Surfaceome profiling enables isolation of cancer-specific exosomal cargo in liquid biopsies from pancreatic cancer patients.

Background: Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling. Patients and methods: Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis. Results: Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation. Conclusion: Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients.

Background: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). Patients and methods: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. Results: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. Conclusions: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

Rapid real-time recirculating PCR using localized surface plasmon resonance (LSPR) and piezo-electric pumping.

Rapid detection and characterization of pathogens in patients with bloodstream infections (BSIs) is a persistent problem for modern medicine, as current techniques are slow or provide incomplete diagnostic information. Real-time polymerase chain reaction (qPCR) allows specific detection of a wide range of targets and quantification of pathogenic burdens to aid in treatment planning. However, new technological advances are required for a rapid and multiplex implementation of qPCR in clinical applications. In this paper, the feasibility of a novel microfluidic platform for qPCR is presented, integrating highly sensitive, label-free localized surface plasmon resonance (LSPR) imaging of DNA hybridization into a recirculating chip design for real-time analysis. Single target and multiplex detection of DNA target amplification are demonstrated, with a limit of detection of 5 fg µL-1 of E. coli DNA for single target PCR, correlating with approximately 300 bacteria per mL. The results of this study demonstrate the potential of this platform for simultaneous real-time detection of multiple target genes within 15 minutes that could provide live saving benefits in patients with BSIs.

Isolation and concentration of bacteria from blood using microfluidic membraneless dialysis and dielectrophoresis.

A microfluidic system that combines membraneless microfluidic dialysis and dielectrophoresis to achieve label-free isolation and concentration of bacteria from whole blood is presented. Target bacteria and undesired blood cells are discriminated on the basis of their differential susceptibility to permeabilizing agents that alter the dielectrophoretic behavior of blood cells but not bacteria. The combined membraneless microdialysis and dielectrophoresis system isolated 79 ± 3% of Escherichia coli and 78 ± 2% of Staphylococcus aureus spiked into whole blood at a processing rate of 0.6 mL h-1. Collection efficiency was independent of the number of target bacteria up to 105 cells. Quantitative PCR analysis revealed that bacterial 16S rDNA levels were enriched more than 307-fold over human DNA in the fraction recovered from the isolation system compared with the original specimen. These data demonstrate feasibility for an instrument to accelerate the detection and analysis of bacteria in blood by first isolating and concentrating them in a microchamber.

Electron spin resonance studies of the interaction of oxidoreductases with 2,6-dimethoxy-p-quinone and semiquinone.

Previous electron spin resonance studies have demonstrated that the decay of ascorbyl plus semiquinone radicals, produced in an aqueous mixture of ascorbate and 2,6-dimethoxy-p-quinone, is accelerated by ascites cells. This effect was concluded to involve a sulfhydryl-containing NAD(P)H-enzyme, and work on cultured cell lines showed that on neoplastic transformation the activity against the radicals was increased. We show here that at least three disulfide-oxidoreductases are able to quench the radicals in a similar way to that of viable cells. Glutathione reductase (EC 1.6.4.2) in the presence of NADPH and oxidised glutathione, and dihydrolipoamide dehydrogenase (EC 1.8.1.4) with NADH and lipoamide, are found to accelerate the radical decay by reducing the quinone or semiquinone. DT-diaphorase (EC 1.6.99.2) in the presence of NAD(P)H can also achieve this by reducing the quinone directly. Lipoamide dehydrogenase and glutathione reductase are also capable of reducing nitroxide spin labels, a finding considered of relevance to the reported reduction of such spin labels by neuroblastoma cells.

Role of peroxide in AC electrical field exposure effects on friend murine erythroleukemia cells during dielectrophoretic manipulations.

