Receptors for epidermal growth factor (urogastrone) and insulin in primary cultures of rat hepatocytes maintained in serum-free medium

We have analyzed the receptors for epidermal growth factor (urogastrone) (EGF-URO) and insulin in primary cultures of adult rat hepatocytes maintained for up to 3 weeks on human placental cell matrix in serum-free defined medium. Cross-link labeling experiments revealed that the insulin receptor, partially damaged by the collagenase isolation procedure, was rapidly regenerated to yield an intact receptor. In contrast, cross-link labeling of the EGF-URO receptor revealed that, upon prolonged culture, there was a progressive disappearance of the high molecular mass (175 kilodaltons (kDa)) receptor form, and an appearance of low molecular mass receptor species (130 and 105 kDa). After 3 weeks of culture, the low molecular mass receptor forms accounted for all of the labeled EGF-URO receptor present in the cells. Measurements of EGF-URO binding indicated that the number of EGF-URO binding sites per cell (2.0 x 10(5) +/- 0.3 x 10(5)) did not change during the 3 weeks of culture. However, there was a decrease in EGF-URO binding affinity, reflected by an increase in the KD from 0.6 to 3.0 nM. At zero time and after 3 weeks in culture, Scatchard plots of the binding data were linear; at intermediate time points, the plots were curvilinear. Despite the changes in the EGF-URO receptor that occurred, cells were still responsive to EGF-URO in terms of the inhibition of acetate incorporation into lipid. The ED50 for EGF-URO (about 0.2 nM) was the same for short-term cultures (48 h) as for cells maintained in culture for 3 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of epidermal growth factor (urogastrone) on gluconeogenesis, glucose oxidation, and glycogen synthesis in isolated rat hepatocytes

Using isolated rat hepatocytes, we studied the effect of epidermal growth factor (urogastrone) (EGF-URO) on the incorporation of [3-14C]pyruvate into glucose and glycogen, on the incorporation of [U-14C]glucose into glycogen, and on the oxidation of [U-14C]glucose to 14CO2. The effects of EGF-URO were compared with those of glucagon and insulin. EGF-URO, with an EC50 of 0.2 nM, enhanced by 34% (maximal stimulation) the conversion of [3-14C]pyruvate into glucose; no effect was observed on the oxidation of glucose to CO2 and on the incorporation of either pyruvate or glucose into glycogen. The effect of EGF-URO on pyruvate conversion to glucose was observed only when hepatocytes were preincubated with EGF-URO for 40 min prior to the addition of substrate. Glucagon (10 nM) increased the incorporation of [3-14C]pyruvate into glucose (44% above control); however, unlike EGF-URO, glucagon stimulated gluconeogenesis better without than with a preincubation period. Neither insulin nor EGF-URO (both 10 nM) affected the incorporation of [U-14C]glucose into glycogen during a 20-min incubation period. However, at longer time periods of incubation with the substrate (60 instead 20 min), insulin (but not EGF-URO) increased the incorporation of [14C]glucose into glycogen; EGF-URO counteracted this stimulatory effect of insulin. In contrast with previous data, our work indicates that EGF-URO can, under certain conditions, counteract the effects of insulin and, like glucagon, promote gluconeogenesis in isolated rat hepatocytes.     

Inhibition of crossed caudal interneurons by lateral interneurons in lamprey spinal cord during fictive locomotion

Templates of the membrane potential profiles from lateral (LI) interneurons and motoneurons during glutamate- and N-methyl-D-aspartate (NMDA)-induced fictive locomotion showed pronounced plateau phases. In contrast, crossed caudal (CC) interneurons had a less obvious and steeper plateau region that was followed by a clear notch coinciding with the end of the lateral interneuron plateau phase. These results indicate a significant inhibitory input from LI to CC interneurons.     

Delineation of amplification,hybridization and location effects in microarray data yields better-quality normalization

Background  

Oligonucleotide arrays have become one of the most widely used high-throughput tools in biology. Due to their sensitivity to experimental conditions, normalization is a crucial step when comparing measurements from these arrays. Normalization is, however, far from a solved problem. Frequently, we encounter datasets with significant technical effects that currently available methods are not able to correct.     

Erratum

Neurotransmitter receptor trafficking and the regulation of synaptic strength. Traffic 2001:2(7):437–448.     

Characterization of the Rac guanine nucleotide exchange factor P-Rex1 in platelets

Background

Blood platelets undergo a carefully regulated change in shape to serve as the primary mediators of hemostasis and thrombosis. These processes manifest through platelet spreading and aggregation and are dependent on platelet actin cytoskeletal changes orchestrated by the Rho GTPase family member Rac1. To elucidate how Rac1 is regulated in platelets, we captured Rac1-interacting proteins from platelets and identified Rac1-associated proteins by mass spectrometry.

Findings

Here, we demonstrate that Rac1 captures the Rac guanine nucleotide exchange factor P-Rex1 from platelet lysates. Western blotting experiments confirmed that P-Rex1 is expressed in platelets and associated with Rac1. To investigate the functional role of platelet P-Rex1, platelets from P-Rex1 ^-/- -deficient mice were treated with platelet agonists or exposed to platelet activating surfaces of fibrinogen, collagen and thrombin. Platelets from P-Rex1 ^-/- mice responded to platelet agonists and activating surfaces similarly to wild type platelets.

Conclusions

These findings suggest that P-Rex1 is not required for Rac1-mediated platelet activation and that the GEF activities of P-Rex1 may be more specific to GPCR chemokine receptor mediated processes in immune cells and tumor cells.     

Hybrid metabolic flux analysis: combining stoichiometric and statistical constraints to model the formation of complex recombinant products

Background  

Stoichiometric models constitute the basic framework for fluxome quantification in the realm of metabolic engineering. A recurrent bottleneck, however, is the establishment of consistent stoichiometric models for the synthesis of recombinant proteins or viruses. Although optimization algorithms for in silico metabolic redesign have been developed in the context of genome-scale stoichiometric models for small molecule production, still rudimentary knowledge of how different cellular levels are regulated and phenotypically expressed prevents their full applicability for complex product optimization.