Mimicking exercise in vitro: effects of myotube contractions and mechanical stretch on omics.

The number of studies using skeletal muscle (SkM) cell culture models to study exercise in vitro are rapidly expanding. Progressively, more comprehensive analysis methods, such as different omics approaches including transcriptomics, proteomics, and metabolomics have been used to examine the intra- and extracellular molecular responses to exercise mimicking stimuli in cultured myotubes. Among other techniques, exercise-like electrical pulse stimulation (EL-EPS) and mechanical stretch of SkM cells are the two most commonly used methods to mimic exercise in vitro. In this mini-review, we focus on these two approaches and their effects on the omics of myotubes and/or cell culture media. Furthermore, besides traditional two-dimensional (2-D) methods, the use of three-dimensional (3-D) SkM approaches are increasing in the field of in vitro exercise mimicry. Our aim with this mini-review is to provide the reader with an up-to-date overview of the 2-D and 3-D models and the use of omics approaches to study the molecular response to exercise in vitro.

DNA methylation across the genome in aged human skeletal muscle tissue and muscle-derived cells: the role of HOX genes and physical activity.

Skeletal muscle tissue demonstrates global hypermethylation with age. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 h, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alterations in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P. Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and HDMCs and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

Pembrolizumab: Role of Modeling and Simulation in Bringing a Novel Immunotherapy to Patients With Melanoma.

Recently, immunotherapy has yielded promising results in several cancer types. Contrary to the established classical chemotherapy-dosing paradigm, a maximum tolerated dose approach does not always produce better clinical outcomes for novel targeted therapies, as their efficacy is frequently robust at pharmacologically active doses below the maximum tolerated dose. Integrated safety and efficacy assessments are needed to inform clinical dose and trial design, and to support an early identification of potentially safe and efficacious combination treatments.

Population Pharmacokinetic/Pharmacodynamic Modeling of Tumor Size Dynamics in Pembrolizumab-Treated Advanced Melanoma.

Pembrolizumab is a potent immune-modulating antibody active in advanced melanoma, as demonstrated in the KEYNOTE-001, -002, and -006 studies. Longitudinal tumor size modeling was pursued to quantify exposure-response relationships for efficacy. A mixture model was first developed based on an initial dataset from KEYNOTE-001 to describe four patterns of tumor growth and shrinkage. For subsequent analyses, tumor size measurements were adequately described by a single consolidated model structure that captured continuous tumor size with a combination of growth and regression terms, as well as a fraction of tumor responsive to therapy. This revised model structure provided a framework to efficiently evaluate the impact of covariates and pembrolizumab exposure. Both models indicated that exposure to the drug was not a significant predictor of tumor size response, demonstrating that the dose range evaluated (2 and 10 mg/kg every 3 weeks) is likely near or at the plateau of maximal response.

Translational Pharmacokinetic-Pharmacodynamic Modeling and Simulation: Optimizing 5-Fluorouracil Dosing in Children With Pediatric Ependymoma.

We previously investigated novel therapies for pediatric ependymoma and found 5-fluorouracil (5-FU) i.v. bolus increased survival in a representative mouse model. However, without a quantitative framework to derive clinical dosing recommendations, we devised a translational pharmacokinetic-pharmacodynamic (PK-PD) modeling and simulation approach. Results from our preclinical PK-PD model suggested tumor concentrations exceeded the 1-hour target exposure (in vitro IC90), leading to tumor growth delay and increased survival. Using an adult population PK model, we scaled our preclinical PK-PD model to children. To select a 5-FU dosage for our clinical trial in children with ependymoma, we simulated various 5-FU dosages for tumor exposures and tumor growth inhibition, as well as considering tolerability to bolus 5-FU administration. We developed a pediatric population PK model of bolus 5-FU and simulated tumor exposures for our patients. Simulations for tumor concentrations indicated that all patients would be above the 1-hour target exposure for antitumor effect.

Systematic evaluation of pembrolizumab dosing in patients with advanced non-small-cell lung cancer.

