Differential selection of Golgi proteins by COPII Sec24 isoforms in procyclic Trypanosoma brucei

The Sec24 subunit of the coat protein complex II (COPII) has been implicated in selecting newly synthesized cargo from the endoplasmic reticulum (ER) for delivery to the Golgi. The protozoan parasite, Trypanosoma brucei, contains two paralogs, TbSec24.1 and TbSec24.2, which were depleted using RNA interference in the insect form of the parasite. Depletion of either TbSec24.1 or TbSec24.2 resulted in growth arrest and modest inhibition of anterograde transport of the putative Golgi enzyme, TbGntB, and the secretory marker, BiPNAVRG-HA9. In contrast, depletion of TbSec24.1, but not TbSec24.2, led to reversible mislocalization of the Golgi stack proteins, TbGRASP and TbGolgin63. The latter accumulated in the ER. The localization of the COPI coatomer subunit, TbεCOP, and the trans Golgi network (TGN) protein, TbGRIP70, was largely unaffected, although the latter was preferentially lost from those Golgi that were not associated with the bilobe, a structure previously implicated in Golgi biogenesis. Together, these data suggest that TbSec24 paralogs can differentiate among proteins destined for the Golgi.     

Change in Body Size and Mortality: Results from the Melbourne Collaborative Cohort Study

Background

The association between change in weight or body mass index, and mortality is widely reported, however, both measures fail to account for fat distribution. Change in waist circumference, a measure of central adiposity, in relation to mortality has not been studied extensively.

Methods

We investigated the association between mortality and changes in directly measured waist circumference, hips circumference and weight from baseline (1990–1994) to wave 2 (2003–2007) in a prospective cohort study of people aged 40–69 years at baseline. Cox regression, with age as the time metric and follow-up starting at wave 2, adjusted for confounding variables, was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for change in body size in relation to mortality from all causes, cardiovascular disease and cancer.

Results

There were 1465 deaths (109 cancer, 242 cardiovascular disease) identified during an average 7.7 years of follow-up from 21 298 participants. Compared to minimal increase in body size, loss of waist circumference (HR: 1.26; 95% CI: 1.09–1.47), weight (1.80; 1.54–2.11), or hips circumference (1.35; 1.15–1.57) were associated with an increased risk of all-cause mortality, particularly for older adults. Weight loss was associated with cardiovascular disease mortality (2.40; 1.57–3.65) but change in body size was not associated with obesity-related cancer mortality.

Conclusion

This study confirms the association between weight loss and increased mortality from all-causes for older adults. Based on evidence from observational cohort studies, weight stability may be the recommended option for most adults, especially older adults.     

Dynamics of a Cytokine Storm

Six volunteers experienced severe inflammatory response during the Phase I clinical trial of a monoclonal antibody that was designed to stimulate a regulatory T cell response. Soon after the trial began, each volunteer experienced a “cytokine storm”, a dramatic increase in cytokine concentrations. The monoclonal antibody, TGN1412, raised serum concentrations of both pro- and anti-inflammatory cytokines το very hiγh values during the first day, while lymphocyte and monocyte concentrations plummeted. Because the subjects were healthy and had no prior indications of immune deficiency, this event provided an unusual opportunity to study the dynamic interactions of cytokines and other measured parameters. Here, the response histories of nine cytokines have been modeled by a set of linear ordinary differential equations. A general search procedure identifies parameters of the model, whose response fits the data well during the five-day measurement period. The eighteenth-order model reveals plausible cause-and-effect relationships among the cytokines, showing how each cytokine induces or inhibits other cytokines. It suggests that perturbations in IL2, IL8, and IL10 have the most significant inductive effect, while IFN-γ and IL12 have the greatest inhibiting effect on other cytokine concentrations. Although TNF-α is a major pro-inflammatory factor, IFN-γ and three other cytokines have faster initial and median response to TGN1412 infusion. Principal-component analysis of the data reveals three clusters of similar cytokine responses: [TNF-α, IL1, IL10], [IFN-γ, IL2, IL4, IL8, and IL12], and [IL6]. IL1, IL6, IL10, and TNF-α have the highest degree of variability in response to uncertain initial conditions, exogenous effects, and parameter estimates. This study illuminates details of a cytokine storm event, and it demonstrates the value of linear modeling for interpreting complex, coupled biological system dynamics from empirical data.     

