Microsystem for transfection of exogenous molecules with spatio-temporal control into adherent cells

Several non-viral techniques involving the use of liposomes, particle bombardment and electroporation have been used for efficient transfection of plasmids and other molecules into cells. Current approaches target whole or bulk regions of tissue, lacking the desired spatial control over the transfection process. In this study, we present a novel approach using microsystems to achieve spatial and temporal control over the transfection process in adherent cells. A 6x6 MEA (microelectrode array) with 100 microm microelectrode dimension was developed on a silicon substrate using standard microfabrication procedures and passivated with a biocompatible layer. Using finite element models, electric field intensities were simulated and locations of optimal electroporation zones in the cell culture on the microelectrode surface were predicted. The MEA was subsequently tested using 3T3 fibroblasts cultured on the MEA surface for 96 h and stimulation voltages in the range of 2-5 V in the presence of propidium iodide (PI), a cell impermeant dye. Maximum electric field intensities in the z-direction were estimated to be in the range of 320-820 V/cm for applied differential voltages in the range of 2-5 V. Cells directly on the top and on the edges of the stimulating microelectrodes in the MEA were preferentially transfected with PI as predicted by the simulations. The results of these experiments demonstrate that spatial and temporal control of desired regions of transfection in vitro can be achieved using MEAs and electroporation.     

Concentration of macroergic phosphates and oxidative potential of skeletal muscles

It is known that a long-duration decline of high-energy phosphate (HP) level in skeletal muscles, induced by administration of beta-guanidinpropionic acid (beta-GPA), is followed by an increase in mitochondrial enzyme activities (MEA). The same increase in MEA was observed in the course of physical exercise training. Under gravitational inloading decrease in MEA and increase in the level of high-energy phosphates occurred. If changes in (HP) level are believed to trigger the alterations in MEA, the increase in high-energy phosphate levels in muscles is to lead to a decline in MEA as well. The present work was purposed to reveal if changes in HP level under different contractile activity levels may be associated with changes in oxidative potential in the skeletal muscles.     

DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation

MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.     

Acute effects of two melanocytolytic agents, hydroquinone and beta-mercaptoethanolamine, upon tyrosinase activity and cyclic nucleotide levels in murine melanomas

The acute in vitro actions of two potent melanocytolytic agents, hydroquinone (HQ) and beta-mercaptoethanolamine (MEA), were determined in the B-16, Cloudman S-91 and Harding-Passey (HP) murine melanomas grown in vivo. Drug treated melanoma dice (5--480 min) were analyzed for tyrosinase activity and cyclic nucleotide levels (cAMP, cGMP). HQ and MEA effects on tyrosinase activity are complex and vary with tumor type, duration of treatment and agent tested. MEA or HQ inhibited B-16 tyrosinase activity. With combined drug therapy, low concentrations of MEA plus HQ stimulate B-16 tyrosinase activity while high concentrations of the drugs have little effect on enzymatic activity. MEA depresses tyrosinase activity while HQ elevates enzymatic activity in the S-19 melanoma. Both high and low concentrations of the combined drugs (MEA plus HQ) elicit the same response, stimulation at 10 min followed by continued depression of tyrosinase activity for the remainder of the 4 h study period. MEA initially stimulates HP tyrosinase activity followed by depression of enzymic activity. In contrast, HQ initially depresses HP tyrosinase activity followed by stimulation of enzyme activity. In combination the drugs inhibit HP tyrosinase activity. The effects of MEA and/or HQ on murine melanoma cyclic nucleotide levels are equally complex. MEA or HQ elevate cAMP and cGMP levels in all three tumors with the exception of S-91 cGMP levels which are not altered. In combination the drugs increase cyclic nucleotide levels in each of the three tumor types but at different times. No correlation is present between cyclic nucleotide levels and tyrosinase activity. Thus, the action of increased cyclic nucleotide levels in melanogenesis can not be separated from the direct actions of MEA and HQ upon melanogenesis. The divergent effects of MEA and/or HQ on tyrosinase activity and cyclic nucleotide levels in these melanomas are not correlated with the known in vivo melanocytolytic activity of these drugs. Thus, these parameters appear to be inadequate indicators of melanoma cell viability in chemotherapeutic screening of drugs effective in destroying malignant melanoma.     

