Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

A comparison of the changes in the non‐neuronal cell populations of the superior cervical ganglia following decentralization and axotomy

Transecting the axons of neurons in the adult superior cervical ganglion (SCG; axotomy) results in the survival of most postganglionic neurons, the influx of circulating monocytes, proliferation of satellite cells, and changes in neuronal gene expression. In contrast, transecting the afferent input to the SCG (decentralization) results in nerve terminal degeneration and elicits a different pattern of gene expression. We examined the effects of decentralization on macrophages in the SCG and compared the results to those previously obtained after axotomy. Monoclonal antibodies were used to identify infiltrating (ED1+) and resident (ED2+) macrophages, as well as macrophages expressing MHC class II molecules (OX6+). Normal ganglia contained ED2+ cells and OX6+ cells, but few infiltrating macrophages. After decentralization, the number of infiltrating ED1+ cells increased in the SCG to a density about twofold greater than that previously seen after axotomy. Both the densities of ED2+ and OX6+ cells were essentially unchanged after decentralization, though a large increase in OX6+ cells occurred after axotomy. Proliferation among the ganglion's total non‐neuronal cell population was examined and found to increase about twofold after decentralization and about fourfold after axotomy. Double‐labeling experiments indicated that some of these proliferating cells were macrophages. After both surgical procedures, the percentage of proliferating ED2+ macrophages increased, while neither procedure altered the proliferation of ED1+ macrophages. Axotomy, though not decentralization, increased the proliferation of OX6+ cells. Future studies must address what role(s) infiltrating and/or resident macrophages play in regions of decentralized and axotomized neurons and, if both are involved, whether they play distinct roles. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 68–79, 2002     

Schiffs bases and derived secondary amines as plant growth inhibitors

Seventy-two Schiffs bases, 44 corresponding secondary amines, and 12 N-acetylated compounds were tested on their growth activity. Eighty-one compounds were active as growth inhibitors in at least one of three bioassays.     

Effect of Lysine to Arginine Mutagenesis in the V3 Loop of HIV-1 gp120 on Viral Entry Efficiency and Neutralization

HIV-1 infection is characterized by an ongoing replication leading to T-lymphocyte decline which is paralleled by the switch from CCR5 to CXCR4 coreceptor usage. To predict coreceptor usage, several computer algorithms using gp120 V3 loop sequence data have been developed. In these algorithms an occupation of the V3 positions 11 and 25, by one of the amino acids lysine (K) or arginine (R), is an indicator for CXCR4 usage. Amino acids R and K dominate at these two positions, but can also be identified at positions 9 and 10. Generally, CXCR4-viruses possess V3 sequences, with an overall positive charge higher than the V3 sequences of R5-viruses. The net charge is calculated by subtracting the number of negatively charged amino acids (D, aspartic acid and E, glutamic acid) from the number of positively charged ones (K and R). In contrast to D and E, which are very similar in their polar and acidic properties, the characteristics of the R guanidinium group differ significantly from the K ammonium group. However, in coreceptor predictive computer algorithms R and K are both equally rated. The study was conducted to analyze differences in infectivity and coreceptor usage because of R-to-K mutations at the V3 positions 9, 10 and 11. V3 loop mutants with all possible RRR-to-KKK triplets were constructed and analyzed for coreceptor usage, infectivity and neutralization by SDF-1α and RANTES. Virus mutants R₉R₁₀R₁₁ showed the highest infectivity rates, and were inhibited more efficiently in contrast to the K₉K₁₀K₁₁ viruses. They also showed higher efficiency in a virus-gp120 paired infection assay. Especially V3 loop position 9 was relevant for a switch to higher infectivity when occupied by R. Thus, K-to-R exchanges play a role for enhanced viral entry efficiency and should therefore be considered when the viral phenotype is predicted based on V3 sequence data.     

Characterization of the Fc gamma receptor on human platelets

IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits.     

Distribution of transferrin synthesis in brain and other tissues in the rat

Levels of transferrin mRNA were measured by hybridization to transferrin cDNA in extracts from various areas of rat brain and other tissues. The highest concentrations of transferrin mRNA were found in the liver and the choroid plexus of the lateral and third ventricles. Lower concentrations were observed in the medulla and thalamus, choroid plexus of the fourth ventricle, cortex, hypothalamus, cerebellum, pituitary, testis, placenta, stomach, spleen, kidney, muscle, and heart. Yolk sac, small intestine, and adrenal glands did not contain detectable transferrin mRNA levels. The size of transferrin mRNA was the same in liver, brain, and testis. Upon incubation of choroid plexus pieces with [14C]leucine in vitro, about 4% of the radioactive protein secreted into the medium was found to be transferrin. Together with previous data (Dickson, P.W., Howlett, G.J., and Schreiber, G. (1985) J. Biol. Chem. 260, 8214-8219; Dickson, P.W., Aldred, A.R., Marley, P.D., Bannister, D., and Schreiber (1986) J. Biol. Chem. 261, 3475-3478) the obtained data suggest that the choroid plexus plays a role in maintenance of homeostasis in the microenvironment of the central nervous system by synthesizing and secreting plasma proteins.     

A T cell-independent mechanism of macrophage activation by interferon-gamma

A primary interest in immunity to intracellular pathogenic microorganisms and tumors is to understand the mechanisms by which macrophages are activated for various functions. Two parameters of macrophage activation are the expression of the class II histocompatibility proteins or Ia molecules (1), and cytotoxic activity. The ability of T cells to induce these responses has been extensively documented and occurs via their secretion of interferon-gamma (IFN-gamma) after interaction with antigen (2-6). However, in a recent study using mice with the severe combined immunodeficiency (scid) mutation (7) which have no detectable T or B cell functions (7-9), we were surprised to find the induction of Ia expression on macrophages and the partial inhibition of bacterial growth after infection with Listeria monocytogenes (10). We have now utilized neutralizing monoclonal antibodies specific for murine IFN-gamma to investigate the mechanism of macrophage activation in scid mice. We show here that IFN-gamma can be produced by scid mice in the absence of lymphocyte-mediated immunity, and this IFN-gamma is important for macrophage activation during infection with Listeria. These results indicate the presence of an important T lymphocyte-independent mechanism of macrophage activation and IFN-gamma production in response to infection.     

Hepatic metabolism and secretion of a cholesterol-enriched lipoprotein fraction

A potentially important source of cholesterol secreted in bile is cholesterol-rich lipoproteins. However, the fate of the cholesterol carried in these lipoproteins after hepatic uptake has not been investigated. We harvested an apoE- and cholesterol-rich lipoprotein fraction (d 1.02-1.06 g/ml) from hypercholesterolemic rats and examined the acute effects of these lipoproteins on hepatic cholesterol metabolism, very low density lipoprotein (VLDL) secretion, and biliary lipid secretion. Administration of a lipoprotein bolus (20 mg of cholesterol) to rats resulted in a significant decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and a significant increase in acyl-coenzyme A:cholesterol acyltransferase activity over controls at 1 hr. Hepatic cholesteryl ester content increased 400% with no change in hepatic free cholesterol content or biliary cholesterol secretion. These cholesterol-rich lipoproteins delivered in the isolated perfused liver effected a fivefold increase in hepatic VLDL secretion with no change in composition. Therefore, cholesterol-rich lipoproteins do not acutely alter biliary cholesterol secretion. Rather, the majority of the cholesterol delivered to the liver in these lipoproteins is either esterified and stored as cholesteryl ester or resecreted as free and esterified cholesterol in hepatic VLDL.