The effects of AC field exposure on the viability and proliferation of mammalian cells under conditions appropriate for their dielectrophoretic manipulation and sorting were investigated using DS19 murine erythroleukemia cells as a model system. The frequency range 100 Hz-10 MHz and medium conductivities of 10 mS/m, 30 mS/m and 56 mS/m were studied for fields generated by applying signals of up to 7V peak to peak (p-p) to a parallel electrode array having equal electrode widths and gaps of 100 micrometer. Between 1 kHz and 10 MHz, cell viability after up to 40 min of field exposure was found to be above 95% and cells were able to proliferate. However, cell growth lag phase was extended with decreasing field frequency and with increasing voltage, medium conductivity and exposure duration. Modified growth behavior was not passed on to the next cell passage, indicating that field exposure did not cause permanent alterations in cell proliferation characteristics. Cell membrane potentials induced by field exposure were calculated and shown to be well below values typically associated with cell damage. Furthermore, medium treated by field exposure and then added to untreated cells produced the same modifications of growth as exposing cells directly, and these modifications occurred only when the electrode polarization voltage exceeded a threshold of approximately 0.4 V p-p. These findings suggested that electrochemical products generated during field exposure were responsible for the changes in cell growth. Finally, it was found that hydrogen peroxide was produced when sugar-containing media were exposed to fields and that normal cell growth could be restored by addition of catalase to the medium, whether or not field exposure occurred in the presence of cells. These results show that AC fields typically used for dielectrophoretic manipulation and sorting of cells do not damage DS19 cells and that cell alterations arising from electrochemical effects can be completely mitigated.

Membrane changes associated with the temperature-sensitive P85gag-mos-dependent transformation of rat kidney cells as determined by dielectrophoresis and electrorotation.

Conventional dielectrophoresis (cDEP) and electrorotation (ROT) measurements have been used to determine the dielectric properties of a clone of normal rat kidney cells, designated 6m2, that exhibits a transformed phenotype at 33 degrees C and a non-transformed phenotype at 39 degrees C. cDEP measurements of the crossover frequencies at which individual 6m2 cells experienced zero cDEP force performed as a function of the conductivity of the suspension medium revealed that, in response to a temperature shift from 33 degrees C to 39 degrees C for 24 h, the mean specific cell membrane capacitance and conductance fell significantly (P < 0.01) from 42.3 (+/-1.3) to 30.3 (+/-2.9) mF/m2 and 743 (+/-422) to 567 (+/-326) S/m2, respectively. ROT analyses demonstrated a similar reduction for the membrane capacitance from 37.2 (+/-7.3) to 27.4 (+/-6.1) mF/m2, and also showed that accompanying changes in the mean internal electrical conductivity and dielectric permittivity of the cells were insignificant. Scanning electron microscopy was used to examine the surface morphology of the cells and, in agreement with our previous reports for leukemia cells, the observed membrane capacitance values correlated closely with the morphological complexity of the cell membrane surface. The observed changes in the membrane dielectric properties are discussed in terms of their biological significance and their relationship to previously-detected changes in cell surface charge.

Membrane changes accompanying the induced differentiation of Friend murine erythroleukemia cells studied by dielectrophoresis.

Dielectrophoresis measurements obtained using an image processing technique are reported over the frequency range 1 Hz to 100 kHz for the Friend murine erythroleukemia cell lines DS19 and R1 before and after treatment with hexamethylene bisacetamide and dimethylsulfoxide, agents that induce terminal differentiation in DS19 but not in R1 cells. Data are analyzed according to the single shell dielectric model of the cell. The membrane capacitance was found to fall by 30% and membrane conductivity by a factor of at least 5 when DS19 cells were induced to differentiate. R1 cells showed no such response. While the theoretical model was found to be useful for comparing differences in data for the different cell lines, several significant discrepancies between its predictions and the experimental data were observed, including positive dielectrophoretic collection at frequencies below 20 Hz and a smaller than predicted response to the membrane permeabilizing agents saponin and valinomycin. Factors that may have accounted for these discrepancies include surface charge effecgs, conduction parallel to the plasma membrane surface, and intracellular compartments.