BACKGROUND: In the phase I KEYNOTE-001 study, pembrolizumab demonstrated durable antitumor activity in patients with advanced non-small-cell lung cancer (NSCLC). We sought to characterize the relationship between pembrolizumab dose, exposure, and response to define an effective dose for these patients. PATIENTS AND METHODS: Patients received pembrolizumab 2 mg/kg every 3 weeks (Q3W) (n = 55), 10 mg/kg Q3W (n = 238), or 10 mg/kg Q2W (n = 156). Response (RECIST v1.1) was assessed every 9 weeks. The relationship between the estimated pembrolizumab area under the concentration-time curve at steady state over 6 weeks (AUCss-6weeks) and the longitudinal change in tumor size (sum of longest diameters) was analyzed by regression and non-linear mixed effects modeling. This model was simultaneously fit to all tumor size data, then used to simulate response rates, normalizing the trial data across dose for prognostic covariates (tumor PD-L1 expression and EGFR mutation status). The exposure-safety relationship was assessed by logistic regression of pembrolizumab AUCss-6weeks versus occurrence of adverse events (AEs) of interest based on their immune etiology. RESULTS: Overall response rates were 15% [95% confidence interval (CI) 7%-28%] at 2 mg/kg Q3W, 25% (18%-33%) at 10 mg/kg Q3W, and 21% (95% CI 14%-30%) at 10 mg/kg Q2W. Regression analyses of percentage change from baseline in tumor size versus AUCss-6weeks indicated a flat relationship (regression slope P > 0.05). Simulations showed the exposure-response relationship to be similarly flat, thus indicating that the lowest evaluated dose of 2 mg/kg Q3W to likely be at or near the efficacy plateau. Exposure-safety analysis showed the AE incidence to be similar among the clinically tested doses. CONCLUSIONS: No significant exposure dependency on efficacy or safety was identified for pembrolizumab across doses of 2-10 mg/kg. These results support the use of a 2 mg/kg Q3W dosage in patients with previously treated, advanced NSCLC. CLINICALTRIALSGOV REGISTRY: NCT01295827.

Phase I dose escalation and pharmacokinetic study of oral gefitinib and irinotecan in children with refractory solid tumors.

PURPOSE: This phase I study endeavored to estimate the maximum tolerated dose and describe the dose-limiting toxicities (DLTs) of oral irinotecan with gefitinib in children with refractory solid tumors. METHODS: Oral irinotecan was administered on days 1-5 and 8-12 with oral gefitinib (fixed dose, 150 mg/m(2)/day) on days 1-12 of a 21-day course. The escalation with overdose control method guided irinotecan dose escalation (7 dose levels, range 5-40 mg/m(2)/day). RESULTS: Sixteen of 19 patients were evaluable, with serial pharmacokinetic studies in ten patients. Diagnoses included osteosarcoma (N = 5), neuroblastoma (N = 3), sarcoma (N = 3), and others (N = 5). Patients received a median of two courses (range 1-20), with at least two patients treated on dose levels 2-7. Three patients had five DLTs; the most common being metabolic (hypokalemia, N = 2 and hypophosphatemia, N = 1) at dose levels two (10 mg/m(2)) and four (20 mg/m(2)). One patient experienced grade 3 diarrhea (40 mg/m(2)). Irinotecan bioavailability was 2.5-fold higher when co-administered with gefitinib, while the conversion rate of irinotecan to SN-38 lactone was unaffected. The study closed due to poor accrual before evaluation of the next recommended irinotecan dose level (35 mg/m(2)). Of 11 patients receiving at least two courses of therapy, three had stable disease lasting two to four courses and one patient maintained a complete response through 18 courses. CONCLUSIONS: The combination of oral gefitinib and irinotecan has acceptable toxicity and anti-tumor activity in pediatric patients with refractory solid tumors. Pharmacokinetic analysis confirms that co-administration of gefitinib increases irinotecan bioavailability leading to an increased SN-38 lactone systemic exposure.

Deriving therapies for children with primary CNS tumors using pharmacokinetic modeling and simulation of cerebral microdialysis data.

