Recent insights in phosphatidylinositol signaling

Studies of phosphatidylinositol signaling pathways are entering a new phase in which molecular genetic techniques are providing powerful tools to dissect the functions of various metabolites and pathways. Studies with phospholipase C are most advanced and clearly indicate that phosphatidylinositol turnover is critical for vision in Drosophila and cell proliferation in various cultured cells. Expression of cDNA constructs and microinjection of PLC or antibodies against it clearly establish a role for PtdIns signaling distinct from its role in calcium mobilization and protein kinase C activation. The importance of inositol cyclic phosphates is also beginning to be realized from the study of cyclic hydrolase using similar techniques. Elucidation of the function of the 3-phosphate inositol phospholipid pathway awaits similar studies. The recent cDNA cloning of inositol monophosphatase (Diehl et al., 1990), Ins(1,4,5)P3 3-kinase (Choi et al., 1990), and inositol polyphosphate 1-phosphatase (York and Majerus, 1991) should provide tools to define further the cell biology of the phosphatidylinositol signaling pathway.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Twin studies demonstrate a host cell genetic effect on productive human immunodeficiency virus infection of human monocytes and macrophages in vitro.

Biological and genetic variability is a prominent feature of human immunodeficiency virus (HIV) strains, especially in tropism, syncytium formation, and replicative capacity. To determine whether there were variable host cell effects on HIV replication in monocytes, three different strains of low-passage-number monocytotropic blood isolates of HIV and the laboratory-adapted strain Ba-L were inoculated into panels of adherent monocytes drawn from 44 different donors, and peak extracellular HIV p24 antigen titers were compared. The clinical HIV strains showed patterns of either moderate or low-level replication in most donor monocytes (20 to 4,000 pg/ml). However, within this range there was marked variation in peak titers in most donors. HIV type 1 Ba-L replicated in all donor monocytes to much higher levels with less variability (30 to 40 ng/ml). Furthermore, replication of 21 clinical blood-derived strains of HIV in blood monocytes and monocyte-derived macrophages (MDM) from pairs of identical twins and age-matched unrelated donors (URD) of the same sex were compared. In all of the seven pairs of identical twins, the kinetics of replication (measured by extracellular HIV p24 antigen) of panels of four clinical HIV type 1 isolates in monocytes were similar within pairs. However, marked and significant differences in kinetics of HIV production occurred within 10 of the 12 unrelated donor pairs (P = 0.0007). The remaining two URD pairs showed similar kinetic patterns, but only one pair had the same HLA-DR genotype. Similar results were observed with monocytes/MDMs obtained from a second bleed of the same donor. Hence, discordant patterns of HIV replication kinetics between URD monocyte pairs contrasted with concordant patterns in identical twin monocytes. These data strongly suggest a host cell genetic effect on productive viral replication in monocytes and MDMs. So far, no consistent genetic linkage of HIV replication pattern with HLA-DR genotype has been observed.     

Combining data in phylogenetic analysis

Systematists have access to multiple sources of character information in phylogenetic analysis. For example, it is not unusual to have nucleotide sequences from several different genes, or to have molecular and morphological data. How should diverse data be analyzed in phylogenetic analysis? Several methods have been proposed for the treatment of partitioned data: the total evidence, separate analysis, and conditional combination approaches. Here, we review some of the advantages and disadvantages of the different approaches, with special concentration on which methods help us to discern the evolutionary process and provide the most accurate estimates of phylogeny.     

Preparative-scale purification of RNA using an efficient method which combines gel electrophoresis and column chromatography.

Here we describe a reliable method for purifying large amounts of RNA of any sequence and length with comparable efficiency and resolution of gel electrophoresis and with capacity approaching that of column chromatography. The RNA mixture of interest is separated on a cylindrical denaturing polyacrylamide gel, eluted by a peristaltic pump, detected by a UV-vis detector, and collected by a fraction collector. Using this method, we were able to separate one third of a 100 ml in vitro transcribed 34mer hammerhead ribozyme (approximately 6.2 mg) in a single run. The entire 100 ml transcribed RNA (approximately 18.5 mg) was separated after consecutive runs using one single gel preparation.     

Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments inE. coli

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.     

Effects of Motility and Adsorption Rate Coefficient on Transport of Bacteria through Saturated Porous Media

Three strains of Pseudomonas fluorescens with different motility rates and adsorption rate coefficients were injected into porous-medium reactors packed with l-mm-diameter glass spheres. Cell breakthrough, time to peak concentration, tailing, and cell recovery were measured at three interstitial pore velocities (higher than, lower than, and much lower than the maximal bacterial motility rate). All experiments were done with distilled water to reduce the effects of growth and chemotaxis. Contrary to expectations, motility did not result in either early breakthrough or early time to peak concentration at flow velocities below the motility rate. Bacterial size exclusion effects were shown to affect breakthrough curve shape at the very low flow velocity, but no such effect was seen at the higher flow velocity. The tendency of bacteria to adsorb to porous-medium surfaces, as measured by adsorption rate coefficients, profoundly influenced transport characteristics. Cell recoveries were shown to be correlated with the ratio of advective to adsorptive transport in the reactors. Adsorption rate coefficients were found to be better predictors of microbial transport phenomena than individual characteristics, such as size, motility, or porous-medium hydrodynamics.