Constitutive and agonist-induced dimerizations of the P2Y1 receptor: relationship to internalization and scaffolding

In living cells, P2Y(1) receptor dimerization was quantitated by an improved version of fluorescence resonance energy transfer donor photobleaching analysis. 44% of the P2Y(1) receptors expressed in HEK293 cell membranes exist as dimers in the resting state, inducible by agonist exposure to give 85-100% dimerization. Monomer and constitutive dimers are fully active. Agonist-induced dimerization follows desensitization and is fully reversible upon withdrawal of agonist. Receptor dimers are required for internalization at 37 degrees C but are not sufficient; at 20 degrees C dimerization also occurs, but endocytosis is abolished. Removal of the C-terminal 19 amino acids abolished both dimerization and internalization, whereas full activation by agonists was retained up to a loss of 39 amino acids, confirming active monomers. This receptor is known to bind through its last four amino acids (DTSL) to a scaffolding protein, Na/H exchanger regulatory factor-2, which was endogenous here, and DTSL removal blocked constitutive dimerization specifically. Distinction should therefore be made between the following: 1) constitutive dimers tethered to a scaffolding protein, together with effector proteins, within a signaling micro-domain, and 2) free dimers in the cell membrane, which here are inducible by agonist exposure. For the class A G-protein-coupled receptors, we suggest that the percentages of free monomers, and in many cases of induced free dimers, commonly become artifactually increased; this would arise from an excess there of the receptor over its specific scaffold and from a lack of the native targeting of the receptor to that site.     

Structure and thermotropic behavior of the Staphylococcus aureus lipid lysyl-dipalmitoylphosphatidylglycerol

We have characterized the structural and thermotropic properties of one of the most important lipids in the cell membrane of Staphylococcus aureus, lysyl-dipalmitoylphosphatidylglycerol (lysyl-DPPG). applying differential scanning calorimetry and small- and wide-angle x-ray scattering. Microcalorimetry revealed that under physiological conditions (phosphate buffer, 20 mM NaPi, 130 mM NaCl, pH 7.4), the synthetic lysyl-DPPG resembles the features of the parent dipalmitoylphosphatidylglycerol (DPPG) with respect to its melting behavior. However, in contrast to DPPG, lowering the pH did not significantly affect the main transition temperature (∼40°C) of lysyl-DPPG, which can be explained by its difference in protonization because of the lysine group. X-ray experiments yielded the first information on chain packing and morphology of lysyl-DPPG. We found that lysyl-DPPG forms an interdigitated lamellar phase below the chain-melting transition. This can be explained by the large headgroup area of lysyl-DPPG as a result of its charged lysine group, especially if the headgroup is arranged parallel to the bilayer plane. Additionally, lysyl-DPPG degradation products, such as lysine and free fatty acids, had significant influences on the melting behavior and led to a multicomponent melting transition. Our results indicate that the degradation of lysyl-DPPG takes place mainly during the hydration process but also depends on lipid storage time, pH, and thermal treatment. Detailed temperature-resolved experiments at pH 5.0 demonstrated the formation of a lamellar gel phase with tilted hydrocarbon chains and a ripple phase, coexisting with the interdigitated lysyl-DPPG bilayers.     

Production of recombinant proteins in clonal root cultures using episomal expression vectors

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.     

Activation of immobilized lipase in non-aqueous systems by hydrophobic poly-DL-tryptophan tethers

Many industrially important reactions use immobilized enzymes in non-aqueous, organic systems, particularly for the production of chiral compounds such as pharmaceutical precursors. The addition of a spacer molecule ("tether") between a supporting surface and enzyme often substantially improves the activity and stability of enzymes in aqueous solution. Most "long" linkers (e.g., polyethylene oxide derivatives) are relatively hydrophilic, improving the solubility of the linker-enzyme conjugate in polar environments, but this provides little benefit in non-polar environments such as organic solvents. We present a novel method for the covalent immobilization of enzymes on solid surfaces using a long, hydrophobic polytryptophan tether. Candida antarctica lipase B (CALB) was covalently immobilized on non-porous, functionalized 1-microm silica microspheres, with and without an intervening hydrophobic poly-DL-tryptophan tether (n approximately 78). The polytryptophan-tethered enzyme exhibited 35 times greater esterification of n-propanol with lauric acid in the organic phase and five times the hydrolytic activity against p-nitrophenol palmitate, compared to the activity of the same enzyme immobilized without tethers. In addition, the hydrophobic tethers caused the silica microspheres to disperse more readily in the organic phase, while the surface-immobilized control treatment was less lipophilic and quickly settled out of the organic phase when the suspensions were not vigorously mixed.     