Dielectrophoretic detection of changes in erythrocyte membranes following malarial infection.

The dielectric properties of normal erythrocytes were compared to those of cells infected with the malarial parasite Plasmodium falciparum. Normal cells provided stable electrorotation spectra which, when analyzed by a single-shelled oblate spheroid dielectric model, gave a specific capacitance value of 12 +/- 1.2 mF/m2 for the plasma membrane, a cytoplasmic permittivity of 57 +/- 5.4 and a cytoplasmic conductivity of 0.52 +/- 0.05 S/m. By contrast, parasitized cells exhibited electrorotation spectra with a time-dependency that suggested significant net ion outflux via the plasma membrane and it was not possible to derive reliable cell parameter values in this case. To overcome this problem, cell membrane dielectric properties were instead determined from dielectrophoretic crossover frequency measurements made as a function of the cell suspending medium conductivity. The crossover frequency for normal cells depended linearly on the suspension conductivity above 20 mS/m and analysis according to the single-shelled oblate spheroid dielectric model yielded values of 11.8 mF/m2 and 271 S/m2, respectively, for the specific capacitance and conductance of the plasma membrane. Unexpectedly, the crossover frequency characteristics of parasitized cells at high suspending medium conductivities were non-linear. This effect was analyzed in terms of possible dependencies of the cell membrane capacitance, conductance or shape on the suspension medium conductivity, and we concluded that variations in the membrane conductance were most likely responsible for the observed non-linearity. According to this model, parasitized cells had a specific membrane capacitance of 9 +/- 2 mF/m2 and a specific membrane conductance of 1130 S/m2 that increased with increasing cell suspending medium conductivity. Such conductivity changes in parasitized cells are discussed in terms of previously observed parasite-associated membrane pores. Finally, we conclude that the large differences between the dielectrophoretic crossover characteristics of normal and parasitized cells should allow straightforward sorting of these cell types by dielectrophoretic methods.

Electrorotation of liposomes: verification of dielectric multi-shell model for cells.

The reliability of multi-shell dielectric models, used to describe the ac electrokinetic behaviour of cells, has been tested by performing electrorotation and dielectrophoretic measurements on unilamellar, oligolamellar, and multilamellar liposomes of diameters ranging from 5 to 24 microm. Fluorescence microscopy, flow cytometry and electron spin resonance were used to characterise the morphology and membranes of the liposomes. The dielectric properties of the various types of liposomes, based on appropriate dielectric shell models, were then analysed using a general purpose, recursive, algorithm. Through simulations, the confidence levels that can be assigned to parameters derived through application of simple shell models are estimated. From this, we confirm that electrorotation data enable accurate determinations to be made of the dielectric properties of the outermost membrane of liposomes, and provide good indications of the level of complexity of the shells and internal compartments. We also demonstrate that, used with sufficient additional information, such as that provided by dielectrophoresis, electrorotation data yields unique solutions for the dielectric parameters of liposome-like particles.

Membrane dielectric responses of human T-lymphocytes following mitogenic stimulation.

