Lysophospholipid regulates release and activation of latent TGF-beta1 from chondrocyte extracellular matrix

Transforming growth factor beta-1 (TGF-beta1) is released from the extracellular matrix of rat growth plate chondrocytes and activated by stromelysin-1 (matrix metalloproteinase 3, MMP-3), an enzyme that is stored in matrix vesicles. MMP-3 is released from these extracellular organelles by the direct action of 1alpha,25(OH)2D3 via activation of phospholipase A2 (PLA2), resulting in local production of lysophospholipids and matrix vesicle membrane destabilization. This effect of 1alpha,25(OH)2D3 is greater in matrix vesicles from growth zone chondrocyte cultures and PLA2 activity is higher in the growth zone in vivo, suggesting that it may depend on chondrocyte maturation state in the endochondral lineage. Previous studies have shown that latent TGF-beta1 can be activated by mild detergents in vitro, suggesting that lysophospholipids may act in vivo in a similar manner. To test this hypothesis, we determined if rat costochondral growth plate cartilage cells produce lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) in a maturation state-dependent manner and if LPC or LPE could release and activate latent TGF-beta1 from the extracellular matrix produced by these cells. Rat growth plate chondrocytes produced both lysophospholipids, with growth zone cells producing higher levels of LPE via PLA1, and resting zone cells producing higher levels of LPC via PLA2. LPC and LPE directly increased activation of recombinant human latent TGF-beta1 in a biphasic manner with a peak at 2 microg/ml. Phosphatidylcholine, phosphatidylethanolamine, and LPE plasmalogen (LPEP), but not choline, also activated TGF-beta1. Latent TGF-beta1 incubated with LPC or LPE, but neither lysophospholipid alone, stimulated [3H]-thymidine incorporation of resting zone cells, indicating the TGF-beta1 released was biologically active. LPC and LPE also released TGF-beta1 in a dose- and time-dependent manner when incubated with cell-free extracellular matrices produced by the cells. These results indicate that LPC and LPE have important roles as regulators of rat growth plate chondrocytes by directly and indirectly activating TGF-beta1 stored in the extracellular matrix.     

Live cell imaging of the assembly, disassembly, and actin cable-dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae

Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64–labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement. Rather, linear actin patch movement occurs at the same velocity and direction as the movement of actin cables. Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement. Our studies support a mechanism whereby actin cables serve as “conveyor belts” for retrograde movement and delivery of actin patches/endosomes to FM4-64–labeled internal compartments.     

The ectoparasitic wasp Eulophus pennicornis (Hymenoptera: Eulophidae) uses instar-specific endocrine disruption strategies to suppress the development of its host Lacanobia oleracea (Lepidoptera: Noctuidae)

To successfully complete its development, the gregarious ectoparasitoid Eulophus pennicornis must inhibit the moult of its host, Lacanobia oleracea. In the present study, we examined the possibility that moult- and metamorphosis-associated endocrine events may be disrupted in caterpillars parasitized as newly moulted last (sixth) instars. Juvenile hormone (JH) titres on days 2 and 5 of the final stadium were significantly higher (> 100 fold) in parasitized than in non-parasitized hosts, in which JH was essentially absent. Elevated JH levels were associated with reduced haemolymph JH esterase (JHE) activity (down by 99.8%) and enhanced in vitro JH biosynthesis by the corpora allata (CA) (up to 4.5 fold). Wasp adults and/or larvae, in which we measured high levels of JH III (up to 2.7 ng/g), but little or no JH I or JH II, were not seen as likely sources of JH in parasitized hosts, in which we found mostly JH I and JH II. In addition, removal of parasitoid eggs or larvae after oviposition did not prevent the rise in JH titres seen in parasitoid-laden hosts, suggesting that wasp venom may be responsible for the observed hormonal dysfunction. Host haemolymph 20-hydroxyecdysone (20-E) levels were largely unaffected by parasitism during the final stadium although they were observed to increase earlier and decrease more rapidly in parasitized insects. We compare these results with those reported earlier for L. oleracea larvae parasitized by E. pennicornis as penultimate (fifth) instars, which display significantly depressed 20-E titres relative to control larvae. We conclude that E. pennicornis employs host endocrine-disruption strategies that differ according to whether the host is parasitized as a penultimate or final-stadium larva.     

