Complement activation by phospholipids: the interplay of factor H and C1q

Complement proteins in blood recognize charged particles. The anionic phospholipid (aPL) cardiolipin binds both complement proteins C1q and factor H. C1q is an activator of the complement classical pathway, while factor H is an inhibitor of the alternative pathway. To examine opposing effects of C1q and factor H on complement activation by aPL, we surveyed C1q and factor H binding, and complement activation by aPL, either coated on microtitre plates or in liposomes. Both C1q and factor H bound to all aPL tested, and competed directly with each other for binding. All the aPL activated the complement classical pathway, but negligibly the alternative pathway, consistent with accepted roles of C1q and factor H. However, in this system, factor H, by competing directly with C1q for binding to aPL, acts as a direct regulator of the complement classical pathway. This regulatory mechanism is distinct from its action on the alternative pathway. Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range. Thus factor H, which is regarded as a down-regulator only of the alternative pathway, has a distinct role in downregulating activation of the classical complement pathway by aPL. A factor H homologue, β2-glycoprotein-1, also strongly inhibits C1q binding to cardiolipin. Recombinant globular domains of C1q A, B and C chains bound aPL similarly to native C1q, confirming that C1q binds aPL via its globular heads.     

Quirks of dye nomenclature. 8. Methylene blue,azure and violet

Methylene blue was synthesized in 1877 and soon found application in medicine, staining for microscopy and as an industrial dye and pigment. An enormous literature has accumulated since its introduction. Early on, it was known that methylene blue could be degraded easily by demethylation; consequently, the purity of commercial samples often was low. Therefore, demethylation products, such as azures and methylene violet, also are considered here. The names and identity of the components, their varying modes of manufacture, analytical methods and their contribution to biological staining are discussed.     

Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system

P-type, E-type, and K-type tachykinin binding sites have been identified in the mammalian CNS. These sites may be tachykinin receptors for which the mammalian neuropeptides substance P, neuromedin K, and substance K are the preferred natural agonists, respectively. In the present investigation, we have compared the pharmacology and the autoradiographic distribution of CNS binding sites for the iodinated (¹²⁵I-Bolton-Hunter reagent) tachykinins substance P, eledoisin, neuromedin K, and substance K. Iodinated eledoisin and neuromedin K exhibited an E-type binding pattern in cortical membranes. Iodinated eledoisin, neuromedin K, and substance K each labeled sites that had a similar distribution but one that was considerably different from that of sites labeled by iodinated substance P. CNS regions where there were detectable densities of binding sites for iodinated eledoisin, neuromedin K, and substance K and few or no sites for iodinated substance P included cortical layers IV–VI, mediolateral septum, supraoptic and paraventricular nuclei, interpeduncular nucleus, ventral tegmental area, and substantia nigra pars compacta. Binding sites for SP were generally more widespread in the CNS. CNS regions where there was a substantial density of binding sites for iodinated substance P and few or no sites for iodinated eledoisin, neuromedin K, and substance K included cortical layers I and II, olfactory tubercle, nucleus accumbens, caudate-putamen, globus pallidus, medial and lateral septum, endopiriform nucleus, rostral thalamus, medial and lateral preoptic nuclei, arcuate nucleus, dorsal raphe nucleus, dorsal parabrachial nucleus, parabigeminal nucleus, cerebellum, inferior olive, nucleus ambiguus, retrofacial and reticular nuclei, and spinal cord autonomic and somatic motor nuclei. In the brainstem, iodinated substance P labeled sites in both sensory and motor nuclei whereas iodinated eledoisin, neuromedin K, and substance K labeled primarily sensory nuclei. Our results are consistent with either of two alternatives: (1) that iodinated eledoisin, neuromedin K, and substance K bind to the same receptor site in the rat CNS, or (2) that they bind to multiple types of receptor sites with very similar distribution.     

A tritrophic signal that attracts parasitoids to host-damaged plants withstands disruption by non-host herbivores

Background  

Volatiles emitted by herbivore-infested plants are highly attractive to parasitoids and therefore have been proposed to be part of an indirect plant defense strategy. However, this proposed function of the plant-provided signals remains controversial, and it is unclear how specific and reliable the signals are under natural conditions with simultaneous feeding by multiple herbivores. Phloem feeders in particular are assumed to interfere with plant defense responses. Therefore, we investigated how attack by the piercing-sucking cicadellid Euscelidius variegatus influences signaling by maize plants in response to the chewing herbivore Spodoptera littoralis.     

Empirical and deterministic accuracies of across-population genomic prediction

Background

Differences in linkage disequilibrium and in allele substitution effects of QTL (quantitative trait loci) may hinder genomic prediction across populations. Our objective was to develop a deterministic formula to estimate the accuracy of across-population genomic prediction, for which reference individuals and selection candidates are from different populations, and to investigate the impact of differences in allele substitution effects across populations and of the number of QTL underlying a trait on the accuracy.

Methods

A deterministic formula to estimate the accuracy of across-population genomic prediction was derived based on selection index theory. Moreover, accuracies were deterministically predicted using a formula based on population parameters and empirically calculated using simulated phenotypes and a GBLUP (genomic best linear unbiased prediction) model. Phenotypes of 1033 Holstein-Friesian, 105 Groninger White Headed and 147 Meuse-Rhine-Yssel cows were simulated by sampling 3000, 300, 30 or 3 QTL from the available high-density SNP (single nucleotide polymorphism) information of three chromosomes, assuming a correlation of 1.0, 0.8, 0.6, 0.4, or 0.2 between allele substitution effects across breeds. The simulated heritability was set to 0.95 to resemble the heritability of deregressed proofs of bulls.

Results

Accuracies estimated with the deterministic formula based on selection index theory were similar to empirical accuracies for all scenarios, while accuracies predicted with the formula based on population parameters overestimated empirical accuracies by ~25 to 30%. When the between-breed genetic correlation differed from 1, i.e. allele substitution effects differed across breeds, empirical and deterministic accuracies decreased in proportion to the genetic correlation. Using a multi-trait model, it was possible to accurately estimate the genetic correlation between the breeds based on phenotypes and high-density genotypes. The number of QTL underlying the simulated trait did not affect the accuracy.

Conclusions

The deterministic formula based on selection index theory estimated the accuracy of across-population genomic predictions well. The deterministic formula using population parameters overestimated the across-population genomic accuracy, but may still be useful because of its simplicity. Both formulas could accommodate for genetic correlations between populations lower than 1. The number of QTL underlying a trait did not affect the accuracy of across-population genomic prediction using a GBLUP method.     

Substance P as a baro- and chemoreceptor afferent neurotransmitter: immunocytochemical and neurochemical evidence in the rat

The possibility that substances P (SP) is a neurotransmitter of baro- and chemoreceptor afferents in the rat was investigated. SP-like immunoreactivity (SP-I) was analyzed quantitatively by radioimmunoassay in various levels of the nucleus tractus solitarius (NTS), the site of termination of these afferents while SP-containing afferent neurons were studied in various portions of the peripheral pathways by immunocytochemistry. It was found that the NTS contained significant amounts of SP-I and that unilateral removal of the nodose ganglia reduces the SP-I content of those portions of the NTS known to receive vagal afferents. In addition, SP-I was visualized in discrete fibers in the tunica adventitia of the aortic arch and carotid sinus regions, the vagus nerve and nodose ganglia. These results in the rat are consistent with our previous studies in the cat and provide further evidence that SP is contained within baro- and chemoreceptor afferent nerves.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

2D proteome analysis initiates new Insights on the Salmonella Typhimurium LuxS protein

Background  

Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2.