Cholesterol-transfer protein located in the intestinal brush-border membrane. Partial purification and characterization

Cholesterol absorption by small intestinal brush border membrane vesicles from taurocholate mixed micelles is a second-order reaction. From a comparison of reaction rates and order before and after proteinase K treatment of brush-border membrane vesicles, it is concluded that cholesterol absorption is protein-mediated. It is shown that the desorption of cholesterol from taurocholate mixed micelles is by a factor of about 10(4) faster than that from egg phosphatidylcholine bilayers. When brush border membrane vesicles are stored at room temperature, intrinsic proteinases are activated and proteins are liberated from the brush border membrane. These proteins collected in the supernatant catalyze cholesterol and phosphatidylcholine exchange between two populations of small unilamellar phospholipid vesicles. One of the active proteins present in the supernatant is purified by a two-step procedure involving gel filtration on Sephadex G-75 SF and affinity chromatography on a Nucleosil-phosphatidylcholine column. The protein thus obtained is pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has an apparent molecular weight of slightly less than 14,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a value of 11,500 determined by gel filtration on Sephadex G-75 SF.     

Direct Measurement of the Cortical Tension during the Growth of Membrane Blebs

Mechanics is at the heart of many cellular processes and its importance has received considerable attention during the last two decades. In particular, the tension of cell membranes, and more specifically of the cell cortex, is a key parameter that determines the mechanical behavior of the cell periphery. However, the measurement of tension remains challenging due to its dynamic nature. Here we show that a noninvasive interferometric technique can reveal time-resolved effective tension measurements by a high-accuracy determination of edge fluctuations in expanding cell blebs of filamin-deficient melanoma cells. The introduced technique shows that the bleb tension is ∼10–100 pN/μm and increases during bleb growth. Our results directly confirm that the subsequent stop of bleb growth is induced by an increase of measured tension, possibly mediated by the repolymerized actin cytoskeleton.     

Amino acid sequences of a-factor mating peptides from Saccharomyces cerevisiae

The molecular structure of a-factor, the mating hormone produced by mating type a cells of Saccharomyces cerevisiae, has been investigated. In culture filtrates of a cells four oligopeptides (a1 to a4) exhibiting a-factor activity have been found. These peptides have been isolated and their amino acid sequences have been determined. The a-factor peptides comprise two (apparently identical) pairs, a1/a2 and a3/a4, which differ in an interchange at position 6 of a valine in a1/a2 for a leucine in a3/a4. a1 and a4, which can be obtained by oxidation with H2O2 of purified a2 and a3, respectively, obviously represent oxidation artifacts formed under the conditions of culture. The amino acid sequences determined for the a-factor peptides are Tyr-Ile-Ile-Lys-Gly-Val Leu-Phe-Trp-Asp-Pro-Ala-Cys. Several lines of evidence suggest that the carboxyl-terminal cysteine residue is S-alkylated by a hydrophobic moiety.     

Messenger RNA-controlled Increase of Phenylalanine Ammonia-Lyase Activity in Parsley: Light-Independent Induction by Dilution of Cell Suspension Cultures into Water

A specific antiserum, raised against purified phenylalanine ammonialyase from irradiated cell suspension cultures of parsley (Petroselinum hortense Hoffm.), was used to compare the enzyme species induced either by dilution or by irradiation of the cell suspensions, to investigate the effect of dilution on the rate of synthesis of the enzyme protein in vivo, and to analyze the changes in specific activity of polyribosomal mRNA for the enzyme subunits in vitro. The mRNA activity in vitro was measured by translation of the polyribosomal RNA in a rabbit reticulocyte lysate.     