The treatment of children with primary central nervous system (CNS) tumors continues to be a challenge despite recent advances in technology and diagnostics. In this overview, we describe our approach for identifying and evaluating active anticancer drugs through a process that enables rational translation from the lab to the clinic. The preclinical approach we discuss uses tumor subgroup-specific models of pediatric CNS tumors, cerebral microdialysis sampling of tumor extracellular fluid (tECF), and pharmacokinetic modeling and simulation to overcome challenges that currently hinder researchers in this field. This approach involves performing extensive systemic (plasma) and target site (CNS tumor) pharmacokinetic studies. Pharmacokinetic modeling and simulation of the data derived from these studies are then used to inform future decisions regarding drug administration, including dosage and schedule. Here, we also present how our approach was used to examine two FDA approved drugs, simvastatin and pemetrexed, as candidates for new therapies for pediatric CNS tumors. We determined that due to unfavorable pharmacokinetic characteristics and insufficient concentrations in tumor tissue in a mouse model of ependymoma, simvastatin would not be efficacious in further preclinical trials. In contrast to simvastatin, pemetrexed was advanced to preclinical efficacy studies after our studies determined that plasma exposures were similar to those in humans treated at similar tolerable dosages and adequate unbound concentrations were found in tumor tissue of medulloblastoma-bearing mice. Generally speaking, the high clinical failure rates for CNS drug candidates can be partially explained by the fact that therapies are often moved into clinical trials without extensive and rational preclinical studies to optimize the transition. Our approach addresses this limitation by using pharmacokinetic and pharmacodynamic modeling of data generated from appropriate in vivo models to support the rational testing and usage of innovative therapies in children with CNS tumors.

A comparison of attitudes towards animals between the German- and French-speaking part of Switzerland.

A comparison of attitudes towards animals between German- and French-speaking Swiss adults is of particular interest, given the often invoked cultural barrier, the <>. We sent questionnaires to 3000 randomly chosen Swiss adults in both language regions. 319 German and 293 French questionnaires were returned. Participants had to express their opinion regarding 29 statements on nature conservation, wild animals, farm animals, meat eating, animal feelings and cognition, and pets. In 19 items we found a significant difference in responses between the German- and the French-speaking participants. It is important to note that the direction of the responses was identical in all cases, the only difference being the degree of agreement. In general, the Swiss agreed that nature conservation is important. They agreed also that animals have feelings, but that these are different from the feelings of humans. Pets were viewed as beneficial to humans. Both cats and dogs were seen as likeable animals, and there was agreement that dogs need more time to care for than cats. Strays were not viewed as a problem in Switzerland, despite the fact that there are numerous stray cat colonies.

An analysis of the relationship between fatty acid composition and the lamellar gel to liquid-crystalline and the lamellar to inverted nonlamellar phase transition temperatures of phosphatidylethanolamines and diacyl-alpha-D-glucosyl glycerols.

The lamellar gel to lamellar liquid-crystalline (Lbeta/Lalpha) and lamellar liquid-crystalline to inverted hexagonal (Lalpha/H(II)) phase transitions of a number of phosphatidylethanolamines (PEs) and diacyl-alpha-D-glucosyl-sn-glycerols (alpha-D-GlcDAGs) containing linear saturated, linear unsaturated, branched or alicyclic hydrocarbon chains of various lengths were examined by differential scanning calorimetry and low-angle X-ray diffraction. As reported previously, for each homologous series of PEs or alpha-D-GlcDAGs, the Lbeta/Lalpha phase transition temperatures (Tm) increase and the Lalpha/H(II) phase transition temperatures (Th) decrease with increases in hydrocarbon chain length. The Tm and the especially the Th values for the PEs are higher than those of the corresponding alpha-D-GlcDAGs. For PEs having the same effective hydrocarbon chain length but different chain configurations, the Tm and Th values vary markedly but with an almost constant temperature interval (deltaT(L/NL)) between the two phase transitions. Moreover, although the Tm and Th values of the PEs and alpha-D-GlcDAGs are equally sensitive on the temperature scale to variations in the length and chemical configuration of the hydrocarbon chains, the deltaT(L/NL) values are generally larger in the PEs and vary less with the hydrocarbon chain structure. This suggests that the PE headgroup has a greater ability to counteract variations in the packing properties of different hydrocarbon chain structures than does the alpha-D-GlcDAG headgroup. With decreasing chain length, this ability of the PE headgroup to counteract the hydrocarbon chain packing properties increases, significantly expanding the temperature interval over which the Lalpha phase is stable relative to the corresponding regions in the alpha-D-GlcDAGs. Overall, these findings indicate that the PEs have a smaller propensity to form the H(II) phase than do the alpha-D-GlcDAGs with an identical fatty acid composition. In contrast to our previous report, there is some variation in the d-spacings of these various PEs (and alpha-D-GlcDAGs) in both the Lalpha and H(II) phases when the hydrocarbon chain structure is changed while the effective chain length is kept constant. These hydrocarbon chain structural modifications produce different d-spacings in the Lalpha and H(II) phases, but those changes are consistent between the PEs and alpha-D-GlcDAGs, probably reflecting differences in the hydrocarbon chain packing constraints in these two phases. Overall, our experimental observations can be rationalized to a first approximation by a simple lateral stress model in which the primary bilayer strain results from a mismatch between the actual and optimal headgroup areas and the primary strain in the H(II) phase arises from a simple hydrocarbon chain packing term.

