Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

Immunization of Rhesus monkeys with a SialylTn–mAb17-1A conjugate vaccine co-formulated with QS-21 induces a temporary systemic cytokine release and NK cytotoxicity against tumor cells

Tumor-associated antigens resulting from aberrant glycosylation, such as the SialylTn carbohydrate antigen, are frequently over-expressed on cancer cells and provide potential targets for cancer vaccination. Immunization of Rhesus monkeys with SialylTn coupled to a highly immunogenic carrier molecule and formulated on aluminum hydroxide induced a strong immune response against the carrier protein but only a moderate IgM immune response against the SialylTn carbohydrate antigen. Co-formulation with QS-21 adjuvant dramatically enhanced the anti-SialylTn immune response and resulted in a SialylTn-specific IgG switch. The kinetics of the carbohydrate-specific IgG response correlated with a temporary release of cytokines such as IFNγ, IL-2, IL-1β, TNFα and GM-CSF which was measurable in the immune serum by xMAP Multiplex technology. Furthermore, tumor cell killing by activated natural killer cells was induced. These data demonstrate that immunization with a tumor-associated carbohydrate antigen in a highly immunogenic formulation results in a temporary release of type 1 cytokines which may be required for the induction of a specific IgG immune response against the carbohydrate antigen as well as for activation of effector cells against tumor cells.     

Preventing aneuploidy: the contribution of mitotic checkpoint proteins

Aneuploidy, an abnormal number of chromosomes, is a trait shared by most solid tumors. Chromosomal instability (CIN) manifested as aneuploidy might promote tumorigenesis and cause increased resistance to anti-cancer therapies. The mitotic checkpoint or spindle assembly checkpoint is a major signaling pathway involved in the prevention of CIN. We review current knowledge on the contribution of misregulation of mitotic checkpoint proteins to tumor formation and will address to what extent this contribution is due to chromosome segregation errors directly. We propose that both checkpoint and non-checkpoint functions of these proteins contribute to the wide array of oncogenic phenotypes seen upon their misregulation.     

Effect of Antimicrobial Consumption and Production Type on Antibacterial Resistance in the Bovine Respiratory and Digestive Tract

The aim of this study was to investigate the relationship between antimicrobial use and the occurrence of antimicrobial resistance in the digestive and respiratory tract in three different production systems of food producing animals. A longitudinal study was set up in 25 Belgian bovine herds (10 dairy, 10 beef, and 5 veal herds) for a 2 year monitoring of antimicrobial susceptibilities in E. coli and Pasteurellaceae retrieved from the rectum and the nasal cavity, respectively. During the first year of observation, the antimicrobial use was prospectively recorded on 15 of these farms (5 of each production type) and transformed into the treatment incidences according to the (animal) defined daily dose (TI_ADD) and (actually) used daily dose (TI_UDD). Antimicrobial resistance rates of 4,174 E. coli (all herds) and 474 Pasteurellaceae (beef and veal herds only) isolates for 12 antimicrobial agents demonstrated large differences between intensively reared veal calves (abundant and inconstant) and more extensively reared dairy and beef cattle (sparse and relatively stable). Using linear mixed effect models, a strong relation was found between antimicrobial treatment incidences and resistance profiles of 1,639 E. coli strains (p<0.0001) and 309 Pasteurellaceae (p≤0.012). These results indicate that a high antimicrobial selection pressure, here found to be represented by low dosages of oral prophylactic and therapeutic group medication, converts not only the commensal microbiota from the digestive tract but also the opportunistic pathogenic bacteria in the respiratory tract into reservoirs of multi-resistance.     

Genetical metabolomics of flavonoid biosynthesis in Populus: a case study

Genetical metabolomics [metabolite profiling combined with quantitative trait locus (QTL) analysis] has been proposed as a new tool to identify loci that control metabolite abundances. This concept was evaluated in a case study with the model tree Populus. Using HPLC, the peak abundances were analyzed of 15 closely related flavonoids present in apical tissues of two full-sib poplar families, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24, and correlation and QTL analysis were used to detect flux control points in flavonoid biosynthesis. Four robust metabolite quantitative trait loci (mQTL), associated with rate-limiting steps in flavonoid biosynthesis, were mapped. Each mQTL was involved in the flux control to one or two flavonoids. Based on the identities of the affected metabolites and the flavonoid pathway structure, a tentative function was assigned to three of these mQTL, and the corresponding candidate genes were mapped. The data indicate that the combination of metabolite profiling with QTL analysis is a valuable tool to identify control points in a complex metabolic pathway of closely related compounds.     

Bax Inhibitor-1-mediated Ca leak is decreased by cytosolic acidosis

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca²⁺ levels. Recently, we elucidated BI-1's Ca²⁺-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca²⁺-channel pore-dead mutant BI-1 (BI-1^D213R) was developed. We determined whether BI-1 behaves as a bona fide H⁺/Ca²⁺ antiporter or as an ER Ca²⁺-leak channel by investigating the effect of pH on unidirectional Ca²⁺-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1^−/− cells increased the ER Ca²⁺-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1^D231R expression in BI-1^−/−, despite its ER localization, did not increase the ER Ca²⁺-leak rate. However, at pH < 6.8, the BI-1-mediated ER Ca²⁺ leak was blocked. Finally, a peptide representing the Ca²⁺-channel pore of BI-1 promoting Ca²⁺ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca²⁺ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca²⁺-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.     

