Phosphoinositides alter lipid bilayer properties

Phosphatidylinositol-4,5-bisphosphate (PIP₂), which constitutes ∼1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP₂ regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid–protein interactions, but the mechanisms by which PIP₂ regulates many of its various targets remain to be fully elucidated. Because many PIP₂ targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states—and thereby protein function. Taking advantage of the gramicidin A (gA) channels’ sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP₂s (brain PIP₂, which is predominantly 1-stearyl-2-arachidonyl-PIP₂, and dioleoyl-PIP₂) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP₂s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP₂ was a more potent modifier than dioleoyl-PIP₂. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC₈-phosphoinositol (PI), PI(4,5)P₂, PI(3,5)P₂, PI(3,4)P₂, and PI(3,4,5)P₃. The diC₈ phosphoinositides, except for PI(3,5)P₂, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC₈ phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP₂ levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP₂, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments.     

Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

ABSTRACT: BACKGROUND: Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. RESULTS: Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse[trade mark sign]). CONCLUSIONS: A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.     

Differential expression of LacdiNAc sequences (GalNAc{beta}1-4GlcNAc-R) in glycoproteins synthesized by Chinese hamster ovary and human 293 cells

The lacdiNAc sequence GalNAcß1     

Identification of screwworm species by polymerase chain reaction-restriction fragment length polymorphism

Abstract. Restriction fragment length polymorphisms in polymerase chain reaction amplified fragments (PCR-RFLP) of mitochondrial DNA were used to differentiate species of New World screwworms (Diptera: Calliphoridae). Twenty-seven restriction enzymes were screened on five regions of mtDNA. Eleven restriction fragment length patterns differentiated New World screwworm, Cochliomyia hominivorax (Coquerel), from secondary screwworm, Cochliomyia macellaria (R). Five restriction fragment length patterns were polymorphic in C. hominivorax while all fragment patterns were fixed in C. macellaria. Diagnostic restriction fragment length patterns were used for species diagnosis, whereas intraspecific variable patterns were used to characterize field samples and laboratory strains. The PCR-RFLP technique is flexible with regard to developmental stage of the sample and method of preservation. We were able to characterize specimens of all life stages from egg to adult including larvae preserved in alcohol and pinned adults. PCR-RFLP is rapid and inexpensive, enabling specimens to be characterized within 24 h for less than 2.50.     

Classification of the Environment of Protein Residues

We have studied the classification of the environment of residues within protein structures. Eisenberg's original idea created environmental categories to discriminate between similar residues [Bowie et al., Science (1991), 253, 164–170]. These environments grouped residues based upon their buried surface area, polarity of the surrounding environment, and secondary structure element in which the residue is found. However, Eisenberg's original categories led to incomplete discrimination between residues that only partially substitute for each other. We have expanded on Eisenberg's original idea of environmental categories, by both considering additional contacts in the calculation of the solvent-accessible molecular surface area and by subdividing the environmental plot into regions based upon its theoretical features. Our alternative surface area calculations were used in conjunction with the polarity of the environment of the residue to define a new set of environmental categories. These new categories were able to discriminate between residues such as threonine, valine, and aspartic acid while reflecting the propensity of these residues to substitute for each other.