Micropreparative isoelectric focusing protein separation in a suspended drop

IEF protein binary separations were performed in a 12-μL drop suspended between two palladium electrodes, using pH gradients created by electrolysis of simple buffers at low voltages (1.5-5 V). The dynamics of pH gradient formation and protein separation were investigated by computer simulation and experimentally via digital video microscope imaging in the presence and absence of pH indicator solution. Albumin, ferritin, myoglobin, and cytochrome c were used as model proteins. A drop containing 2.4 μg of each protein was applied, electrophoresed, and allowed to evaporate until it splits to produce two fractions that were recovered by rinsing the electrodes with a few microliters of buffer. Analysis by gel electrophoresis revealed that anode and cathode fractions were depleted from high pI and low pI proteins, respectively, whereas proteins with intermediate pI values were recovered in both fractions. Comparable data were obtained with diluted bovine serum that was fortified with myoglobin and cytochrome c.

Suspended sediment pulse effects in rainbow trout (Oncorhynchus mykiss) - relating apical and systemic responses

To provide an integrated perspective on mineral particle effects in salmonids, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to daily mica particle pulses for 8 and 24 days. On day 8, increased immature erythrocyte proportions indicated a previous stress response. This response was absent on day 24, on which condition factor as well as plasma protein and aspartate aminotransferase activity decreased. The latter two related negatively to the hepato-somatic index, suggesting metabolic adaptations. The hepato-somatic index increased on days 8 and 24, while spleen-somatic index increased on day 24. No histopathological damage occurred in gills, liver, spleen, or kidney. However, splenic melano-macrophages increased on both days, and hyaline degenerations of kidney tubular cells were apparent on day 24. Overall, particle pulses affected rainbow trout more via turbidity rather than by physical damage. We conclude that (i) rainbow trout may adapt to sediment pulses as early as 8 days of exposure and (ii) particle pulses over 24 days can cause structural and metabolic changes in rainbow trout, even when gill damage is absent and apical effects on condition are moderate.

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.