Available lysine content in human milk: stability during manipulation prior to ingestion

Available lysine content is an indicator of protein quality and nutritional value of milk. Many studies have examined the effects of extraction, treatment and storage of human milk upon its components, though no references are found regarding the possible changes in milk quality as defined by its content in essential amino acids such as lysine. The present study investigates the available lysine content in human milk and the variations in lysine resulting from milk manipulation as follows: (a) Cold storage (refrigeration at 4 degrees C for 48 hours, and frozen for 15 days at -20 degrees C); (b) Thermal treatment under conditions of low (Holder)(63 degrees C/30 minutes) and high pasteurization (75 degrees C/15 seconds). The results obtained show a decrease in milk lysine concentration after storage in both refrigerated and frozen samples. Pasteurization causes a highly significant loss of available lysine. The lysine losses were greater on applying low pasteurization versus the more gentle conditions of high pasteurization. CONCLUSIONS: While manipulation through cold storage or thermal treatment does not affect the protein content of human milk, its protein quality is modified. When human milk must be subjected to hygienization, it is preferable to apply high temperature treatment (75 degrees C, 15 seconds) than habitual pasteurization (63 degrees C, 30 minutes).     

The auxin-inducible GH3 homologue Pp-GH3.16 is downregulated in Pinus pinaster root systems on ectomycorrhizal symbiosis establishment

In an attempt to determine whether auxin-regulated plant genes play a role in ectomycorrhizal symbiosis establishment, we screened a Pinus pinaster root cDNA library for auxin-upregulated genes. This allowed the identification of a cDNA, Pp-GH3.16, which encodes a polypeptide sharing extensive homologies with GH3 proteins of different plants. Pp-GH3.16 was specifically upregulated by auxins and was not affected by cytokinin, gibberellin, abscisic acid or ethylene, or by heat shock, water stress or anoxia. Pp-GH3.16 mRNAs were quantified in pine roots inoculated with two ectomycorrhizal fungi, Hebeloma cylindrosporum and Rhizopogon roseolus. Surprisingly, Pp-GH3.16 was downregulated following inoculation with both fungal species. The downregulation was most rapid on establishment of symbiosis with an indole-3-acetic acid (IAA)-overproducing mutant of H. cylindrosporum, which overproduced mycorrhizas characterized by a hypertrophic Hartig net. This indicates that, despite being auxin-inducible, Pp-GH3.16 can be downregulated on establishment of symbiosis with a fungus that releases auxin. By contrast, Pp-GH3.16 was not downregulated in pine root systems inoculated with a nonmycorrhizal mutant of H. cylindrosporum, suggesting that the downregulation we observed in mycorrhizal root systems was a component of the molecular cross-talk between symbiotic partners at the origin of differentiation of symbiotic structures.     

Theriogenology and developments in epidemiology: future opportunities?

The concepts and methods of the different branches of epidemiology, particularly clinical epidemiology, have much to offer the discipline of theriogenology. As with theriogenology, epidemiologic methods evolve when technological innovation enables new approaches to old problems. The recent emergence, from clinical epidemiology, of the evidence-based medicine paradigm in human medicine, and the associated developments of systematic reviews and meta-analysis, present new opportunities for collaboration and synergy between the two disciplines.     

Using DNA microarrays to identify library-independent markers for bacterial source tracking

Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers.     

Toll-like receptors as molecular switches

Members of the Toll family of single-pass transmembrane receptors are key mediators of innate immunity in both vertebrates and invertebrates. They respond to various pathogen-associated stimuli and transduce the complex signalling responses that are required for inflammation and for the subsequent development of adaptive immunity. Here, we propose a molecular mechanism for signalling by the Toll and Toll-like receptors that involves a series of protein conformational changes initiated by dimerization of their extracellular domains. The initial dimerization event, which is triggered by the interaction of the receptor with its ligand, might disrupt a pre-formed but non-functional dimer. Formation of a stable receptor-ligand complex then relieves constitutive autoinhibition, enabling receptor-receptor association of the extracellular juxtamembrane regions and cytoplasmic signalling domains. This activation process constitutes a tightly regulated, unidirectional molecular switch.     

Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels

The ATP-sensitive potassium (K(ATP)) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity ～100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in K(ATP) channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.