Combining optical and electrical impedance techniques for quantitative measurement of confluence in MDCK-I cell cultures

A new method combining optical and electrical impedance measurements is described that enables submicroscopic cell movements to be monitored. The cells are grown on small gold electrodes that are transparent to light. This modified electrical cell-substrate impedance sensor (ECIS) allows simultaneous microscopic recording of both growth and motility, thus enabling cell confluence on the electrodes to be systematically correlated to the impedance in regular time intervals of seconds and for extended periods of time. Furthermore, the technique provides an independent measure of monolayer cell densities that we compare to calculated values from a theoretical model. We have followed the attachment and spreading behavior of epithelial Madin-Darby canine kidney strain I (MDCK-I) cell cultures on microelectrodes for up to 40 h. The studies reveal a high degree of correlation between the measured resistance at 4 kHz and the corresponding cell confluence in 4- to 6-h intervals with typical linear cross-correlation factors of r equaling approximately 0.9. In summary, the impedance measured with the ECIS technique provides a good quantitative measure of cell confluence.     

An in vivo method for measuring turbulence in mechanical prosthesis leakage jets

This work introduces a method for the in vivo measurement and analysis of turbulence within the leakage of a mechanical heart valve. Several analysis techniques were applied to ultrasound measurements acquired within the atrium of a pig, and error associated with these techniques was analyzed. The technique chosen applies cyclic averaging to mean and maximum velocity measurements within small, normalized phase windows to calculate Reynolds normal stresses in the direction of the ultrasound beam. Maximum shear stresses are estimated from these normal stresses using an analytical technique. The stresses observed were smaller than those reported from previous in vitro simulations.     

'One bead two compound libraries' for detecting chemical and biochemical conversions

When combinatorial chemistry was introduced 13 years ago, the expectations were high for the delivery of results, particularly in the pharmaceutical industry. However, combinatorial chemistry was implemented independently of the application for which the products were going to be used. Resins developed only for efficient solid-phase synthesis were used and products were employed in existing assays developed for traditional solution studies. There was almost no assay or technology development and the use of real combinatorial methods soon had to give way to high-throughput synthesis and traditional screening. However, during recent years more sophisticated resins and assay techniques have been developed that may result in a second and more successful implementation of real integrated combinatorial chemistry. The first in this line of new developments is the 'one bead two compound' assay, in which the resin bead in addition to a combinatorial library member contains a reporter compound that can act as a beacon to monitor the activity of the library member. This powerful concept can be generally applied in all fields of combinatorial chemistry including drug, catalysts and material development.     

Recombinant human serum amyloid P component from Pichia pastoris: production and characterization

Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.     

Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation

To study the effect of extracellular acidosis on apoptosis and necrosis during ischemia and reoxygenation, we exposed human post-mitotic NT2-N neurones to oxygen and glucose deprivation (OGD) followed by reoxygenation. In some experiments, pH of the cell medium was lowered to 5.9 during either OGD or reoxygenation or both. Staurosporine, used as a positive control for apoptosis, caused Poly(ADP-ribose)-polymerase (PARP) cleavage and nuclear fragmentation, but no PARP cleavage and little fragmentation were seen after OGD. Low molecular weight DNA fragments were found after staurosporine treatment, but not after OGD. No protective effect of caspase inhibitors was seen after 3 h of OGD and 21 h of reoxygenation, but after 45 h of reoxygenation caspase inhibition induced a modest improvement in 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) cleavage. While acidosis during OGD accompanied by neutral medium during reoxygenation protected the neurones (MTT: 228 +/- 117% of neutral medium, p < 0.001), acidosis during reoxygenation only was detrimental (MTT: 38 +/- 25%, p < 0.01). We conclude that apoptotic mechanisms play a minor role after OGD in NT2-N neurones. The effect of acidosis on neuronal survival depends on the timing of acidosis, as acidosis was protective during OGD and detrimental during reoxygenation.     

The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data indicating a survival-promoting effect of NCAM-stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA-fragmentation and caspase-3 activation. The survival-promoting effect of NCAM-stimulation was also shown to be dependent on PI3K.     

ADAM12/syndecan-4 signaling promotes beta 1 integrin-dependent cell spreading through protein kinase Calpha and RhoA

The ADAMs (a disintegrin and metalloprotease) comprise a large family of multidomain proteins with cell-binding and metalloprotease activities. The ADAM12 cysteine-rich domain (rADAM12-cys) supports cell attachment using syndecan-4 as a primary cell surface receptor that subsequently triggers beta(1) integrin-dependent cell spreading, stress fiber assembly, and focal adhesion formation. This process contrasts with cell adhesion on fibronectin, which is integrin-initiated but syndecan-4-dependent. In the present study, we investigated ADAM12/syndecan-4 signaling leading to cell spreading and stress fiber formation. We demonstrate that syndecan-4, when present in significant amounts, promotes beta(1) integrin-dependent cell spreading and stress fiber formation in response to rADAM12-cys. A mutant form of syndecan-4 deficient in protein kinase C (PKC)alpha activation or a different member of the syndecan family, syndecan-2, was unable to promote cell spreading. GF109203X and G?6976, inhibitors of PKC, completely inhibited ADAM12/syndecan-4-induced cell spreading. Expression of syndecan-4, but not syn4DeltaI, resulted in the accumulation of activated beta(1) integrins at the cell periphery in Chinese hamster ovary beta1 cells as revealed by 12G10 staining. Further, expression of myristoylated, constitutively active PKCalpha resulted in beta(1) integrin-dependent cell spreading, but additional activation of RhoA was required to induce stress fiber formation. In summary, these data provide novel insights into syndecan-4 signaling. Syndecan-4 can promote cell spreading in a beta(1) integrin-dependent fashion through PKCalpha and RhoA, and PKCalpha and RhoA likely function in separate pathways.