Human peripheral blood T-lymphocytes, normally resting at the G0 phase, were stimulated with phytohemagglutinin (PHA) and interleukin-2 (IL-2) to induce the cell division cycle. The cells were examined at 24-h intervals for up to 96 h by flow cytometry to determine cell cycle distributions and by electrorotation to determine dielectric properties. The average membrane specific capacitance was found to vary from 12 (+/-1.5) mF/m2 prior to stimulation to 10 (+/-1.5) and 16 (+/-3.5) mF/m2 at 24 and 48 h after stimulation, respectively, and to remain unchanged up to 96 h after stimulation. Scanning electron microscopy studies of the cells revealed an increased complexity in cell membrane morphology following stimulation, suggesting that the observed change in the membrane capacitance was dominated by the alteration of cell surface structures. The average electrical conductivity of the cell interior decreased from approximately 1.1 S/m prior to stimulation to approximately 0.8 S/m at 24 h after stimulation and showed little change thereafter. The average dielectric permittivity of the cell interior remained almost unchanged throughout the course of the cell stimulation. The percentage of T-lymphocytes in the S and G2/M phases increased from approximately 4% prior to stimulation to approximately 11 and approximately 34% at 24 and 48 h after stimulation, respectively. The large change in membrane specific capacitance between the 24 and 48 h time period coincided with the large alteration in the cell cycle distribution where the S and G2/M populations increased by approximately 23%. These data, together with an analysis of the variation of the membrane capacitance during the cell cycle based on the cell cycle-dependent membrane lipid accumulation, show that there is a correlation between membrane capacitance and cell cycle phases that reflects alterations in the cell plasma membrane.

Dielectrophoretic characterisation of Friend murine erythroleukaemic cells as a measure of induced differentiation.

Dielectrophoresis measurements, the study of the motion of particles in non-uniform a.c. electrical fields, have been made on three cell lines (DS19, R1 and DR1) of Friend murine erythroleukaemia cells as a function of hexamethylene bisacetamide (HMBA) treatment. The effects of saponin treatment on R1 cells and neuraminidase on human red blood cells were also studied. It is shown that the dielectrophoretic behaviour can be interpreted in terms of cell surface charge and cell membrane conductivity effects. HMBA reduces the cell surface charge on all three cell lines, and in lines DS19 and DR10, where the cells are induced to differentiate, there is an increase in effective cell conductivity. This gain in conductivity is concluded to be associated with either an enhanced lateral electrophoretic motion of delocalised ions or of the polarisability of dipoles at the membrane surface.

Non-uniform spatial distributions of both the magnitude and phase of AC electric fields determine dielectrophoretic forces.

It is well known that the conventional dielectrophoretic force acting on a polarised particle in a non-uniform AC electric field is proportional to the in-phase component of the induced dipole moment and the non-uniformity of the field strength. In contrast, the travelling-wave-dielectrophoretic force that acts on a particle subjected to a travelling electric field is proportional to the out-of-phase component of the induced dipole moment. We derive a theory that unifies the description and interpretation of conventional dielectrophoretic and travelling-wave-dielectrophoretic forces. We show that a particle in a non-uniform AC electric field experiences a dielectrophoretic force due to spatial non-uniformities of the magnitude and the phase of the field interacting, respectively, with the in-phase and out-of-phase components of the induced dipole moment. The theory is used to explain the translational effects observed for particles in the presence of standing, travelling and rotating fields in several experimental electrode configurations. The good agreement found between the experimental observations and the theoretical predictions validate the theory.

Changes in Friend murine erythroleukaemia cell membranes during induced differentiation determined by electrorotation.

We used electrorotation measurements to investigate alterations in the plasma membranes of DS19 murine erythroleukaemia cells that accompanied erythropoietic differentiation induced by hexamethylene bisacetamide (HMBA). Following 3 days of HMBA treatment, the mean cell membrane specific capacitance determined from electrorotation spectra of individual, viable cells at physiological tonicity (300 mosmol/kg) fell from 1.74 to 1.53 microF/cm2, in agreement with trends observed earlier by dielectrophoretic measurements on bulk cell populations. Scanning and transmission electron microscopy revealed that the relatively high values found for cell membrane capacitance (> 1 microF/cm2) reflected the large area of plasma membrane associated with complex surface morphology including numerous microvilli. Furthermore, it demonstrated that the fall in membrane capacitance during HMBA treatment correlated with a reduction in the density of these complex surface features. Differences in the mechanical characteristics of the cell membranes of untreated and treated cells were then examined by exposing cells to osmotic stress. The intricacy of membrane morphology intensified with increasing osmolality of the suspending medium and this was reflected in higher specific capacitance values. When the osmolality was increased from 210 to 450 mosmol/kg, the mean membrane capacitance of untreated DS19 cells changed from 1.58 to 2.05 microF/cm2 while that for HMBA-treated cells changed from 1.47 to 1.72 microF/cm2, a significantly smaller response. This demonstrated that cells exposed to 72 h of differentiation treatment had an enhanced mechanical resilience as compared with their untreated counterparts, evidencing the early stages of the development of the membrane skeleton which becomes fully developed in mature erythrocytes. Our findings demonstrate the value of electrorotation measurements as a method for the non-invasive characterisation of viable leukaemic cells and their responses to stimuli and show that the membrane capacitance values so derived reflect membrane morphology.