Partial purification and characterization of monomethylethanolamine kinase and dimethylethanolamine kinase activities from rat liver.

Monomethylethanolamine (MEA) kinase and dimethylethanolamine (DEA) kinase activities were purified 950 and 750 fold respectively from rat liver by conventional procedures. Certain properties of the partially purified enzyme preparation suggest that they are different from both choline kinase activity and ethanolamine kinase activity and differ from one another. This is based upon the following observations: 1. The heat stabilities of MEA kinase and DEA kinase activities are significantly different from one another and are different from the stability of choline kinase and ethanolamine kinase activities. 2. K+ in the presence of Mg2+ increases MEA kinase activity by 100% but has no effect on DEA kinase activity. 3. Different Ki values and the types of inhibition by several structurally related amino alcohols were found for MEA kinase and DEA kinase activities. 4. The purification fold of MEA kinase and DEA kinase are different from each other and from that of choline kinase and ethanolamine kinase.     

Morphological changes (including filamentation) in Escherichia coli grown under starvation conditions on silicon wafers and other surfaces

Using a scanning electron microscope, pleomorphism (notably filamentation) was seen when Escherichia coli was grown under starvation conditions for 14 d on microporous silicon wafers, titanium, glass and plastic discs. Under these conditions, the 'standard', rod shaped cell (1-3 microns) failed to separate after division and filaments developed, some as long as 50 microns, with many showing bulbous tips. Filamentation began to occur 5 d after the imposition of starvation conditions. Dumbbell shaped cells were also observed, although apparent 'Y' and 'V'-shaped cells proved to be artefacts, caused by overlapping rods. The implications of the appearance of pleomorphism in E. coli, when grown under starvation conditions, is discussed in relation to its pathogenicity and growth in the environment.     

Spreading and orientation of epithelial cells on grooved substrata

The spreading and orientation of epithelial (E) cells was studied on titanium-coated grooved substrata by light, transmission (TEM) and scanning electron microscopy (SEM). Vertical-walled grooves and V-shaped grooves, 3-60 microns deep, were produced in silicon wafers by micromachining, a process which was developed for the fabrication of micro-electronic components, and the grooved substrata were replicated in Epon. Photolithography was used to prepare photoresist-based and silicon dioxide-silicon substrata with grooves of approximately 2 and approximately 0.5 micron deep, respectively. Cell clusters were markedly oriented by all the grooved substrata examined, with the orientation index being highest for substrata with grooves of the smallest repeat spacing. Time-lapse cinemicrography showed that the grooves directed the migration of E cells, but the control was not absolute, as some cells crossed over the ridges and descended into the grooves. The 0.5 micron grooves appeared less effective than the deeper grooves in directing cell locomotion. SEM and TEM of E cells spreading on the grooved substrata demonstrated that cell processes, including lamellae and filopodia, were capable of bending around and closely adapting to groove edges. E cells did not flatten as extensively on a substratum with 22 microns deep V-shaped grooves as on a smooth surface, although some cells were markedly elongated. One mechanism proposed to explain contact guidance of fibroblasts is that linear elements of the locomotory system, such as microfilament bundles, are unable to operate when bent. The observed flexibility of epithelial cell processes and the ability of substrata with shallow grooves to orient E cells indicate that contact guidance of E cells on micromachined substrata cannot be explained by the mechanical stiffness of long linear cytoskeletal elements.     

Metabolic Pathway Involved in 2-Methyl-6-Ethylaniline Degradation by Sphingobium sp. Strain MEA3-1 and Cloning of the Novel Flavin-Dependent Monooxygenase System meaBA

2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02′ in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase.     

