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A common benchmark in the brain tissue mechanics literature is that the properties of acute brain slices should be measured within 8 h of the experimental animal being sacrificed. The core assumption is that—since there is no substantial protein degradation during this time—there will be no change to elastic modulus. This assumption overlooks the possibility of other effects (such as osmotic swelling) that may influence the mechanical properties of the tissue. To achieve consistent and accurate analysis of brain mechanics, it is important to account for or mitigate these effects. Using atomic force microscopy (AFM), tissue hydration and volume measurements, we find that acute brain slices in oxygenated artificial cerebrospinal fluid (aCSF) with a standard osmolarity of 300 mOsm/l experience rapid swelling, softening, and increases in hydration within the first 2 hours after slicing. Reductions in elastic modulus can be partly mitigated by addition of chondroitinase ABC enzyme (CHABC). Increasing aCSF osmolarity to 400 mOsm/l does not prevent softening but may hasten equilibration of samples to a point where measurements of relative elastic modulus are consistent across experiments.

Examines the careers of Carl and Gerty Cori, and the other eminent scientists who trained in their laboratory.

Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.

Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of α from βγ subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of Gβγ. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked Gβγ stimulation of adenylyl cyclase 2, phospholipase C-β3, potassium channels, and β-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between Gβγ and G$\\alpha_o$. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and β-adrenergic receptor kinases that participate in Gβγ interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from Gβγ.

The ability of the Ras oncogene to transform normal cells has been well established. One downstream effector of Ras is the lipid hydrolyzing enzyme phospholipase D. Recent evidence has emerged indicating a role for phospholipase D in cell proliferation, membrane trafficking, and migration. To study the potential importance of phospholipase D in the oncogenic ability of Ras, we used Rat-2 fibroblasts with reduced phospholipase D1 activity (Rat-2V25). Here, we show that H-Ras transformation of Rat-2 fibroblasts requires normal phospholipase D1 activity. WT Rat-2 fibroblasts transfected with the H- RasV12oncogene grew colonies in soft agar and tumors in nude mice. However, Rat-2V25 cells when transfected with the H- RasV12oncogene did not form colonies in soft agar or produce tumors when xenografted onto nude mice. Interestingly, in the presence of phosphatidic acid, the product of phospholipase D, growth in soft agar and tumor formation was restored. We also observed a dramatic increase in the expression of phospholipase D1 in colorectal tumors when compared with adjacent normal mucosa. Our studies identify phospholipase D1 as a critical downstream mediator of H-Ras-induced tumor formation.

PTEN, a tumor suppressor phosphatase, is important in the regulation of cell migration and invasion. Physiological regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) by cell surface receptors has not been described. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P), which acts through the S1P2 receptor (S1P2R) G protein-coupled receptor (GPCR) to inhibit cell migration, utilizes PTEN as a signaling intermediate. S1P2R inhibition of cell migration is abrogated by dominant-negative PTEN expression. S1P was unable to efficiently inhibit the migration of PtenΔ loxP/Δ loxPmouse embryonic fibroblasts; however, the antimigratory effect was restored upon the expression of PTEN. S1P2R activation of Rho GTPase is not affected in PtenΔ loxP/Δ loxPcells, and dominant-negative Rho GTPase reversed S1P inhibition of cell migration in WT cells but not in PtenΔ loxP/Δ loxPcells, suggesting that PTEN acts downstream of the Rho GTPase. Ligand activation of the S1P2R receptor stimulated the coimmunoprecipitation of S1P2R and PTEN. Interestingly, S1P2R signaling increased PTEN phosphatase activity in membrane fractions. Furthermore, tyrosine phosphorylation of PTEN was stimulated by S1P2R signaling. These data suggest that the S1P2R receptor actively regulates the PTEN phosphatase by a Rho GTPase-dependent pathway to inhibit cell migration. GPCR regulation of PTEN maybe a general mechanism in signaling events of cell migration and invasion.

To study the physiological function of diacylglycerol (DAG) kinase ι (DGKι), which converts DAG to phosphatidic acid, we deleted this gene in mice. In contrast to previous studies showing that DGK isoforms decrease Ras activity, signaling downstream of Ras in embryonic fibroblasts was significantly reduced in cells lacking DGKι. DGKs regulate Ras signaling by attenuating the function of the DAG-dependent Ras guanyl nucleotide-releasing proteins (RasGRPs). We tested whether DGKι inhibited the four known RasGRPs and found that it inhibited only RasGRP3. In addition to activating Ras, RasGRP3 also activates Rap1, which in some cases can antagonize the function of Ras. We demonstrate that DGKι bound to RasGRP3 and inhibited its activation of Rap1 by metabolizing DAG. This inhibition consequently affected Ras signaling. We tested the physiological consequence of deleting DGKι by crossing wild-type or DGKι-deficient mice with mice carrying a v-Ha-Ras transgene, and then we assessed tumor formation. We observed significantly fewer tumors in DGKι-deficient mice. Because Rap1 can antagonize the function of Ras, our data are consistent with a model in which DGKι regulates RasGRP3 with a predominant effect on Rap1 activity. Additionally, we found that DGKζ, which is structurally similar to DGKι, inhibited RasGRPs 1, 3, and 4 and predominantly affected Ras signaling. Thus, type IV DGKs regulate RasGRPs, but the downstream effects differ depending on the DGK.

Phosphatidate-dependent protein phosphorylation was observed in soluble extracts from rat liver, brain, lung, and testis. The phosphorylation was stimulated by free Ca2+in the range of 360-800 nM. Incubation mixtures containing phosphatidate provided markedly different profiles of protein phosphorylation from those with phosphatidylserine plus 1,2-diolein. Phosphatidate-dependent phosphorylation of a 30-kDa protein in the soluble fraction from heart was also observed. This phosphorylation did not require Ca2+. Soluble fractions from liver, testis, brain, and lung phosphorylated the 30-kDa heart protein in a phosphatidate-dependent Ca2+-independent manner. We propose that part of the action of phosphatidate in cells may be mediated by a protein kinase(s).