Molecular Dynamics Simulations of PIP2 and PIP3 in Lipid Bilayers: Determination of Ring Orientation, and the Effects of Surface Roughness on a Poisson-Boltzmann Description

Molecular dynamics (MD) simulations of phosphatidylinositol (4,5)-bisphosphate (PIP₂) and phosphatidylinositol (3,4,5)-trisphosphate (PIP₃) in 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) bilayers indicate that the inositol rings are tilted ∼40° with respect to the bilayer surface, as compared with 17° for the P-N vector of POPC. Multiple minima were obtained for the ring twist (analogous to roll for an airplane). The phosphates at position 1 of PIP₂ and PIP₃ are within an Ångström of the plane formed by the phosphates of POPC; lipids in the surrounding shell are depressed by 0.5-0.8 Å, but otherwise the phosphoinositides do not substantially perturb the bilayer. Finite size artifacts for ion distributions are apparent for systems of ∼26 waters/lipid, but, based on simulations with a fourfold increase of the aqueous phase, the phosphoinositide positions and orientations do not show significant size effects. Electrostatic potentials evaluated from Poisson-Boltzmann (PB) calculations show a strong dependence of potential height and ring orientation, with the maxima on the −25 mV surfaces (17.1 ± 0.1 Å for PIP₂ and 19.4 ± 0.3 Å for PIP₃) occurring near the most populated orientations from MD. These surfaces are well above the background height of 10 Å estimated for negatively charged cell membranes, as would be expected for lipids involved in cellular signaling. PB calculations on microscopically flat bilayers yield similar maxima as the MD-based (microscopically rough) systems, but show less fine structure and do not clearly indicate the most probable regions. Electrostatic free energies of interaction with pentalysine are also similar for the rough and flat systems. These results support the utility of a rigid/flat bilayer model for PB-based studies of PIP₂ and PIP₃ as long as the orientations are judiciously chosen.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Growth of acetotrophic,methane-producing bacteria in a pH auxostat

Three acetotrophicMethanosarcina species, which included marine, nonmarine, and thermophilic strains, were grown on acetate in a 10-liter pH auxostat. Specific growth rates and molar growth yields were constant throughout growth. Cell yields were up to 18-fold greater than previously reported. These properties of the pH auxostat indicate that it is a preferred culture method for the biochemical study of methanogenesis from acetate.     

Molecular Identification of Aeromonas and Coliform Bacteria Isolated on m Endo Media from Lake Erie Waters

Summary Bacteria from recreational waters collected from two Lake Erie beaches in Dunkirk, New York were plated onto m Endo LES media. The 16S rRNA gene was then amplified from coliform and non-coliform bacteria using the polymerase chain reaction. The PCR products were characterized by restriction fragment length polymorphism (RFLP) analysis. A total of 8 RFLP groups were identified from the analysis of 920 samples and selected PCR products from each group were sequenced. The DNA sequence analysis indicated that more than half of the bacteria identified as coliforms on the m Endo plates belonged to the genus Aeromonas from the family Aeromonadaceae. Most of the remaining coliforms were from the Enterobacteriaceae. The data indicate that m Endo agar plates allow the growth of non-coliform bacteria, especially Aeromonas species.     

Multipotent Cancer Stem Cells Derived from Human Malignant Peritoneal Mesothelioma Promote Tumorigenesis