Poly(ethylene glycol) interfaces: an approach for enhanced performance of microfluidic systems

Microfluidic systems are extensively used platform for analytical and therapeutic applications. One of the major problems encountered in these systems is the loss of material due to non-specific surface interactions. When biological solutions are flowed through microchannels, they tend to adsorb on the surface due to the negative charge of the surface. This results in a reduced efficiency of the system which can be critical in sensitive biological analysis. Poly(ethylene glycol) (PEG) is known to form non-fouling interfaces on silicon and glass which are common materials used in microfluidic systems. The most common approach for modifying silicon/glass with PEG involves a solution phase protocol. Since the micro/nanofluidic systems have channel sizes ranging from hundreds of microns to submicron with variety of complicated network, this surface modification approach is not sufficient in forming uniform, conformal, and ultrathin films on the surface. Due to the enclosed features in these systems, the properties of liquids such as viscosity and surface tension play an important role in the clogging and eventually biofouling of these microchannels. Hence, we have developed a solvent-free vapor deposition protocol for modifying silicon/glass surfaces with PEG. Various concentrations of protein solutions were flowed through unmodified and PEG-modified glass microcapillaries of different lengths at different flow rates. PEG surfaces formed on silicon have shown 80% reduction in protein adsorption in static conditions.     

Blood pressure responses of conscious rats to intravenous administration of enkephalin derivatives (D-ala methionine and leucine enkephalinamide, and methionine and leucine enkephalinamide)

The present study was designed to determine the blood pressure (BP) responses of conscious rats given intravenous (IV) injections of enkephalin derivatives (D-ala²-methionine enkephalinamide, DAMEA; D-ala²-leucine enkephalinamide, DALEA; methionine enkephalinamide, MEA; leucine enkephalinamide, LEA) and the receptor mechanisms mediating the resultant change in BP. IV injection of 1.6–16.0 nmoles of DAMEA or DALEA caused a transient but potent decrease in mean arterial pressure (MAP) and mean heart rate (MHR). LEA and MEA (16.0 nmoles) given IV produced slight pressor responses, which were not associated with concomitant tachycardia whereas 48 nmoles of MEA elicited a hypotensive effect accompanied by a fall in MHR. Pretreatment studies whereby various receptor antagonists (naloxone, diprenorphine, phentolamine, D-L-propranolol or atropine) were given IV 5 min before subsequent IV administration of DAMEA, DALEA, MEA or LEA (16 nmoles) showed that naloxone, diprenorphine and atropine blocked the depressor and bradycardic effects of DALEA and DAMEA. Naloxone and phentolamine suppressed the pressor reponse of both MEA and LEA (16.0 nmoles) while diprenorphine blocked the rise in MAP to only MEA. The results show that DAMEA and DALEA mediate their depressor actions in conscious rats via a negative chronotropic effect through an interaction of muscarinic cholinergic receptors on the myocardium. It is suggested that the pressor response of MEA and LEA may be produced via an -receptor mediated effect on the peripheral vasculature to cause vasoconstriction.     

Overexpression of a gibberellin inactivation gene alters seed development, KNOX gene expression, and plant development in Arabidopsis

We have examined the role of gibberellins (GAs) in plant development by expression of the pea GA 2-oxidase2 ( PsGA2ox2 ) cDNA, which encodes a GA inactivating enzyme, under the control of the MEDEA (MEA) promoter. Expression of MEA:PsGA2ox2 in Arabidopsis caused seed abortion, demonstrating that active GAs in the endosperm are essential for normal seed development. MEA:PsGA2ox2 plants had reduced ovule number per ovary and exhibited defects in phyllotaxy and leaf morphology which were partly suppressed by GA treatment. The leaf architecture and phyllotaxy defects of MEA:PsGA2ox2 plants were also restored by sly1-d which reduces DELLA protein stability to increase GA response. MEA:PsGA2ox2 seedlings had increased expression of the KNOTTED1 -like homeobox (KNOX) genes, BP , KNAT2 and KNAT6 , which are known to control plant architecture. The expression of KNOX genes is also altered in wild-type plants treated with GA. These results support the conclusion that GAs can suppress the effects of elevated KNOX gene expression, and raise the possibility that localized changes in GA levels caused by PsGA2ox2 alter the expression of KNOX genes to modify plant architecture.     

