Small Pipette Research Articles

Pressure in the Cochlea During Infrared Irradiation.

The purpose of the study is to demonstrate laser-evoked pressure waves in small confined volumes such as the cochlea. Custom-fabricated pressure probes were used to determine the pressure in front of the optical fiber in a small dish and patch pipettes to measure temperature changes. Pressure probes were inserted into scala tympani (ST) or vestibuli during laser stimulation. With a sensitive microphone the pressure was measured in the outer ear canal. Heating was spatially confined. The heat relaxation time was 35 ms. During laser stimulation in the cochlea at 17 μJ/pulse, the pressure in the outer ear canal (EC) was 43.5 dB (re 20 μPa). The corresponding intracochlear pressure was calculated to be about 78.5 dB (re 20 μPa) using the middle ear reverse transfer function of -35 dB. At 164 μJ/pulse, the pressure in the EC was on average 63 dB (re 20 μPa) and the intracochlear pressure was estimated to be 98 dB (re 20 μPa), which is similar to the value obtained with the pressure probe, 100 dB (re 20 μPa). Side-emitting optical fibers were used to steer the beam path. The pressure values were independent of the orientation of the beam path. Evoked compound action potentials of the auditory nerve were maximum when spiral ganglion neurons were in the beam path. Pressure waves are generated during infrared laser stimulation. The intracochlear pressure was independent from the orientation of the beam path. Neural responses required the spiral ganglion neurons to be directly irradiated.

EVALUATION OF INTRA-CYTOPLASMIC SPERM INJECTION (ICSI) OUTCOME AFTER OOCYTES DENUDATION WITH DIFFERENT DIAMETERS OF CAPILLARY PIPETTE

The cumulus-oocyte complex (COC) is a structure that connects the oocyte (germline) with the surrounding cumulus cells. The diameter of fully grown human egg is 110 to 120 microns with a volume of approximately 900,000 cubic microns. The COC must be removed (denuded) before intra-cytoplasmic sperm injection (ICSI) in order to evaluate the mature oocytes and to be able for injection, the denudation of COC is done by enzymatic treatment and mechanical stripping. The vigorous mechanical stripping or using small diameter pipette may cause; displacement of the polar body and impair the microtubule frame. The actin filament organization causes the break of cellular bridge that connects the polar body to the oocyte, deformation and damage of the oocyte cytoskeleton impairing its ability to sustain correct pronuclear formation or may result in zona pellucida fraction and oocyte degeneration. In this study, 1756 oocytes were collected from 90 cycles, and oocytes divided into 2 groups. The first group of oocytes was mechanically denuded with pipette diameter 150 µm, and the second group of oocytes was denuded with pipette diameter 200 µm. This manipulation showed no statistical differences in the number of oocytes that degenerated during denudation, normal and abnormal fertilization and in the rate of blastocyst formation when using denudation pipette of 150 µm over that of 200 µm (p >0.05).

Nanopipette delivery: influence of surface charge.

In this report, transport through a nanopipette is studied and the interplay between current rectification and ion delivery for small pipettes is examined. First, surface charge dependence of concentration polarization effects in a quartz nanopipette was investigated. Electrical characterization was performed through current-potential (I-V) measurements. In addition, fluorescein (an anionic fluorescent probe) was utilized to optically map ion enrichment and ion depletion in the nanopipette tip. Bare nanopipettes and polyethylenimine (PEI)-modified nanopipettes were examined. Results confirm that concentration polarization is a surface charge dependent phenomenon and delivery can be controlled through modification of surface charge. The relationship between concentration polarization effects and voltage-driven delivery of charged electroactive species was investigated with a carbon ring/nanopore electrode fabricated from pyrolyzed parylene C (PPC). Factors such as surface charge polarity of the nanopipette, electrolyte pH, and electrolyte concentration were investigated. Results indicate that with modification of surface charge, additional control over delivery of charged species can be achieved.

Juvenile mossy fibres: two pipers in the same pub?