Separation and subtyping of planarian neoblasts by density-gradient centrifugation and staining

A method has been devised to isolate neoblasts from planarians in high purity and high yield. Specimens of Dugesia polychroa and Dugesia tahitiensis were disintegrated mechanically. After several prepurification steps consisting in sequential filtering through increasingly fine meshes and differential centrifugation, the resulting cell suspension was separated by centrifugation in discontinuous density gradients formed from Percoll solutions. Isosmotic conditions were applied. Two gradients are recommended, one isopycnic four-step gradient (densities 1.03, 1.05, 1.07, 1.09) to obtain one single fraction with a high yield of neoblasts in high purity and one six-step gradient for preparing subfractions of neoblasts somewhat less pure, but in still greater amounts. This latter gradient (densities 1.04, 1.05, 1.06, 1.065, 1.07, 1.09) was run under conditions of rate zonal centrifugation and used for further subtyping of neoblasts by specific staining with azure A – eosin B. A first survey of tentative types based on morphological criteria is given. The viability of neoblasts isolated in the way described was tested in primary cell cultures. In current experiments, 6-week-old cultures had a viability of roughly 50%, with mitoses up to 1 week after the isolation of neoblasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

Circulating surfactant protein D as a potential lung-specific biomarker of health outcomes in COPD: a pilot study

Background

There is a paucity of surrogate lung-specific biological markers that can be used to track disease progression and predict clinical outcomes in chronic obstructive pulmonary disease (COPD). The principal aim of this pilot study was to determine whether circulating surfactant protein D (SPD) or Clara Cell protein-16 (CC16) levels are associated with lung function or health status in patients with severe COPD.

Methods

We studied 23 patients with advanced COPD. Lung function measurements, Chronic Respiratory Disease Questionnaire (CRQ) scores, and serum levels of SPD, CC16, and C-reactive protein (CRP) were determined at baseline and at 3 months.

Results

At baseline, FEV₁ was inversely associated with serum SPD levels (P = 0.045) but not with CC16 (P = 0.675) or CRP levels (P = 0.549). Over a 3 month period, changes in SPD levels correlated significantly with changes in CRQ scores (adjusted P = 0.008) such that patients who had the largest declines in serum SPD levels experienced the largest gains in health status. The association was particularly notable between circulating SPD level and the dyspnea domain of the CRQ score (P = 0.018). Changes in CC16 or CRP levels did not correlate with changes in CRQ scores.

Conclusion

Changes in serum SPD levels tracked well with changes in health status over a 3 month period in patients with severe COPD. These data suggest that circulating SPD levels may be useful biomarkers to track health outcomes of COPD patients.     

Genomics: success or failure to deliver drug targets?

For decades, the entire pharmaceutical industry has focused on a limited number of drug targets. Owing to advances in molecular biology and genome technology at the beginning of the 1990s, discovery and isolation of a large number of genes from the human genome became feasible. This triggered a multi billion US dollars investment by both biotechnology and pharmaceutical companies to gain access to and patent as many potential drug targets as possible. Although the combined effort of publicly funded projects and private investments resulted in rapid identification of essentially all genes of the human genome, harnessing this information to enable drug discovery has turned out to be more challenging and time consuming than initially anticipated.     

Expression,purification, and characterization of subunit E,an essential subunit of the vacuolar ATPase

A recombinant form of subunit E (Vma4p) from yeast vacuolar ATPases (V-ATPases) has been overexpressed in Escherichia coli, purified to homogeneity, and explored by mass spectrometry. Analysis of the secondary structure of Vma4p by circular dichroism spectroscopy indicated 32% alpha-helix and 23% beta-sheet content. Vma4p formed a hybrid-complex with the nucleotide-binding subunits alpha and beta of the closely related F(1) ATPase of the thermophilic bacterium PS3 (TF(1)). The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF(1). By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF(1) ATPase activity. This is the first demonstration of a hydrolytically active hybrid-complex consisting of F(1) and V(1) subunits. The arrangement of subunit E in V(1) has been probed using the recombinant Vma4p, the alpha(3)beta(3)E-hybrid-complex together with V(1) and an A(3)B(3)HEG-subcomplex of the V(1) ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V(1).