Interaction of ascorbate with the radioprotective effect of mercaptoethylamin. An exploratory study in mice, whole animals and cell cultures.

The injection of ascrobate together with cysteamine (beta-mercaptoethylamin or MEA) was shown to cause a partial reversion of the radioprotective action of MEA in mice, and simultaneously of the suppressive action of MEA on RNA synthesis in bone marrow cells. In mouse spleen lymphocytes stimulated by concanavalin A in vitro, MEA and ascorbate exhibited a strong antagonism, neutralizing each other's inhibitory action on RNA synthesis. The latter effect failed to appear after chelation of trace metals, and it is indicated that the ability of ascorbate to counteract the effects of MEA on radiosensitivity and metabolism requires the formation of oxidized products, probably monodehydroascorbate, in agreement with previous observations on bacteria.     

Enhancement by cysteamine of N-methyl-N-nitrosourea mutagenesis in E. coli

Cysteamine (MEA) is comutagenic to methylnitrosourea (MNU) in E. coli AB 1157 but not in the nonadaptable mutant derivative ada-6 of that strain. The comutagenic action of MEA was eliminated by cysteine at low concentrations, which also lowered mutation frequencies in AB1157 but not in ada-6. In model experiments it was shown that cysteine counteracted the inhibition by MEA of beta-galactosidase induction in both bacterium strains. The comutagenic action of MEA is interpreted as being due to an inhibition of induction of methyltransferase during treatment with MNU.     

Rheology of myocardium. The relation between force, velocity, sarcomere length and activation in rat cardiac muscle

The relations between force, shortening velocity and sarcomere length (F-V-SL) during cardiac contraction, underlie Starling's Law of the Heart. F-V-SL were investigated in isolated, intact and skinned trabeculae and myocytes from rat heart. SL and V were measured with laser diffraction techniques; F was measured with a silicon strain gauge. The "ascending" F-SL relation appeared to result from both length dependent sensitivity of the contractile system to activator calcium ions and the presence of restoring forces (Fr), residing in the collagen skeleton of the muscle. Fr increased exponentially with decreasing SL below slack length to 25% of maximal twitch force (Ft) at SL = 1.60 microns. V was inversely proportional to the load and attained a maximum at zero load (Vo). Vo increased with factors that increased F: [Ca++], SL, and time during the twitch. Vo reached a maximum and remained constant (13.5 microns/s) when F attained or exceeded 50% of its maximum value. Viscous force in the passive muscle increased with V to a maximum of 4% of Ft at V = 40 microns/s. The relation between Vo and these factors could be predicted by a model of contraction in which the measured visco-elastic properties of myocardium were incorporated, while the truly unloaded maximal velocity of sarcomere shortening was assumed to be independent of the level of activation of the contractile filaments. A model of the cardiac cycle which explains the relation between Frank's and Starling's laws is presented.     

Biodegradation of amine waste generated from post-combustion CO2 capture in a moving bed biofilm treatment system

Nitrogen and organic matter removal from reclaimer waste of a monoethanolamine (MEA) based CO₂-capture plant was demonstrated in a pre-denitrification biofilm system. The reclaimer waste was generated from a 30 % (w/w) MEA solvent used for capturing CO₂ from flue gas from a coal-fired power plant. MEA, N-(2-hydroxylethyl)glycine (HEGly) and 2-hydroxyethylformamide (HEF) were the major contaminants treated. Hydrolysis of MEA to ammonia and further oxidation of organic intermediates readily occurred in the pre-denitrification system with a hydraulic retention time of 7 h. The biofilm system achieved 98 ± 1 % removal of MEA and 72 ± 16 % removal of total nitrogen. This is the first demonstration of efficient biodegradation of real amine waste from a post-combustion CO₂ capture facility by pre-denitrification without external electron donor.     