Juvenile mossy fibres: two pipers in the same pub?

Biting midges (Diptera: Ceratopogonidae) from Martín García Island, Argentina

Nearly 230 species of biting midges have been recorded or described from Argentina; 38 of them are known from the Buenos Aires province and only one is cited from Martín García Island. This paper presents the results raised from six collecting trips which took place on the island during spring 2005, summer 2006 and autumn 2009. Diverse sampling sites including permanent and temporary aquatic environments were chosen, most of the ten sampling sites were ponds of diverse origin, some of these environments were covered with floating vegetation as Lemna gibba, Lemna minuscule, Salvinia biloba, Salvinia minima, Azolla filiculoides, Limnobium laevigatum, Pistia stratiotes, Spirodela intermedia, Wolffiella oblonga and Wolffia columbiana. Other sites were placed in urban and suburban areas. Adults were collected with sweep nets at sunrise and sunset and with light traps at intervals of four to five hours at night, depending on electricity availability on the island. Larvae and pupae were collected with different implements depending on characteristics of each surveyed aquatic habitat. In free standing water, they were captured with small sieves or hand pipettes and micropipettes, flotation techniques were utilized for sampling vegetated areas, free and rooted floating hydrophytes were extracted for removing insects among them. Thirteen species of Ceratopogonidae were collected, three of Atrichopogon Kieffer, three of Forcipomyia Meigen, two of Dasyhelea Kieffer, four of Culicoides Latreille, and one of Bezzia Kieffer, all representing new records from the island.

89 HIGH PREGNANCY RATES AFTER TRANSFER OF LARGE EQUINE BLASTOCYSTS COLLAPSED VIA MICROMANIPULATION BEFORE VITRIFICATION

Whereas small equine embryos may be successfully cryopreserved, pregnancy rates after cryopreservation of large equine blastocysts (>300 μm) are low. This has been attributed to their large diameter, failure of cyoprotectants to diffuse into the blastocoele, and/or impedance from the equine embryonic capsule. Previously, we successfully biopsied the trophoblast of equine in vivo-recovered expanded blastocysts (>300 μm; Choi et al. 2009 Reprod. Fertil. Dev. 21, 166-167). In that study, we observed embryonic growth in vitro after warming of a blastocyst vitrified immediately after biopsy. An efficient vitrification method was recently described for ferret embryos, which, like horse embryos, contain a large amount of lipid. This method utilizes very small (250 μm inner diameter) pipettes (Femtotips, Eppendorf; Sun et al. 2008 Biol. Reprod. 79, 832-840). We utilized this technique, with minor modifications, to vitrify equine embryos. In Study 1, the vitrification procedure was performed on equine in vitro-produced blastocysts (<200 μm diameter). All 11 embryos vitrified with the procedure and subsequently warmed grew in culture, showing an increase in diameter over the first 24 h, followed by protrusion of embryonic tissue through holes in the zona pellucida. In Study 2, 10 in vivo-recovered expanded blastocysts, >300 μm diameter, were biopsied using a Piezo drill, causing blastocoele collapse, and were then immediately vitrified. Three of these vitrified blastocysts (346, 399, and 514 μm in diameter) were warmed and transferred to culture; all 3 expanded within 8 h and subsequently proliferated through holes in the embryonic capsule. Seven vitrified in vivo-recovered blastocysts were warmed at the laboratory, shipped to a transfer facility, and transferred transcervically to recipient mares. Five out of 7 transferred blastocysts (71%) resulted in pregnancy at Day 14. Diameters of these embryos before biopsy and vitrification were 360, 398, 415, 434, and 599 μm. The 2 biopsied and vitrified embryos that did not result in pregnancy after transfer were originally 376 and 515 μm in diameter. To our knowledge, this represents the first report of normal (>50%) pregnancy rates achieved after transfer of cryopreserved expanded equine blastocysts. Our findings suggest that biopsy before cryopreservation, which punctures the embryonic capsule and causes blastocoele collapse, combined with vitrification in a small-diameter pipette, may provide an efficient procedure for cryopreservation of large equine blastocysts. Further studies are needed to explore the different factors associated with success of this procedure. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