Oxidative damage of albumin in advanced liver disease

Albumin has a number of biological functions and the serum albumin level is related to prognosis in advanced liver disease. Oxidative stress is believed to play an important role in the pathogenesis of liver failure. The aim of the present study was to characterize oxidative modification of albumin in patients with various degrees of liver failure and to investigate implications for its binding function. Patients with liver cirrhosis (n=10), acute-on-chronic liver failure (n=8) and healthy controls (n=15) were included in the study. Three fractions of albumin were separated by HPLC according to the redox state of cysteine-34 and detected by fluorescence as well as UV absorption. Carbonyl groups were measured as a marker of oxidative modification in plasma proteins and, by western blotting, on albumin. Progressive oxidative modification of albumin was found with increasing severity of liver failure indicated by an increased content of carbonyl groups and oxidation of cysteine-34. Fluorescence properties of albumin were altered by oxidation and, in patients with acute-on-chronic liver failure, by high plasma levels of bilirubin. This alteration of albumin fluorescence by bilirubin provides evidence for a preferred binding of bilirubin to the fully reduced form of albumin.     

Experimental manipulations of afferent immune responses influence efferent immune responses to brain tumors

Tumors grow more readily in the brain than in the periphery, in part due to immune privilege. Differences in both afferent and efferent components of the immune response contribute to this lower level of responsiveness. On the afferent side, despite the lack of lymphatic vessels in the brain, antigens from brain arrive in lymph nodes and spleen by several routes, and the route taken may influence the type of response generated. Work with viruses and soluble antigens in mice has shown that the intracerebral location and the volume of the inoculation influence the strength of the cytotoxic T cell response. We examined whether these factors influence the T cell response against experimental brain tumors in mice. Placement of tumor cells in the cerebral ventricles instead of the parenchyma generated an immune response sufficient to increase survival time. A large volume of an intraparenchymal infusion of tumor cells caused spread of cells to the ventricles, and resulted in longer survival time relative to a small volume infusion. Infusion of the same dose of radiolabeled tumor cells in either a small volume or a large volume allowed tracking of potential tumor antigens to the periphery. Both modes of infusion resulted in similar levels of radioactivity in blood, spleen and kidney. Unexpectedly, cells infused intraparenchymally in a small volume, compared to a large volume, resulted in (1) more radioactivity in cervical lymph nodes (parotid and deep cervical lymph nodes), (2) a greater number of CD11b+/Gr1+ myeloid suppressor cells in the tumors, and (3) fewer CD8+ cells within the tumor mass. Consistent with these observations, providing a stronger afferent stimulus by giving a concurrent subcutaneous injection of the same tumor cells infused into the brain increased CD8+ T cell infiltration of the tumor in the brain. These results suggest that the immune response elicited by antigens that drain predominantly to the cervical lymph nodes may be less effective than responses elicited at other lymph nodes, perhaps due to immunosuppressive cells. Directing therapies to the optimal peripheral sites may improve immune responses against brain tumors.     

Sex differences in circulating and renal angiotensins of hypertensive mRen(2). Lewis but not normotensive Lewis rats

Sex differences in blood pressure are evident in experimental models and human subjects, yet the mechanisms underlying this disparity remain equivocal. The current study sought to define the extent of male-female differences in the circulating and tissue renin-angiotensin aldosterone systems (RAASs) of congenic mRen(2). Lewis and control Lewis rats. Male congenics exhibited higher systolic blood pressure than females [200 +/- 4 vs. 146 +/- 7 mmHg, P < 0.01] or Lewis males and females [113 +/- 2 vs. 112 +/- 2 mmHg, P > 0.05]. Plasma ANG II levels were twofold higher in male congenics [47 +/- 3 vs. 19 +/- 3 pM, P < 0.01] and fivefold higher than in male or female Lewis rats [6 +/- 1 vs. 6 +/- 1 pM]. ANG I levels were also highest in the males; however, plasma ANG-(1-7) was higher in female congenics. Male congenics exhibited greater circulating renin and angiotensin-converting enzyme (ACE) activities, as well as angiotensinogen, than female littermates. Renal cortical and medullary ANG II levels were also higher in the male congenics versus all the other groups; ANG I was lower in the males. Cortical ACE2 activity was higher in male congenics, yet neprilysin activity and protein were greater in the females, which may contribute to reduced renal levels of ANG II. These data reveal that sex differences in both the circulating and renal RAAS are apparent primarily in the hypertensive group. The enhanced activity of the RAAS in male congenics may contribute to the higher pressure and tissue injury evident in the strain.