A classification of bioinformatics algorithms from the viewpoint of maximizing expected accuracy (MEA)

Many estimation problems in bioinformatics are formulated as point estimation problems in a high-dimensional discrete space. In general, it is difficult to design reliable estimators for this type of problem, because the number of possible solutions is immense, which leads to an extremely low probability for every solution-even for the one with the highest probability. Therefore, maximum score and maximum likelihood estimators do not work well in this situation although they are widely employed in a number of applications. Maximizing expected accuracy (MEA) estimation, in which accuracy measures of the target problem and the entire distribution of solutions are considered, is a more successful approach. In this review, we provide an extensive discussion of algorithms and software based on MEA. We describe how a number of algorithms used in previous studies can be classified from the viewpoint of MEA. We believe that this review will be useful not only for users wishing to utilize software to solve the estimation problems appearing in this article, but also for developers wishing to design algorithms on the basis of MEA.     

Sexual responses of the male rat medial preoptic area and medial amygdala to estrogen I: site specific suppression of estrogen receptor alpha

Male rat copulation is mediated by estrogen-sensitive neurons in the medial preoptic area (MPO) and medial amygdala (MEA); however, the mechanisms through which estradiol (E(2)) acts are not fully understood. We hypothesized that E(2) acts through estrogen receptor α (ERα) in the MPO and MEA to promote male mating behavior. Antisense oligodeoxynucleotides (AS-ODN) complementary to ERα mRNA were bilaterally infused via minipumps into either brain area to block the synthesis of ERα, which we predicted would reduce mating. Western blot analysis and immunocytochemistry revealed a knockdown of ERα expression in each brain region; however, compared to saline controls, males receiving AS-ODN to the MPO showed significant reductions in all components of mating, whereas males receiving AS-ODN to the MEA continued to mate normally. These results suggest that E(2) acts differently in these brain regions to promote the expression of male rat sexual behavior and that ERα in the MPO, but not in the MEA, promotes mating.     

Sexual responses of the male rat medial preoptic area and medial amygdala to estrogen II: site specific effects of selective estrogenic drugs

In the medial preoptic area (MPO) and medial amygdala (MEA), estradiol (E(2)) aromatized from testosterone (T) may act via either estrogen receptor (ER) α or ERβ to mediate mating in male rats. We tested the hypothesis that, in the MPO, ERα exclusively mediates sexual responses to E(2) by monitoring mating in four groups of castrated male rats administered dihydrotestosterone (DHT) subcutaneously and MPO implants delivering either: cholesterol, E(2), propyl pyrazole triol (PPT, ERα-agonist) or diarylpropionitrile (DPN, ER β-agonist); a fifth group of intact males served as DPN toxicity control, receiving DPN MPO implants. In a follow-up study, either 1-methyl-4-phenyl pyridinium (MPP, ERα-antagonist) or blank MPO cannulae were implanted in castrated male rats receiving T subcutaneously, whereas intact MPP toxicity controls received MPP MEA implants. PPT or E(2) MPO implants maintained mating, but cholesterol or DPN MPO implants did not. Moreover, MPP MPO implants interfered with T reinstatement of mating suggesting that, in the MPO, ERα is necessary and sufficient for mating in androgen-maintained male rats and ERβ is not sufficient. Because it is unknown which ER subtype(s) mediate sexual responses of the MEA to E(2), we examined mating following MEA implants of cholesterol, E(2), PPT or DPN in four groups of castrated male rats administered DHT subcutaneously. E(2) MEA implants maintained mounting but mating was significantly decreased in groups receiving PPT, DPN or cholesterol MEA implants suggesting that, unlike the MPO where ERα alone is essential, sexual responses of the MEA to E(2) require simultaneous interactions among multiple ER subtypes.