During the progression of malignant peritoneal mesothelioma (MPeM), tumor nodules propagate diffusely within the abdomen and tumors are characterized by distinct phenotypic sub-types. Recent studies in solid organ cancers have shown that cancer stem cells (CSCs) play a pivotal role in the initiation and progression of tumors. However, it is not known whether tumorigenic stem cells exist and whether they promote tumor growth in MPeM. In this study, we developed and characterized a CSC model for MPeM using stably expandable tumorigenic stem cells derived from patient tumors. We found morphologically distinct populations of CSCs that divide asymmetrically or symmetrically in MPeM in vitro cell culture. The MPeM stem cells (MPeMSCs) express stem cell markers c-MYC, NES and VEGFR2 and in the presence of matrix components cells form colony spheres. MPeMSCs are multipotent, differentiate into neuronal, vascular and adipose progeny upon defined induction and the differentiating cells express lineage-specific markers such as TUBB3, an early neuronal marker; vWF, VEGFA, VEGFC and IL-8, endothelial markers; and PPARγ and FABP4, adipose markers. Xenotransplantation experiments using MPeMSCs demonstrated early tumor growth compared with parental cells. Limiting dilution experiments using MPeMSCs and endothelial lineage-induced cells derived from a single MPeMSC resulted in early tumor growth in the latter group indicating that endothelial differentiation of MPeMSCs is important for MPeM tumor initiation. Our observation that the MPeM tumors contain stem cells with tumorigenic potential has important implications for understanding the cells of origin and tumor progression in MPeM and hence targeting CSCs may be a useful strategy to inhibit malignant progression.     

Measurement of pharyngeal cross-sectional area by finite element analysis.

A noninvasive measurement of pharyngeal cross-sectional area (CSA) during sleep would be advantageous for research studies. We hypothesized that CSA could be calculated from the measured pharyngeal pressure and flow by finite element analysis (FEA). The retropalatal airway was visualized by using a fiber-optic scope to obtain the measured CSA (mCSA). Flow was measured with a pneumotachometer, and pharyngeal pressure was measured with a pressure catheter at the palatal rim. FEA was performed as follows: by using a three-dimensional image of the upper airway, a mesh of finite elements was created. Specialized software was used to allow the simultaneous calculation of velocity and area for each element by using the measured pressure and flow. In the development phase, 677 simultaneous measurements of CSA, pressure, and flow from one subject during non-rapid eye movement (NREM) and rapid eye movement (REM) sleep were entered into the software to determine a series of equations, based on the continuity and momentum equations, that could calculate the CSA (cCSA). In the validation phase, the final equations were used to calculate the CSA from 1,767 simultaneous measurements of pressure and flow obtained during wakefulness, NREM, and REM sleep from 14 subjects. In both phases, mCSA and cCSA were compared by Bland-Altman analysis. For development breaths, the mean difference between mCSA and cCSA was 0.0 mm2 (95% CI, -0.1, 0.1 mm2). For NREM validation breaths, the mean difference between mCSA and cCSA was 1.1 mm2 (95% CI 1.3, 1.5 mm2). Pharyngeal CSA can be accurately calculated from measured pharyngeal pressure and flow by FEA.     

Identification of a gene, closely linked to dnaK, which is required for high-temperature growth of Escherichia coli.

We have constructed four deletion derivatives of the cloned dnaK gene. Plasmid pDD1, in which the last 10 amino acids of the DnaK protein have been replaced by three different amino acids derived from the pBR322 vector, was as effective as plasmid pKP31, from which it was derived, in restoring the ability of a dnaK null mutant, Escherichia coli BB1553, to plate lambda phage and to grow at high temperatures. The other three mutations, involving much larger deletions of the dnaK gene, did not restore the ability to plate lambda phage or the ability to grow at high temperatures. Plasmid pKUC2, which contains the whole dnaK gene and its promoters, was capable of restoring the ability of E. coli BB1553 to plate lambda phage but, surprisingly, it did not restore the ability to grow at high temperatures, even though it was shown that the DnaK protein was efficiently expressed in these cultures. By transposon mutagenesis and sub-cloning, we have shown the presence of a second gene in plasmid pKP31 which is required for high-temperature growth of E. coli BB1553. This gene, which we call htg A, is presumably also defective in the dnaK null mutant E. coli BB1553. We have also demonstrated that the inability of E. coli K756 to grow above 43.5 degrees C is complemented by sub-clones which contain the htg A gene, but not by plasmid pKUC2.