Comparison of analytical and inverse finite element approaches to estimate cell viscoelastic properties by micropipette aspiration

The viscoelastic properties of cells are important in predicting cell deformation under mechanical loading and may reflect cell phenotype or pathological transition. Previous studies have demonstrated that viscoelastic parameters estimated by finite element (FE) analyses of micropipette aspiration (MA) data differ from those estimated by the analytical half-space model. However, it is unclear whether these differences are statistically significant, as previous studies have been based on average cell properties or parametric analyses that do not reflect the inherent experimental and biological variability of real experimental data. To determine whether cell material parameters estimated by the half-space model are significantly different from those predicted by the FE method, we implemented an inverse FE method to estimate the viscoelastic parameters of a population of primary porcine aortic valve interstitial cells tested by MA. We found that inherent differences between the analytical and inverse FE estimation methods resulted in statistically significant differences in individual cell properties. However, in cases with small pipette to cell radius ratios and short loading periods, model-dependent differences were masked by experimental and cell-to-cell variability. Analytical models that account for finite cell-size and loading rate further relaxed the experimental conditions for which accurate cell material parameter estimates could be obtained. These data provide practical guidelines for analysis of MA data that account for the wide range of conditions encountered in typical experiments.

Myosin II Tailpiece Determines Its Paracrystal Structure, Filament Assembly Properties, and Cellular Localization

Non muscle myosin II (NMII) is a major motor protein present in all cell types. The three known vertebrate NMII isoforms share high sequence homology but play different cellular roles. The main difference in sequence resides in the C-terminal non-helical tailpiece (tailpiece). In this study we demonstrate that the tailpiece is crucial for proper filament size, overcoming the intrinsic properties of the coiled-coil rod. Furthermore, we show that the tailpiece by itself determines the NMII filament structure in an isoform-specific manner, thus providing a possible mechanism by which each NMII isoform carries out its unique cellular functions. We further show that the tailpiece determines the cellular localization of NMII-A and NMII-B and is important for NMII-C role in focal adhesion complexes. We mapped NMII-C sites phosphorylated by protein kinase C and casein kinase II and showed that these phosphorylations affect its solubility properties and cellular localization. Thus phosphorylation fine-tunes the tailpiece effects on the coiled-coil rod, enabling dynamic regulation of NMII-C assembly. We thus show that the small tailpiece of NMII is a distinct domain playing a role in isoform-specific filament assembly and cellular functions.

Effects of near‐infrared laser exposure in a cellular model of wound healing

Clinical studies have demonstrated beneficial outcomes for low-level laser therapy (LLLT) using near-infrared (NIR) wavelengths. It has been hypothesized that the benefits of NIR LLLT are due in part to the thermal effects of NIR exposure. However, it is not clear whether photochemical interactions between NIR light and superficial tissues contribute to beneficial outcomes. To investigate the photochemical effects of NIR exposure, the efficacy of 980 nm NIR LLLT on human fibroblast growth rates is investigated using an in vitro model of wound healing. A small pipette is used to induce a wound in fibroblast cell cultures, which are imaged at specific time intervals over 48 h and exposed to a range of laser doses (1.5-66 J/cm(2)) selected to encompass the range of doses used during other in vivo and in vitro studies. For each image acquired, wound sizes were quantified using a novel application of existing image processing algorithms. Cell growth rates were compared across different laser exposure intensities with the same exposure duration, and across different laser exposure durations with the same exposure intensity. Exposure to low- and medium-intensity laser light accelerates cell growth, whereas high-intensity light negated the beneficial effects of laser exposure. Cell growth was accelerated over a wide range of exposure durations using medium-intensity laser light, with no significant inhibition of cell growth at the longest exposure durations used in this study. Low-level exposure to 980 nm laser light can accelerate wound healing in vitro without measurable temperature increases. However, these results also demonstrate the need for appropriate supervision of laser therapy sessions to prevent overexposure to NIR laser light that may inhibit cell growth rates observed in response to lower intensity laser exposure.