Changes in cell surface charge and transmembrane potential accompanying neoplastic transformation of rat kidney cells.

Free flow electrophoresis measurements have been used to determine the surface charge density of normal rat kidney (NRK) cells and a clone of NRK, designated as 6m2, that exhibit a transformed phenotype at 33 degrees C and a non-transformed phenotype at 39 degrees C. A clone of 6m2, designated 54-5A4, which is transformed at both 33 degrees C and 39 degrees C was also studied. A surface charge density of -1.42 microC/cm2 was obtained for the NRK and non-transformed 6m2 cells at 39 degrees C, whereas at 33 degrees C values of -1.85 and -1.78 microC/cm2 were determined for the transformed 6m2 and 54-5A4 cells, respectively. It was found that 72% of the increased charge that appeared on the transformed 6m2 cells compared with the non-transformed 6m2 cells was RNAase sensitive. The time-dependent decrease in surface charge that accompanied the shift of the 6m2 cells from their transformed to non-transformed state was found to mirror the increase in transmembrane potential previously reported using a fluorescent dye technique, and was also comparable to the reported temporal changes in their morphology and virally-coded protein content.

Electrorotational studies of the cytoplasmic dielectric properties of Friend murine erythroleukaemia cells.

Electrorotation (ROT) spectra of Friend murine erythroleukaemia DS19 cells were measured in the frequency range 10 kHz-100 MHz as a function of suspension osmolality and cell differentiation treatment with hexamethylene bisacetamide (HMBA). A minimization program was employed to curve-fit the measured spectra using a single-shell dielectric model, allowing the derivation of cellular interior conductivity and permittivity (valid for the frequency range 10-100 MHz) and the cytoplasmic membrane capacitance (its dependence on the cell differentiation state and suspension osmolality having been reported earlier). Following HMBA treatment, DS19 cells exhibited a slight increment in average interior permittivity and a decrement in interior conductivity, although the changes were not statistically significant. For both untreated and HMBA treated samples, the average interior conductivity increased and permittivity decreased with increasing suspension osmolality. Of significance was that the average permittivity of cell interiors was larger than that of pure water. The electrorotation spectra of freshly prepared cell nuclei were measured, and the derived nuclear dielectric parameters were employed in numerical simulations to investigate the effects of nuclei on the ROT spectra of intact cells. Other cellular internal structures such as mitochondria were also analysed using theoretical simulations. It was concluded that the derived large permittivity values did not result from cell nuclei or mitochondria, and, instead, we suggest that they may arise from the combined effects of several cytoplasmic organelles.

A High-Voltage SOI CMOS Exciter Chip for a Programmable Fluidic Processor System.

