Control of motile and invasive cell phenotypes by focal adhesion kinase

Cell motility is stimulated by extracellular stimuli and initiated by intracellular signaling proteins that localize to sites of cell contact with the extracellular matrix termed focal contacts. Focal adhesion kinase (FAK) is an intracellular protein-tyrosine kinase (PTK) that acts to regulate the cycle of focal contact formation and disassembly required for efficient cell movement. FAK is activated by a variety of cell surface receptors and transmits signals to a range of targets. Thus, FAK acts as an integrator of cell motility-associated signaling events. We will review the stimulatory and regulatory mechanisms of FAK activation, the different signaling connections of FAK that are mediated by a growing number of FAK-interacting proteins, and the modulation of FAK function by tyrosine and serine phosphorylation. We will also summarize findings with regard to FAK function in vertebrate and invertebrate development as well as recent insights into the mechanistic role(s) of FAK in promoting cell migration. As increased FAK expression and tyrosine phosphorylation have been correlated with the progression to an invasive cell phenotype, there is growing interest in elucidating the important FAK-related signaling connections promoting invasive tumor cell movement. To this end, we will discuss the effects of FAK inhibition via the dominant-negative expression of the FAK C-terminal domain termed FAK-related non-kinase (FRNK) and how these studies have uncovered a distinct role for FAK in promoting cell invasion that may differ from its role in promoting cell motility.     

RNAi-based analysis of CAP, Cbl, and CrkII function in the regulation of GLUT4 by insulin

Stimulation of glucose transport by insulin in cultured adipocytes through translocation of intracellular GLUT4 glucose transporters to the plasma membrane has been suggested to require phosphatidylinositol (PI) 3-kinase-dependent and independent mechanisms. To test the involvement of a PI 3-kinase-independent pathway leading to activation of the TC10 GTPase, the putative intermediates CAP, c-Cbl, Cbl-b, and CrkII were selectively depleted in 3T3-L1 adipocytes using highly efficient small interfering (si) RNAs. Simultaneous depletion of the ubiquitination factors c-Cbl plus Cbl-b in cultured adipocytes had the expected effect of delaying dephosphorylation of EGF receptors upon removal of EGF. However, siRNA-mediated gene silencing of both Cbl isoforms or CAP or CrkII in these cells failed to attenuate insulin-stimulated deoxyglucose transport or Myc-tagged GLUT4-GFP translocation at either sub-maximal or maximal concentrations of insulin. The dose-response relationship for insulin stimulation of deoxyglucose transport in primary adipocytes derived from c-Cbl knock-out mice was also identical to insulin action on adipocytes from wild type mice. These data are consistent with the hypothesis that CAP, Cbl iso-forms, and CrkII are not required components of insulin signaling to GLUT4 transporters.     

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.     

Substrate modulates compound I formation in peroxide shunt pathway of Pseudomonas putida cytochrome P450(cam)

The active oxygenating intermediate, a ferryl-oxo-(II) porphyrin cation radical (compound I), in substrate-bound cytochrome P450(cam) (P450(cam)) has eluded detection and kinetic analysis for several decades. Upon rapid mixing of peroxides-H(2)O(2) and m-CPBA with substrate-bound forms of P450(cam), we observed an intermediate with spectral features characteristic of compound I. Unlike in H(2)O(2), kinetic investigation on the reaction of m-CPBA with various substrate (camphor, adamantone, and norcamphor)-bound P450(cam) and its Y96A mutant shows a preferential binding of the aromatic end group of m-CPBA to the active-site of the enzyme and modulation of compound I formation by the local environment of heme active-site. The results presented in this paper describe the importance of heme environment in modulating formation of compound I, and form the first kinetic analysis of this intermediate in the peroxide shunt pathway of substrate-bound P450(cam).     

A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.     

Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent

The role of signaling pathways including the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) during viral infection has gained much recent attention. Our laboratory reported on an important regulatory role for extracellular signal-regulated kinases (ERK1/2), subfamily members of the MAPKs, during coxsackievirus B3 (CVB3) infection. However, the role of the PI3K pathway in CVB3 infection has not been well characterized. CVB3 is the most common known viral infectant of heart muscle that directly injures and kills infected cardiac myocytes during the myocarditic process. In the present study, we investigated the role of protein kinase B (PKB) (also known as Akt), a general downstream mediator of survival signals through the PI3K cascade, in regulating CVB3 replication and virus-induced apoptosis in a well-established HeLa cell model. We have demonstrated that CVB3 infection leads to phosphorylation of PKB/Akt on both Ser-473 and Thr-308 residues through a PI3K-dependent mechanism. Transfection of HeLa cells with a dominant negative mutant of Akt1 or pretreatment of wild-type HeLa cells with the specific PI3K inhibitor LY294002 significantly suppresses viral RNA expression, as reflected in diminished viral capsid protein expression and viral release. Dominant negative Akt1 and LY294002 also increase apoptosis in infected cells, which can be reversed by addition of the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Interestingly, blocking of apoptosis by zVAD.fmk does not reverse the viral RNA translation blockade, indicating that the inhibitory effect of dominant negative Akt1 on viral protein expression is not caspase dependent. In addition, we showed that the attachment of virus to its receptor-coreceptor complex is not sufficient for PKB/Akt activation and that postentry viral replication is required for Akt phosphorylation. Taken together, these data illustrate a new and imperative role for Akt in CVB3 infection in HeLa cells and show that the PI3K/Akt signaling is beneficial to CVB3 replication.     

Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus

The genome of feline calicivirus (FCV) is an approximately 7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3' end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5' end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.     

Data on the push–out bond strength of three different root canal treatment sealers

It is of interest to document data on the push – out bond strength of three different root canal treatment sealers such as MTA Fillapex (MTA based), AH plus (Epoxy Resin based) and Apexit plus (Calcium hydroxide based). Forty-five freshly extracted human maxillary central incisors with closed apices were selected randomly. All the teeth were sectioned at cement-enamel junction using a diamond disc before starting the root canal preparation to obtain root length of 12 mm. All teeth were instrumented using ProTaper rotary instruments. 5.25% sodium hypochlorite was used for irrigation between instrumentation followed by 17% EDTA, and final rinse by saline. Obturation procedures were done using the gutta-percha single cone technique. 45 roots were randomly assigned to 3 groups of 15 for obturation with gutta-percha cones and 1 of the 3 sealers (n=15). Group 1 = MTA Fillapex sealer + gutta-percha: Group 2 = AH plus sealer + gutta-percha:Group 3 = Apexit plus sealer + gutta-percha. The roots were sectioned horizontally to its canal into 3 sections: Coronal, Mid-root and Apical-thirds using a precision cutting machine, with a thickness of 3 mm. The specimens were subjected to push-out test using a universal testing machine that carried a plunger. The loading speed was 1mm/min until the dislodgment of the material occurred. The independent t- test was used to compare the mean scores among the study groups. The level of significance was set at 5% for all tests. After the push-out bond strength test, each sample was evaluated under stereomicroscope (40x) to determine the mode of failure and recorded as one of the following categories: adhesive, cohesive or mixed. The observations thus obtained were subjected to statistical analysis using Student - t test. AH Plus showed significantly higher values than MTA Fillapex and Apexit plus (p < 0.05). Amongst the push-out bond strength AH Plus sealer showed significant difference from MTA Fillapex and Apexit plus groups. There was no significant difference between MTA Fillapex and Apexit plus however (p>0.05). Microscopic analysis displayed that the majority of the modes were cohesive failures for AH Plus, adhesive failures for MTA Fillapex and mixed failures for Apexit Plus. . Thus, AH Plus had the highest bond strength and MTA Fillapex had the lowest bond strength to root dentin. Mean push-out bond strength values were ranked as follows; AH Plus >Apexit Plus > MTA Fillapex. Microscopic analysis displayed that the majority of the modes were cohesive failures of AH Plus, adhesive failures for MTA Fillapex and mixed failures for Apexit Plus.     

Biomimetic synthesis and anti-HIV activity of dimeric phloroglucinols

Plants are an important source of a variety of bioactive compounds with different modes of action. Anti-HIV agents from plant sources can be useful in developing novel therapies for inhibiting HIV infection. Based on the reported anti-HIV activity of plant derived phloroglucinols, several new dimeric phloroglucinols were synthesized in the present study by varying substitution on aromatic ring and at methylene bridge. Some of the synthesized compounds have shown good HIV inhibitory activity in a human CD4+ T cell line (CEM-GFP) infected with HIV-1 NL_4.3 virus isolate. Structure–activity studies indicate that phenyl, 4-benzyloxy-1-phenyl and cyclohexyl substitution at methylene bridge gave compounds with better anti-HIV activity. Compounds 22 and 24 showed highest anti-HIV activity with an IC₅₀ of 0.28 μM and 2.71 μM, respectively, former was more active than the positive standard AZT in cell based assay.