Monolithic microextraction tips by emulsion photopolymerization

Monoliths formed by photopolymerization are excellent means for fabricating functional elements in miniaturized microdevices such as microextraction tips which are becoming important for sample preparation. Various silica-based and polymer-based materials have been used to fabricate monoliths with through pores of several nm to 4 μm. However, the back pressure created by such methods is still considered to be high for microtips that use suction forces to deliver the liquid. In this study, we demonstrated that emulsion techniques such as oil-in-water can be used to form monoliths with large through pores (>20 μm), and with rigid structures on small (10 μL) and large (200 μL) pipette tips by photopolymerization. We further showed that, with minor modifications, various functionalized particles (5–20 μm) can be added to form stable emulsions and successfully encapsulated into the monoliths for qualitative and quantitative solid-phase microextractions for a diverse application. Due to high permeability and large surface area, quick equilibration can be achieved by pipetting to yield high recovery rates. Using tryptic digests of ovalbumin as the standard, we obtained a recovery yield of 90–109% (RSD: 10–16%) with a loading capacity of 3 μg for desalting tips immobilized with C18 beads. Using tryptic digests of β-casein and α-casein as standards, we showed that phosphopeptides were substantially enriched by tips immobilized with immobilized metal affinity chromatography or TiO 2 materials. Using estrogenic compounds as standards, we obtained a recovery yield of 95–108% (RSD: 10–12%) and linear calibration curves ranging from 5 to 100 ng ( R 2 > 0.99) for Waters Oasis HLB tips immobilized with hydrophilic beads.

Development of the safety cytoplasmic exchange method in pronuclear stage oocyte; prevention of mitochondria coexistence in cytoplasmic that used the pronuclear microinjection

OBJECTIVE: The effectiveness of oocyte cytoplasmic exchange has already been demonstrated for the treatment of the infertility cause of defective cytoplasm and of the prevention of heredity of the mitochondrial diseases (Kuwayama, 58th ASRM meeting, 2002). However, mitochondria is brought in with the nuclear when the cell membrane fusion method (McGrath, et al.Science 1983) is used, and two kinds of mitochondria, derivation from both of donor and recipient mitochondria, exist together consequentially in the donor cytoplasm. Because mitochondria coexistence does not exist in nature, and the safety in the situation is uncertain, these two types of mitochondria coexistence is an obstruction in a clinical use of this technology application. To prevent mitochondria coexistence, we tried the development of the pronuclear microinjection method. DESIGN: Prospective in vitro study. MATERIALS AND METHODS: The pronuclear stage oocytes with informed consent were used for the experiment. A pronuclear was removed from the cell membrane crushed oocyte that was crushed by the pulse of the Piezo micromanipulator (the cell membrane crushing method) in modified P1 medium containing 60% of serum substitute supplement (SSS, Irvine Scientific). To remove the cytoplasm containing mitochondria, the removed pronuclear was washed by the repetitive pipetting with the small glass pipette assembled in the micromanipulator. The cytoplasm-free pronuclear was injected directly to the donor cytoplasm with the glass pipette of the inside diameter 15 or 25 μm. The pronuclear injected oocytes were cultured with Cleavage Medium (SAGE) containing 10% of SSS for 30 minutes 5% CO2 5% O2 and 90% N2 at 37C. The oocyte that the cell membrane had been maintained intact after 30 minutes from the pronuclear microinjection was judged survival and the oocyte that was able to confirm the pronuclear intact was judged the pronuclear microinjection success. RESULTS: The survival rate in the pronuclear micro injected oocyte using a pipette inner diameter 15 or 25 μm were 93% (37 / 40) and 50% (20 / 40) (P<0.01), and the pronuclear microinjection success rate were 78% (29 / 37) and 70% (14 / 20), respectively. CONCLUSIONS: The pronuclear that was removed from the oocyte without cytoplasm is able to inject into the enucleated cytoplasm directly. The result shows the possibility that this pronuclear microinjection method becomes the effective treatment of the infertility cause of defective cytoplasm, and of the prevention of heredity of the mitochondrial diseases.