A high-voltage (HV) integrated circuit has been demonstrated to transport fluidic droplet samples on programmable paths across the array of driving electrodes on its hydrophobically coated surface. This exciter chip is the engine for dielectrophoresis (DEP)-based micro-fluidic lab-on-a-chip systems, creating field excitations that inject and move fluidic droplets onto and about the manipulation surface. The architecture of this chip is expandable to arrays of N X N identical HV electrode driver circuits and electrodes. The exciter chip is programmable in several senses. The routes of multiple droplets may be set arbitrarily within the bounds of the electrode array. The electrode excitation waveform voltage amplitude, phase, and frequency may be adjusted based on the system configuration and the signal required to manipulate a particular fluid droplet composition. The voltage amplitude of the electrode excitation waveform can be set from the minimum logic level up to the maximum limit of the breakdown voltage of the fabrication technology. The frequency of the electrode excitation waveform can also be set independently of its voltage, up to a maximum depending upon the type of droplets that must be driven. The exciter chip can be coated and its oxide surface used as the droplet manipulation surface or it can be used with a top-mounted, enclosed fluidic chamber consisting of a variety of materials. The HV capability of the exciter chip allows the generated DEP forces to penetrate into the enclosed chamber region and an adjustable voltage amplitude can accommodate a variety of chamber floor thicknesses. This demonstration exciter chip has a 32 x 32 array of nominally 100 V electrode drivers that are individually programmable at each time point in the procedure to either of two phases: 0deg and 180deg with respect to the reference clock. For this demonstration chip, while operating the electrodes with a 100-V peak-to-peak periodic waveform, the maximum HV electrode waveform frequency is about 200 Hz; and standard 5-V CMOS logic data communication rate is variable up to 250 kHz. This HV demonstration chip is fabricated in a 130-V 1.0-mum SOI CMOS fabrication technology, dissipates a maximum of 1.87 W, and is about 10.4 mm x 8.2 mm.

Chemical heterogeneity of structural proteins of cancers with a common low electron spin resonance signal.

Electron spin resonance measurements are presented for the separated structural and soluble components of a variety of both normal and cancerous tissues. The signal at g = 2 from the structural part of the normal tissues was at least an order of magnitude stronger than the corresponding signal from the structural part of the cancers. In contrast, no significant difference in radical concentration was found between the soluble fractions of normal and cancerous tissues. Despite their common low signal at g = 2, the structural cancer fractions displayed a remarkable chemical heterogeneity. An important finding was that even cancers of the same histology reacted differently from one another when treated with a variety of chemical probes.

Alterations in electrophoretic mobility, diaphorase activity, and terminal differentiation induced in murine erythroleukemia lines by differentiating agents.

The electrophoretic mobilities (EPMs) and semiquinone reductase activities of two clones of Friend murine erythroleukemia (MEL) cells were investigated as a function of treatment with the inducing agents dimethylsulfoxide (DMSO) and hexamethylene bisacetamide (HMBA). As reported previously by others, the inducible clone DS19 lost its ability to grow in soft agar and expressed hemoglobin as judged by benzidine/H2O2 staining after 96 hours of treatment with 1% DMSO or 4 mM HMBA. In addition, its EPM fell by 14%, its semiquinone reductase activity by 40%, and its mean diameter by 10%. The second clone, R1, retained its ability to grow in soft agar and lacked hemoglobin expression after treatment with HMBA and DMSO, characterizing it as noninducible. However, R1 did demonstrate alterations in EPM, semiquinone reductase activity, and cell diameter that closely paralleled those found in DS19. Such responses were not seen in three non-MEL cell lines exposed to HMBA or DMSO, suggesting that clone R1 responded to these inducing agents in a cell-line specific manner but that its ability to complete the sequences necessary for differentiation may be blocked at an unknown point distal to the block characteristic of untreated cells. The data show that while a reduction in EPM, semiquinone reductase activity, and cell diameter accompany induced differentiation in MEL cells, such changes can occur in the absence of a commitment to terminal differentiation.

Water structure-dependent charge transport in proteins.

Dielectric and conductivity measurements are reported for bovine serum albumin as a function of hydration. Strong evidence is found for the existence of mobile charges whose short- and long-range hopping motion strongly depends on the physical state of the protein-bound water. These charges are considered to be protons. Insights into the nature of the electrical properties of protein-methylglyoxal complexes are provided, and the possibilities for correlated proton-electron motions are outlined.