The effect of catecholamines and prostaglandins upon human and rat erythrocytes.

Both human and rat erythrocytes respond to low doses (10(-11)--10(-9) M) of L-isoproterenol and L-epinephrine with an increased degree of hypotonic hemolysis and a decreased rate of filtration through standardized paper filters. The receptors in both cell types have many of the characteristics of beta-receptors for catecholamines. However, hormone-receptor interaction in the human cell does not lead to an increase in intracellular cyclic AMP concentration, but in the rat cell, hormone-receptor interaction does lead to a significant increase in cyclic AMP content. Thus, catecholamine-beta-receptor interaction, at least in the human red cell, leads to a change in red cell properties which are not mediated by adenylate cyclase activation. Likewise, prostaglandin E2, at 10(-12)--10(-10) M, causes are increased degree of hypotonic hemolysis and a decreased rate of filtration through standardized paper filters, but it also does not increase the cycliC AMP content of the human erythrocyte but does increase that of the rat erythrocyte. Nevertheless, exogenous cyclic AMP, when added at a concentration of 10(-8) M to washed human erythrocytes, increases the degree of hypotonic hemolysis. Conversely, prostaglandin E1, at 10(-12)--10(-10) M, causes a decreased degree of hypotonic hemolysis and an increased rate of filtration through a standard filter. Both prostaglandin E2 and the catecholamines decrease the size of a rapidly exchangeable calcium pool, and prostaglandin E1 increases it.     

Renal uptake and excretion of epidermal growth factor from plasma in the rat

The rat excretes around 2 nmol epidermal growth factor (EGF) in the urine per 24 h. The urinary EGF might be derived from plasma and/or might be synthesized in the kidneys. We have used the rat to study the renal uptake and excretion of homologous EGF from plasma. I.v. injected 125I-EGF was removed from the circulation within a few minutes. 5 min after the injection, the kidneys contained 12% of the 125I-EGF. The kidneys seemed to degrade most of the 125I-EGF which they accumulated from blood, as only 4% of the injected label was excreted as intact 125I-EGF in the urine. The amount of endogenous EGF in plasma was under the detection limit of our enzyme-linked immunosorbent assay (0.03 nmol/l) and it remained so after bilateral nephrectomy. Even if plasma EGF was 0.03 nmol/l excretion of EGF from plasma could account for less than 5% of the urinary EGF. This study shows that the kidneys are able to accumulate EGF from plasma and excrete a part of it as intact EGF in the urine. However, excretion of immunoreactive EGF from plasma can only account for a minor part of the urinary EGF.     

Express Your LOV: An Engineered Flavoprotein as a Reporter for Protein Expression and Purification

In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an “effector” protein normally produced by enterohemorrhagic E. coli strains and “injected” into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.     

Tetraethylammonium and p-aminohippurate as clearance markers for renal plasma flow in the rat during saline and glucose infusion.

Renal plasma clearances (C) of 14C-tetraethylammonium (TEA) and p-aminohippurate (PAH) as estimates of arterial renal plasma flow (ARPF) were evaluated in anesthetized rats during control conditions and during intravenous glucose infusion. Venous renal blood flow was measured directly by means of a servo-controlled pump, keeping the renal venous pressure constant. Arteriovenous extraction fractions (E = 1 - P(renal venous)/P(renal arterial)) for PAH averaged 88.3 +/- (SE) 0.8% in control rats and 82.0 +/- 0.9% in glucose-infused rats (p less than 0.001); E(TEA) averaged 92.0 +/- 0.6 and 90.1 +/- 0.6%, respectively (p less than 0.05). Under both experimental conditions, (C/E)PAH did not differ significantly from ARPF, while (C/E)TEA underestimated ARPF; the rate of extraction of TEA exceeded the rate of excretion by 15-20%, probably due to accumulation of TEA in renal tissue. It is concluded that, when corrected for E, C(PAH) is in general a more accurate estimate for ARPF than C(TEA). However, under conditions involving changes in plasma glucose levels C(TEA) may provide a better estimate of the effective renal plasma flow than C(PAH).