ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed reaction in the pathway of starch synthesis. It was recently shown that potato (Solanum tuberosum) tuber AGPase is subject to redox-dependent posttranslational regulation, involving formation of an intermolecular Cys bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme (A. Tiessen, J.H.M. Hendriks, M. Stitt, A. Branscheid, Y. Gibon, E.M. Farré, P. Geigenberger [2002] Plant Cell 14: 2191-2213). We show here that AGPase is also subject to posttranslational regulation in leaves of pea (Pisum sativum), potato, and Arabidopsis. Conversion is accompanied by an increase in activity, which involves changes in the kinetic properties. Light and sugars act as inputs to trigger posttranslational regulation of AGPase in leaves. AGPB is rapidly converted from a dimer to a monomer when isolated chloroplasts are illuminated and from a monomer to a dimer when preilluminated leaves are darkened. AGPB is converted from a dimer to monomer when sucrose is supplied to leaves via the petiole in the dark. Conversion to monomeric form increases during the day as leaf sugars increase. This is enhanced in the starchless phosphoglucomutase mutant, which has higher sugar levels than wild-type Columbia-0. The extent of AGPB monomerization correlates with leaf sugar levels, and at a given sugar content, is higher in the light than the dark. This novel posttranslational regulation mechanism will allow starch synthesis to be regulated in response to light and sugar levels in the leaf. It complements the well-characterized regulation network that coordinates fluxes of metabolites with the recycling of phosphate during photosynthetic carbon fixation and sucrose synthesis.     

Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point

Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.     

Results of an international round robin for the quantification of serum non-transferrin-bound iron: Need for defining standardization and a clinically relevant isoform

Non-transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. Various methods to measure NTBI were comparatively assessed as part of an international interlaboratory study. Six laboratories participated in the study, using methods based on iron mobilization and detection with iron chelators or on reactivity with bleomycin. Serum samples of 12 patients with hereditary (n=11) and secondary (n=1) hemochromatosis were measured during a 3-day analysis using 4 determinations per sample per day, making a total of 144 measurements per laboratory. Bland-Altman plots for repeated measurements are presented. The methods differed widely in mean serum NTBI level (range 0.12-4.32mumol/L), between-sample variation (SD range 0.20-2.13mumol/L and CV range 49.3-391.3%), and within-sample variation (SD range 0.02-0.45mumol/L and CV range 4.4-193.2%). The results obtained with methods based on chelators correlated significantly (R(2) range 0.86-0.99). On the other hand, NTBI values obtained by the various methods related differently from those of serum transferrin saturation (TS) when expressed in terms of both regression coefficients and NTBI levels at TS of 50%. Recent studies underscore the clinical relevance of NTBI in the management of iron-overloaded patients. However, before measurement of NTBI can be introduced into clinical practice, there is a need for more reproducible protocols as well as information on which method best represents the pathophysiological phenomenon and is most pertinent for diagnostic and therapeutic purposes.     

Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells

HER2, a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we applied a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2 and their downstream activation of ERK to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we could separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrated that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activate ERK were quantitatively equivalent. We found that HER2-mediated effects on EGFR dimerization and trafficking were sufficient to explain the observed HER2-mediated amplification of epidermal growth factor-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared with the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking yielding increased EGFR sparing cause the sustained HER2-mediated enhancement of ERK signaling.     

Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinosylcytosine (ara-C) in leukemia and solid tumor cell lines

Resistance to, the hydrophilic drug ara-C, might be meditated by decreased membrane transport. Lipophilic prodrugs were synthesized to facilitate uptake. These compounds were equally active as ara-C, while the compounds with the shortest fatty-acid group and highest number of double bonds were the more active. These compounds also show a better retention profile, their effect is retained longer than for ara-C.     

Antiproliferative activity and mechanism of action of fatty acid derivatives of gemcitabine in leukemia and solid tumor cell lines and in human xenografts

Gemcitabine is a deoxycytidine analog, which can be inactivated by deamination catalyzed by deoxycytidine deaminase (dCDA). Altered transport over the cell membrane is a mechanism of resistance to gemcitabine. To facilitate accumulation, the fatty acid derivative CP-4125 was synthesized. Since, the fatty acid is acylated at the site of action of dCDA, a decreased deamination was expected. CP-4125 was equally active as gemcitabine in a panel of rodent and human cell lines and in human melanoma xenografts bearing mice. In contrast to gemcitabine, CP-4125 was not deaminated but inhibited deamination of deoxycytidine and gemcitabine. Pools of the active triphosphate of gemcitabine increased for over 20 hr after CP-4125 exposure, while these pools decreased directly after removal of gemcitabine. In conclusion: CP-4125 is an interesting new gemcitabine derivative.     