Electrochemical attosyringe

The ability to manipulate ultrasmall volumes of liquids is essential in such diverse fields as cell biology, microfluidics, capillary chromatography, and nanolithography. In cell biology, it is often necessary to inject material of high molecular weight (e.g., DNA, proteins) into living cells because their membranes are impermeable to such molecules. All techniques currently used for microinjection are plagued by two common problems: the relatively large injector size and volume of injected fluid, and poor control of the amount of injected material. Here we demonstrate the possibility of electrochemical control of the fluid motion that allows one to sample and dispense attoliter-to-picoliter (10(-18) to 10(-12) liter) volumes of either aqueous or nonaqueous solutions. By changing the voltage applied across the liquid/liquid interface, one can produce a sufficient force to draw solution inside a nanopipette and then inject it into an immobilized biological cell. A high success rate was achieved in injections of fluorescent dyes into cultured human breast cells. The injection of femtoliter-range volumes can be monitored by video microscopy, and current/resistance-based approaches can be used to control injections from very small pipettes. Other potential applications of the electrochemical syringe include fluid dispensing in nanolithography and pumping in microfluidic systems.

Donor Centrosome Regulation of Initial Spindle Formation in Mouse Somatic Cell Nuclear Transfer: Roles of Gamma-Tubulin and Nuclear Mitotic Apparatus Protein 11

During the process of spindle-chromosome complex depletion in the oocyte, it is unclear whether both gamma-tubulin and nuclear mitotic apparatus protein 1 (NUMA1), which are required for mitotic organization and spindle assembly, are removed. The role of the donor cell centrosome and donor nuclear NUMA1 in the initial spindle morphogenesis and chromosome remodeling also remains unclear. In the present study, we show that in the mouse, the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only approximately 10% of NUMA1 is removed during spindle-chromosome complex depletion in the recipient oocyte. This process does not impede initial spindle morphogenesis and is regulated by the centrosome of the donor cumulus cell. Retaining the donor cell centrosome establishes a monopolar spindle, whereas prior removal of the centrosome by a narrow-bore micropipette leads to bipolar spindle formation. Our data show that the centrosome of the donor cell regulates initial spindle morphogenesis and that the donor cumulus cell NUMA1 compensates for the deficiency in recipient NUMA1 during the formation of metaphase-like structures after nuclear transfer. Full-term offspring of cloned mice were obtained after injection of donor cells only with a pipette having an inner diameter of 7-8 microm, which retained the donor cell centrosome. In contrast, removing the donor cell centrosome with a small pipette impaired preimplantation development and prevented full-term development. In conclusion, the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell in the mouse.

