71.1 +/- 3.8% and 48.6 +/- 3.8% reduction, respectively), whereas inhibition of endothelial Pyk2 also results in decreased neutrophil transmigration (25.5 +/- 6.0% reduction). Moreover, overexpression of the nonphosphorylatable Y658F or Y731F mutants of VE-cadherin impairs transmigration of neutrophils compared with overexpression of wild-type VE-cadherin (32.7 +/- 7.1% and 38.8 +/- 6.5% reduction, respectively). Our results demonstrate that engagement of ICAM-1 by leukocytes results in tyrosine phosphorylation of VE-cadherin, which is required for efficient neutrophil TEM.     

Infection pressure of human alveolar echinococcosis due to village and small town foxes (<Emphasis Type="Italic">Vuples vulpes</Emphasis>) living in close proximity to residents

This study investigated the epidemiological and ecological factors to assess the infection pressure of alveolar echinococcosis to human which are living in villages and small towns. Foxes and fox faeces were examined for Echinococcus multilocularis and foxes were observed by radio telemetry in Upper Bavaria, Germany. Forty-three percent of the village foxes (n = 65) had been infected with E. multilocularis. This prevalence rate did not differ significantly from the prevalence among rural foxes, which was 39% (n = 33; χ ² = 0.12, df = 1, p = 0.727) determined by the intestinal scraping technique. PCR analyses of fox faeces showed a higher infection rate of 35% (n = 26) among rural foxes than among foxes in villages and small towns (26%, n = 69; χ ² = 0.68, df = 1, p = 0.411). One quarter of the fox faecal samples come from private gardens of residents. The radio-tracking study on 17 foxes showed that foxes preferred the built-up area and grassland outside the villages. Village foxes concentrated their activity within a range of 500 m around the settlement. Sixty-four percent of all bearings for radio-tracked foxes showed positions in areas outside the town, and 36% of bearings were within the settlement. Village foxes, which are infected with E. multilocularis, are able to carry the parasite continuously into settlements and fox faeces present an immediate source of infection to humans, especially within their gardens. Therefore, foxes are responsible for environmental E. multilocularis egg contamination in the vicinity of humans, leading to an infection risk to inhabitants of villages and small towns.     

The capsular polysaccharide of Haemophilus influenzae type B prevents killing by complement and antibodies against outer membrane protein a

Abstract The ability of antibodies, raised in rabbits against purified outer membrane protein a ( M _r 47 000) of Haemophilus influenzae type b, to promote complement-dependent killing of these encapsulated organisms was investigated. Killing of encapsulated strains was not induced by these antibodies in conjunction with either human, mouse, rabbit or guinea-pig complement. Acapsular mutants were effectively killed by complement in the presence of antibodies against protein a . Killing was dependent on the presence of the 47-kDa protein a and was not influenced by the outer membrane protein subtype or lipopolysaccharide serotype of the strain. The killing-promoting activity could be absorbed from the sera with cells of strains with the same protein a , purified protein a , but not by purified lipopolysaccharide and capsular polysaccharide. Binding experiments showed that the encapsulated strain and its acapsular mutant bound antibodies against protein a with the same rate and to the same extent, indicating that the capsule probably interferes with complement activation or insertion of the membrane attack complex into the bacterial cell.     

Adhesion of Biodegradative Anaerobic Bacteria to Solid Surfaces

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe³⁺ on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.