Apolipoprotein E is resistant to intracellular degradation in vitro and in vivo. Evidence for retroendocytosis

Apolipoprotein E (apoE) is an important determinant for the uptake of triglyceride-rich lipoproteins and emulsions by the liver, but the intracellular pathway of apoE following particle internalization is poorly defined. In the present study, we investigated whether retroendocytosis is a unique feature of apoE as compared with apoB by studying the intracellular fate of very low density lipoprotein-sized apoE-containing triglyceride-rich emulsion particles and LDL after LDLr-mediated uptake. Incubation of HepG2 cells with [(3)H]cholesteryl oleate-labeled particles at 37 degrees C led to a rapid release of [(3)H]cholesterol within 30 min for both LDL and emulsion particles. In contrast, emulsion-derived (125)I-apoE was more resistant to degradation (>/=120 min) than LDL-derived (125)I-apoB (30 min). Incubation at 18 degrees C, which allows endosomal uptake but prevents lysosomal degradation, with subsequent incubation at 37 degrees C resulted in a time-dependent release of intact apoE from the cells (up to 14% of the endocytosed apoE at 4 h). The release of apoE was accelerated by the presence of protein-free emulsion (20%) or high density lipoprotein (26%). Retroendocytosis of intact particles could be excluded since little intact [(3)H]cholesteryl oleate was released (<3%). In contrast, the degradation of LDL was complete with virtually no secretion of intact apoB into the medium. The intracellular stability of apoE was also demonstrated after hepatic uptake in C57Bl/6 mice. Intravenous injection of (125)I-apoE and [(3)H]cholesteryl oleate-labeled emulsions resulted in efficient LDLr-mediated uptake of both components by the liver (45-50% of the injected dose after 20 min). At 1 h after injection, only 15-20% of the hepatic (125)I-apoE was degraded, whereas 75% of the [(3)H]cholesteryl oleate was hydrolyzed. From these data we conclude that following LDLr-mediated internalization by liver cells, apoE can escape degradation and can be resecreted. This sequence of events may allow apoE to participate in its hypothesized intracellular functions such as mediator of the post-lysosomal trafficking of lipids and very low density lipoprotein assembly.     

The effect of omitted milking on the behaviour of cows in the context of cluster attachment failure during automatic milking

In robotic milking there is always a slight chance of failure to attach the milking cluster. Attachment failure is most likely for cows whose udder conformation is less convenient for robot attachment. In general, after milking failure cows try to revisit the milking robot if they are not sent to a separate area. Since it is difficult to estimate the effect of milking failure on such a cow and her welfare in conditions of robotic milking, a specific 16-day trial was conducted on 12 cows. These cows were milked in a milking parlour with six milking stalls. Each afternoon milking, three cows were not milked. All the cows were closely observed in the cubicle house for 1 h after the afternoon milking. Thereafter, all cows were brought to the milking parlour the third time and the three unmilked cows were milked. In total, each cow was observed 12 times after milking and four times after omitted milking. The following behavioural traits were registered: time budget for the 1 h, occurrence and time until eating, drinking, lying, urination and defecation, and aggressive interactions. Milking order was defined on the basis of how often a cow came to the milking parlour in the first batch of six cows. Moreover, the data related to the milk yield and the use of the automatic feeding installation with the complete diet were analysed. After omitted milking, only the cows from the first batch stood longer in cubicles (14.2 min of 1 h) and lay less (5.4 min of 1 h) than milked cows of the same batch (respectively 7.0 min and 16.3 min for standing and lying in cubicles) (P<0.01). After omitted milking, cows urinated earlier and more frequently (64.5%) than milked cows (36.3%) (P<0.002) (both batches). There were no statistically significant differences in eating time and feed intake after milking and omitted milking. Milk yield per cow averaged 24.9 kg during days with omitted (delayed by 1 h) milking and 25.3 kg during the days without omitted milking (P<0.05). It was concluded that cows show some signs of discomfort after omitted milking (urination); this discomfort seemed to be greater in cows coming earlier to the milking parlour (afterwards they preferred to stand rather than to lie). The 60% of cases of milk leakage found after omitted milking indicates that failed cluster attachment can be accompanied by an extra risk factor for the occurrence of mastitis. However, omitted milking as a treatment did not influence feeding and aggressive behaviour or milking order when unmilked cows were brought to the milking parlour the third time together with the milked cows. Our methods and results can be useful for estimating the effects of robot milking failures on a cow. Future studies should pay particular attention to high-yielding cows and to longer periods of delayed milking.