347 EFFECTS OF NEWBORN CANINE SERUM ON IN VITRO MATURATION OF CANINE OOCYTES

Research on in vitro maturation (IVM) of canine oocytes has been scant, due to the fact that canine oocytes are at an immature stage (geminal vesicle stage) during ovulation. The most commonly used protein source for IVM of canine oocytes is fetal bovine serum (FBS), estrus bitch serum (EBS), or bovine serum albumin (BSA). However, the IVM rate of canine oocytes is still low. One of the reasons may be that the culture medium is not appropriate enough for canine oocytes in IVM. Therefore, we evaluated the effects of four different protein sources, BSA, FBS, EBS, and newborn canine serum (NBCS), on IVM of canine oocytes. The NBCS was collected from 3 puppies that had induced euthanasia within 30 min after delivery. The entire volume of blood was collected by heart puncture, through which an 18 G needle was attached to a 10-mL sterile plastic syringe (without anticoagulant), and then transferred into a 10-mL plastic conical tube. The samples were stored overnight at 4�C to allow clotting to occur. To separate the serum, the sample was centrifuged for 10 min at 3000 rpm; the NBCS was heat inactivated at 56�C for 30 min and stored at -70�C for later use. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and the oocytes collected were cultured in NCSU37 supplemented with 1 mg mL-1 cysteine, 0.2 mM pyruvic acid, 20 �g mL-1 E2, and various protein sources (0.3% BSA, 10% FBS, 10% EBS, and 10% NBCS) at 38.5�C, 5% CO2 in air for 72 h. Cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were then fixed in 4% paraformaldehyde supplemented with 10 �g mL-1 Hoechst 33342 at room temperature for 40 min. Nuclear status was observed using fluorescence under a UV light. The proportion of oocytes at the MII stage was significantly higher in the NBCS group than in the EBS, FBS, and BSA groups (14.6 vs. 10.4, 8.8, and 3.3%; P &amp;lt; 0.05). These results indicate that the supplementation of NBCS in IVM medium can enhanced the development of canine oocytes to the MII stage. This work was supported by KOSEF (Grant M10525010003-05N2501-00310) and ARPC (Grant 203121-03-2-SB010).

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter &gt;110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

Micropipette: a new technique in emulsion research

Micron-scale studies on emulsions have, to date, been largely limited to the imaging of colloidal structures. In this communication, micropipette techniques are introduced as a progression beyond mere visualization: using small suction pipettes, mechanical experiments are performed on individual emulsion drops, from which interfacial properties can be deduced. To demonstrate this technique, the interfacial tension, emulsion stability and adsorption characteristics are directly assessed at the surfaces of micron-sized water droplets that are dispersed in crude oil.

Oligo(dT)-immobilized Pipette Tip: Efficient New Methodology for mRNA Preparation and Direct Gene Amplification

One powerful gene amplification technology, reverse transcription-PCR (RT-PCR), allows researchers to derive functional genetic information from small amounts of clinical specimens; however, it is labor-intensive, and multiple steps are needed to prepare mRNA in good quality and in good yield. We have been developing rapid and efficient procedures for mRNA purification and detection in microplates (1)(2)(3)(4). The oligo(dT)-immobilized polystyrene microplate (GenePlate) can capture mRNA from crude cell lysates (1), quantify it (2), synthesize sense and antisense strands of cDNA (1)(3), and preserve cDNA (cDNA bank) (4). The most recent oligo(dT)-immobilized polypropylene microplate (GenePlate-PP) has the additional advantage of allowing direct RT-PCR in the plate immediately after mRNA is captured (5). Here, we introduce an oligo(dT)-immobilized pipette tip for not only mRNA purification but also for direct RT-PCR in a single pipette tip. Disposable polypropylene pipette tips (tip volume, 1–200 μL; Fisher Scientific) immobilized with oligo(dT20) via the 5′ end were kindly provided by Advanced Gene Computing Technologies (AGCT, Irvine, CA). Rabbit globin mRNA was purchased from Gibco-BRL, and total RNA was extracted from rat brain with guanidine isocyanate lysis and acid-phenol extraction (6). Rabbit globin mRNA (200 ng) or rat brain total RNA (5 μg) was suspended in hybridization buffer (10 mmol/L Tris, pH 7.6, 1 mmol/L EDTA, 500 mmol/L NaCl, 20 mmol/L vanadyl ribonucleoside complex) in a final volume of 70–100 μL in 10-mL tubes. The oligo(dT)-immobilized tips were attached to small disposable pipettes (length, 9.5 cm; weight, 6.1 g; Tricontinent), and were placed in 10-mL tubes to aspirate a fixed volume (50 μL) of mRNA solution. Each pipette was supported vertically within 10-mL tubes. Because 10-mL tubes contained a larger volume of mRNA solution than the tips can aspirate, the excess solution prevented evaporation from tips during incubation. To …

The pipette aspiration applied to the local stiffness measurement of soft tissues.