Internalization of Human 5-HT<Subscript>4a</Subscript> and 5-HT<Subscript>4b</Subscript> Receptors is Splice Variant Dependent

The family of 5-HT₄ receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT_4a and h5-HT_4b receptors. In the present study, the internalization of these two splice variants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT_4a-GFP and h5-HT_4b-GFP were generated by fusing the coding sequence of the 5-HT₄ receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT_4b-GFP receptor, but not of the h5-HT_4a-GFP receptor. The h5-HT_4b receptor displays a dual coupling to Gαi,o and Gαs proteins, in contrast to the h5-HT_4a receptor, which couples to Gαs proteins only. We investigated whether the difference in internalization of the two splice variant receptors was related to their differential coupling. Therefore, we performed agonist-stimulation of the receptor following inhibition of the Gαi,o protein coupling using PTX. The h5-HT_4b receptor internalization is PTX insensitive. We co-transfected the fluorescent chimeric receptors with other wild-type variants, which did not produce an alteration of the receptor trafficking. These findings provide the first evidence of differential internalization between the two splice variants, 5-HT_4a and 5-HT_4b receptors.     

Distinct severe acute respiratory syndrome coronavirus-induced acute lung injury pathways in two different nonhuman primate species

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), caused by influenza A virus H5N1 and severe acute respiratory syndrome coronavirus (SARS-CoV), supposedly depend on activation of the oxidative-stress machinery that is coupled with innate immunity, resulting in a strong proinflammatory host response. Inflammatory cytokines, such as interleukin 1β (IL-1β), IL-8, and IL-6, play a major role in mediating and amplifying ALI/ARDS by stimulating chemotaxis and activation of neutrophils. To obtain further insight into the pathogenesis of SARS-CoV-associated ALI, we compared SARS-CoV infections in two different nonhuman primate species, cynomolgus macaques and African green monkeys. Viral titers in the upper and lower respiratory tract were not significantly different in SARS-CoV-infected macaques and African green monkeys. Inflammatory cytokines that play a major role in mediating and amplifying ALI/ARDS or have neutrophil chemoattractant activity, such as IL-6, IL-8, CXCL1, and CXCL2, were, however, induced only in macaques. In contrast, other proinflammatory cytokines and chemokines, including osteopontin and CCL3, were upregulated in the lungs of African green monkeys to a significantly greater extent than in macaques. Because African green monkeys developed more severe ALI than macaques, with hyaline membrane formation, some of these differentially expressed proinflammatory genes may be critically involved in development of the observed pathological changes. Induction of distinct proinflammatory genes after SARS-CoV infection in different nonhuman primate species needs to be taken into account when analyzing outcomes of intervention strategies in these species.     

Genetic diversity in adult and seedling populations of <Emphasis Type="Italic">Primula vulgaris</Emphasis> in a fragmented agricultural landscape

Habitat fragmentation is known to generally reduce the size of plant populations and increase their isolation, leading to genetic erosion and increased between-population genetic differentiation. In Flanders (northern Belgium) Primula vulgaris is very rare and declining. Populations have incurred strong fragmentation for the last decades and are now restricted to a few highly fragmented areas in an intensively used agricultural landscape. Previous studies showed that small populations of this long-lived perennial herb still maintained high levels of genetic variation and low genetic differentiation. This pattern can either indicate recent gene flow or represent historical variation. Therefore, we used polymorphic microsatellite loci to investigate genetic variation and structure in adult (which may still reflect historical variation) and seedling (recent generation, thus affected by current processes) life stages. The recent generation (seedlings) showed a significant loss of observed heterozygosity (H _o) together with lower expected heterozygosity (H _e), a trend for higher inbreeding levels (F _IS) and higher differentiation (F _ST) between populations compared to the adult generation. This might result from (1) a reduction in effective population size, (2) higher inbreeding levels in the seedlings, (3) a higher survival of heterozygotes over time due to a higher fitness of heterozygotes (heterosis) and/or a lower fitness of homozygotes (inbreeding depression), (4) overlapping generations in the adult life stage, or (5) a lack of establishment of new (inbred) adults from seedlings due to degraded habitat conditions. Combining restoration of both habitat quality and gene flow between populations may be indispensable to ensure a sustainable conservation of fragmented populations.     

Sex differences in the brain: The relation between structure and function

In the fifty years since the organizational hypothesis was proposed, many sex differences have been found in behavior as well as structure of the brain that depend on the organizational effects of gonadal hormones early in development. Remarkably, in most cases we do not understand how the two are related. This paper makes the case that overstating the magnitude or constancy of sex differences in behavior and too narrowly interpreting the functional consequences of structural differences are significant roadblocks in resolving this issue.