Hepatic adenylate cyclase. Development-dependent coupling to the beta-adrenergic receptor in the neonate

Guanine nucleotide-dependent modulation of agonist binding to the beta-receptor reflects coupling of the receptor to the nucleotide regulatory protein. Similarly, guanine nucleotide-dependent stimulation of adenylate cyclase can be used as an index of coupling between the regulatory protein and the catalytic unit of the cyclase. Using both approaches we have studied coupling in the beta-adrenergic receptor-adenylate cyclase system in rabbit liver during neonatal development. With [3H]dihydroalprenolol as ligand, the Bmax was relatively unchanged (200-300 fmol/mg of protein) between birth and end of day 1 and was similar to adult values. Guanyl-5'-yl imidodiphosphate-dependent shift in agonist (l-isoproterenol) competition curves was biphasic, decreasing from 10-fold in membranes isolated from animals at term to about 6-fold in membranes from 6-h-old neonates, and increasing progressively in older animals to a maximal measurable value of 42-fold in the adult. The ability of guanyl-5'-yl imidodiphosphate, GTP, GTP plus isoproterenol, NaF, or forskolin to activate adenylate cyclase was also biphasic and age-dependent. With Mn2+ the measured activity was not at any time greater than the activity at term. Pretreatment of membranes with cholera toxin resulted in differential levels of enhancement of adenylate cyclase activity wherein much lower enhancement was observed in membranes from neonatal animals. With [32P]NAD as substrate, cholera toxin-catalyzed ADP-ribosylation of membranes indicated development-dependent accumulation of Ns peptides. From these results we suggest that there is a decreased efficiency in the coupling of the beta-adrenergic receptor to hepatic adenylate cyclase in early neonatal life. The molecular basis for the biphasic nature of the coupling is presently unclear.     

Docosahexaenoic acid is selectively enriched in plasma phospholipids during pregnancy in Trinidadian women--results of a pilot study

The fetal demand for docosahexaenoic acid (DHA) has to be satisfied by the mother. We determined the fatty acids in maternal plasma non-esterified fatty acid (NEFA), triacylglycerol (TAG) and phosphatidylcholine (PC), in a cross-sectional study of non-pregnant (n = 10), pregnant (n = 19), and postpartum (n = 9) women. There were lipid class-dependent differences in plasma polyunsaturated fatty acid (PUFA) concentrations between groups. During pregnancy, DHA was most highly enriched in PC, about 230%, with more modest enrichment for linoleic acid (LA) and arachidonic acid (AA), and no enrichment of alpha-linolenic acid (alpha-LNA). There was relative enrichment of LA, AA and alpha-LNA in TAG, but not of DHA. There was no specific enrichment of any PUFA in the NEFA pool. These data accord with the suggestion that the enrichment of alpha-LNA in TAG and of DHA in phospholipids reflects hepatic regulation of n-3 PUFA metabolism which potentially enhances the delivery of DHA to the placenta.     

Motif effects in Affymetrix GeneChips seriously affect probe intensities

An Affymetrix GeneChip consists of an array of hundreds of thousands of probes (each a sequence of 25 bases) with the probe values being used to infer the extent to which genes are expressed in the biological material under investigation. In this article, we demonstrate that these probe values are also strongly influenced by their precise base sequence. We use data from >28 000 CEL files relating to 10 different Affymetrix GeneChip platforms and involving nearly 1000 experiments. Our results confirm known effects (those due to the T7-primer and the formation of G-quadruplexes) but reveal other effects. We show that there can be huge variations from one experiment to another, and that there may also be sizeable disparities between batches within an experiment and between CEL files within a batch.