Diffusion and transfer of antibody proteins from a sugar-based hydrogel.

Diffusion of antibody protein from hydrogel films and hydrogel encapsulated in a microcapillary was studied. Thin hydrogel films were formed by crosslinking 6-acryloyl-B-O-methylgalactoside with N,N'-methylene-bis-acrylamide and the diffusive transport of monoclonal antimouse IgG-FITC into and out of the hydrate films was measured. Diffusion coefficients in 2 and 4% crosslinked hydrogel films were measured. The measured diffusion constants determined for IgG in both the 2 and 4% hydrogel films were comparable to the free diffusion of IgG in bulk water (Dmean approximately 10(-7) cm2/s). In addition, 2% crosslinked hydrogels were prepared in a capillary tube and the transport of antimouse IgG-FITC into and out of the hydrated hydrogel was measured. Kinetic analysis indicated that the protein transport through the capillary hydrogel was faster than would be expected for a simple diffusion process. Finally, by utilizing the diffusion of antibody from the capillary hydrogel, transfer of antibody to a silica surface was demonstrated. A capillary hydrogel loaded with antimouse IgG-FITC was used to transfer the protein to a silica surface forming a 30-micron spot of antibody, which was imaged using fluorescence microscopy. These results may lead to the development of a nonlithographic method of patterning antibodies on surfaces for use in integrated microimmunosensors.

Colon-cancer cell variants producing regressive tumors in syngeneic rats, unlike variants yielding progressive tumors, attach to interstitial collagens through integrin alpha2beta1.

Nine clones of tumor cells, derived from a single rat colon carcinoma, were analyzed for their adhesive properties and in vivo growth patterns. Four clones (denoted REG) gave rise to regressively growing tumors. Cells from the 4 REG clones attached significantly better to collagen types I and III than did cells from the 5 clones (denoted PRO) which grew progressively in vivo. In contrast, REG and PRO clones did not differ in their attachment to collagen type IV, laminin or fibronectin. The attachment of REG cells to collagen was dependent on Mg2+, but not Ca2+. Monospecific rabbit IgG to rat integrin beta 1-chain inhibited REG cell attachment to collagen, demonstrating involvement of a beta 1 integrin in this process. PRO and REG cells expressed an underglycosylated beta 1 chain (Mr approximately 105,000) that was somewhat smaller than beta 1-chains described previously on rat fibroblasts and hepatocytes (Mr approximately 115,000). Monoclonal IgG to rat integrin alpha 2 beta 1, but not to alpha 1 beta 1, readily inhibited REG cell attachment to collagen, demonstrating the involvement of integrin alpha 2 beta 1. However, beta 1 and alpha 2 integrin subunits were found in purified glycoproteins from both PRO and REG cells. This suggests that alpha 2 beta 1 integrin is expressed by both cell variants, but is functional as a collagen receptor on REG cells only. In this system of tumor-cell variants, the clear-cut differences in attachment to interstitial collagens of the 9 clones suggest a possible relationship between this attachment and the capacity to induce progressive or regressive tumors.

Selective adhesion of functional microtubules to patterned silane surfaces.