A simple method of identifying the initial slope of the stress-strain curve (i.e., Young's modulus of the soft tissue) by introducing the pipette aspiration technique is presented. The tissue was assumed to be isotropic and macroscopically homogeneous. Numerical simulations by the linear finite element analysis were performed for the axisymmetric model to survey the effects of friction at the tissue-pipette contact boundary, pipette cross-sectional geometry, relative size of the specimen to the pipette, and the layered inhomogeneity of the specimen tissue. The friction at the contact region had little effect on the measurement of Young's modulus. The configuration of the pipette was shown to affect the measurement for small pipette wall thickness. The measurement also depended on the relative size of the specimen to the pipette for relatively small specimens. The extent of the region contributing to the measurement was roughly twice the inside radius of the pipette. In this region, the maximum stress did not exceed the level of the aspiration pressure, with only minor exceptional locations. Calculation of strain energy components indicated that the major contributions to the deformation under pipette aspiration were by the normal extension and shear deformation in pipette axial direction. Experimental verification of the present method for the isotropic, homogeneous artificial material is also presented.

A patch-clamp study of delayed rectifier currents in skeletal muscle of control and mdx mice.

1. Potassium currents were measured in the extensor digitorum longus muscle of normal and mdx mice, which lack the protein dystrophin, using the cell-attached and inside-out patch clamp techniques, in the presence of asymmetrical K+ concentrations (3 mM in the pipette, 160 mM in the bath). 2. In cell-attached patches, the delayed rectifier was the most commonly found potassium channel, with a density of roughly 8 channels microns-2. Outward macroscopic currents were activated in macropatches depolarized to potentials positive to -60 mV. The probability of opening reached half-maximal values around -40 mV for control patches and -31 mV for patches from mdx mice. 3. Tail currents were linear in the range between -60 and +20 mV, reversing close to -100 mV. The single channel current at 0 mV, estimated from non-stationary analysis of variance, was used in conjunction with the slope of the linear part of the tail current to calculate the single channel conductance, yielding a value of 19 +/- 1 pS. 4. At 0 mV, the delayed rectifier inactivated with two time constants, of 70 +/- 20 ms and 600 +/- 200 ms. Prepulses of 500 ms duration to different potentials produced incomplete inactivation with inactivation reaching 50% of its maximum at -50 mV. 5. Single channel activity was recorded using small pipettes. Both single channel conductance and kinetic behaviour were in agreement with the macroscopic current data. 6. In excised patches, the delayed rectifier current ran down, unmasking other K+ channels. A Ca(2+)-dependent K+ channel of 186 pS (BK-like channel) was found frequently in patches bathed in solutions containing appropriate concentrations of calcium, especially at stronger depolarizations. A K+ channel of 63 pS was unmasked in control excised patches bathed in solutions devoid of ATP. This channel was not observed in patches excised from mdx fibers.

テオフィリン徐放性シロップ製剤の調剤および服薬時における留意点

THEODUR®syrup (TDRsyr) is the first liquid dosage form of a long-acting theophylline in Japan. The dosage form design, a matrix form containing theophylline, is suspended in Dsorbitol solution having a high viscosity. To ensure its efficacy, it is necessary to develop precise dispensing technigue. The content of theophylline in TDRsyr was measured by highperformance liquid chromatography (HPLC) after methanol extraction. The concentration of theophylline, after being shaken in a container of TDRsyr (20mg/ml), was 101.5w/v% of the indication value. And its coefficient of variation was 0.7%(n=5), conforming that the quantitative method has good reproducibility. For reliable dispensing, shaking the commercial TDRsyr for longer than 40 minutes is required, and shaking must be done in an up-and-down manner 8 times (8 cycles) per second. Moreover, prior taking it as a dosage, it must be shaken for longer than 2 minutes for 4 cycles per second. When a patient measures out the TDRsyr dosage, a measuring cup is found to be more suitable for reliable dispensing than a small pipette.