We show that microtubule polymers can be immobilized selectively on lithographically patterned silane surfaces while retaining their native properties. Silane films were chemisorbed on polished silicon wafers or glass coverslips and patterned using a deep UV lithographic process developed at the Naval Research Laboratory. Hydrocarbon and fluorocarbon alkyl silanes, as well as amino and thiol terminal alkyl silanes, were investigated as substrates for microtubule adhesion with retention of biological activity. Microtubules were found to adhere strongly to amine terminal silanes while retaining the ability to act as substrates for the molecular motor protein kinesin. Aminosilane patterns with linewidths varying from 1 to 50 microns were produced lithographically and used to produce patterns of selectively adhered microtubules. Microtubules were partially aligned on the patterned lines by performing the immobilization in a fluid flow field. Patterns were imaged with atomic force microscopy and differential interference contrast microscopy. Motility assays were carried out using kinesin-coated beads and observed with differential interference contrast microscopy. Kinesin bead movement on the patterned microtubules was comparable to movement on microtubule control surfaces.

Thermodynamics of interaction of the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly with dioleoylphosphatidylcholine vesicles: direct calorimetric determination.

The binding of the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly to unilamellar lipid vesicles of dioleoylphosphatidylcholine (DOPC) was investigated by isothermal titration calorimetry (ITC). The peptide Z-D-Phe-L-Phe-Gly is known to inhibit fusion of myxo- and paramyxoviruses with cells as well as cell-cell and vesicle-vesicle fusion in model systems. Calorimetric titrations conducted over a range of temperatures permitted characterization of the thermodynamics of the interaction of Z-D-Phe-L-Phe-Gly with model DOPC lipid membranes. Simultaneous global analysis of 15 ITC binding curves acquired at four different temperatures allowed determination of the equilibrium site association constant (K), stoichiometry of binding (n), binding enthalpy change (delta H), and heat capacity change of binding (delta Cp) in a single set of experiments. The binding affinity and enthalpy change per mole of DOPC bound at 25 degrees C was log K = 2.463 +/- 0.075 and delta H = -1.07 +/- 0.12 kcal/mol DOPC while the binding heat capacity change per mole of DOPC bound was delta Cp = -20.3 +/- 2.8 cal/(K.mol DOPC) with a temperature dependence (from 10-45 degrees C) of d(delta Cp)/dT = 0.37 +/- 0.18 cal/(K2.mol DOPC). A temperature-independent binding stoichiometry was determined to be n = 5.56 +/- 0.33 DOPC molecules per Z-D-Phe-L-Phe-Gly. A comparison of these results with previous peptide-lipid binding studies is discussed as is their relevance to a current model of the interaction of fusion-inhibiting peptides with phospholipid membranes.

[Ethology and good health in pets]. / Ethologie und Wohlbefinden bei Heimtieren.

Ethology, or the comparative study of behaviour and its biological roots, has made significant contributions to our understanding of companion animal behaviour and thereby, the welfare of these animals. Ethological questions, concepts and methods can be readily applied to companion animal studies and the interpretation of results. In particular, comparison of behaviour shown in a 'reference system' with that exhibited in more restricted environments has proven very useful. Although emphasis has been placed on farm animals in the past, more and more animal protection laws require that regulations and guidelines, including those concerning companion animals, be based upon sound ethological studies. It is only through such observations that we can objectively determine what 'species-typical' behaviour is and what the animal's 'needs' are. Based upon these, recommendations for proper housing conditions can be made, which in turn reduce the number of cases where behavioural disturbances tarnish the relationship between humans and their companion animals. Using past research on the domestic cat and human-cat interactions, the author illustrates these points and makes suggestions for future research.

Impairment of integrin-mediated cell-matrix adhesion in oxidant-stressed PC12 cells.

Oxidants are believed to play an important and complex role in neuronal injury and death in the aging process and various neurode generative diseases. We studied the effect of oxidative stress on integrin-mediated cell-extracellular matrix (ECM) interactions using the PC12 neuronal cell line. In assays in which attachment was measured between 30 and 90 min, addition of hydrogen peroxide (H2O2) to the attachment medium resulted in a dose-dependent inhibition of initial cell attachment to collagen. Addition of H2O2 also caused previously attached cells to detach from collagen. The inhibition by H2O2 was specific for integrin-mediated adhesion, since attachment to substrata coated with non-ECM molecules was much less affected. Exposure of cells to H2O2 resulted in a rapid and profound reduction of intracellular ATP, accompanied by only a slight increase in intracellular free Ca2+ concentration ([Ca2+]i). Treatment of cells with the microfilament-disrupting agent, cytochalasin B, like that with H2O2, inhibited cell adhesion to collagen. We propose that integrin-mediated cell adhesion, which requires interactions between cytoplasmic portions of integrin subunits and cytoskeletal microfilaments, is impaired by oxidative stress as a result of the depletion of intracellular ATP and that such depletion is an early event in the process of oxidant-induced neuronal injury.

Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

Studies of the thermotropic phase behavior of phosphatidylcholines containing 2-alkyl substituted fatty acyl chains: a new class of phosphatidylcholines forming inverted nonlamellar phases.

We have synthesized a number of 1,2-diacyl phosphatidylcholines with hydrophobic substituents adjacent to the carbonyl group of the fatty acyl chain and studied their thermotropic phase behavior by differential scanning calorimetry, 31P-nuclear magnetic resonance spectroscopy, and x-ray diffraction. Our results indicate that the hydrocarbon chain-melting phase transition temperatures of these lipids are lower than those of the n-saturated diacylphosphatidylcholines of similar chain length. In the gel phase, the 2-alkyl substituents on the fatty acyl chains seem to inhibit the formation of tightly packed, partially dehydrated, quasi-crystalline bilayers (Lc phases), although possibly promoting the formation of chain-interdigitated bilayers. In the liquid-crystalline state, however, these 2-alkyl substituents destabilize the lamellar phase with respect to one or more inverted nonlamellar structures. In general, increases in the length, bulk, or rigidity of the alkyl substituent result in an increased destabilization of the lamellar gel and liquid-crystalline phases and a greater tendency to form inverted nonlamellar phases, the nature of which depends upon the size of the 2-alkyl substituent. Unlike normal non-lamella-forming lipids such as the phosphatidylethanolamines, increases in the length of the main acyl chain stabilize the lamellar phases and reduce the tendency to form nonlamellar structures. Our results establish that with a judicious choice of a 2-alkyl substituent and hydrocarbon chain length, phosphatidylcholines (and probably most other so-called "bilayer-preferring" lipids) can be induced to form a range of inverted nonlamellar structures at relatively low temperatures. The ability to vary the lamellar/nonlamellar phase preference of such lipids should be useful in studies of bilayer/nonbilayer phase transitions and of the molecular organization of various nonlamellar phases. Moreover, because the nonlamellar phases can easily be induced at physiologically relevant temperatures and hydration levels while avoiding changes in polar headgroup composition, this new class of 2-alkyl-substituted phosphatidylcholines should prove valuable in studies of the physiological role of non-lamella-forming lipids in reconstituted lipid-protein model membranes.

Expression of integrin alpha 1 beta 1 is regulated by nerve growth factor and dexamethasone in PC12 cells. Functional consequences for adhesion and neurite outgrowth.

PC12 cells respond to nerve growth factor by differentiating into sympathetic neuron-like cells that employ the integrin alpha 1 beta 1 to attach to, and extend neurites on, substrata coated with collagen or laminin. In one PC12 subline, PC12i, prolonged treatment with nerve growth factor results in a marked increase in synthesis of alpha 1 subunits and in the level of alpha 1 mRNA, with a corresponding increase in alpha 1 beta 1 expressed on the cell surface. These changes are accompanied by substantial increases in initial cell attachment to collagen and in the fraction of neurite-bearing cells and average neurite length. Integrin beta 1-subunits are constitutively expressed, so that alpha 1 synthesis controls the amount of alpha 1 beta 1 heterodimer expressed on PC12i cells. Acidic fibroblast growth factor also induces alpha 1 beta 1 in PC12i cells, with consequent enhancement of neurite outgrowth; treatment with epidermal growth factor or dibutyryl cyclic AMP does not have these effects. Another subline, PC12c, expresses high levels of alpha 1 mRNA and alpha 1 protein constitutively. With or without nerve growth factor pretreatment, these cells adhere well to collagen and a majority extend neurites when replated in the presence of nerve growth factor. Dexamethasone treatment of PC12c cells reduces expression of alpha 1 mRNA and alpha 1 protein, with consequent reduction in attachment to collagen. In both sublines, then, there is a direct relationship between the level of a specific matrix receptor and cell-matrix adhesion. Moreover, our results suggest that induced expression of this matrix receptor is an essential aspect of the regulation of neurite extension by nerve growth factor in PC12i cells.