电阻抗法在植物根系生物学研究中的应用

因受限于检测方法,对埋藏于土壤中植物根系研究的深入程度远低于其地上部分。传统获取根系的方法不仅费时费力,而且对根系原位分布和生存微环境具有明显的扰动破坏,故随着根系研究的不断深入,迫切需要寻求一种非破坏性的根系检测方法。电阻抗法是在一定频率的外加电源下,测量电路中的根系电学特征(电容、电阻和电阻抗图谱),并且电学特征与根系生物量和形态指标之间存在较好的相关性,然而,由于对电阻抗法基于电路中电流流向等的关键机理尚不清楚,有些学者对该方法的大范围应用提出了质疑。本文首先对生物电阻抗法测量根系的原理和模型进行阐述,然后重点综述根系电阻抗法研究的不同理论方法及存在的问题,最后提出电阻抗法研究植物根系应该解决的重要科学问题,并展望电阻抗法更加广泛的研究和应用前景,以期为获取非破坏性的根系研究技术提供参考。     

Ion and water balance in the epithelium of the abdominal skin of the frogLeptodactylus ocellatus

Summary Epithelia from the abdominal skin of the South American frogLeptodactylus ocellatus were isolated by a method consisting of trypsination and dissection. When mounted between two chambers containing Ringer's solution they show electrical properties similar to those found in whole skin. Oxygen consumption was measured. The effects of amiloride, ouabain and low temperature are studied. An analysis of the ionic distribution in the epithelium is performed. The study demonstrates that, regardless of other effects that trypsin could introduce, it constitutes a valuable tool to analyze the basic mechanisms of transepithelial transport at epithelia, cellular and subcellular levels.     

Simplified and versatile method for isolation of high-quality RNA from pancreas

Isolation of high-quality RNA from pancreas is challenging because the organ contains large quantities of RNases and undergoes autolysis upon harvest. Here we present a simplified perfusion method of the pancreas using an RNase inhibitor. The technique consistently yields high-quality RNA from stored pancreas samples suitable for molecular biology applications, including quantitative RT-PCR. Yields are comparable to RNA isolated from pancreas immediately, but superior to RNA isolated from stored samples that were snap-frozen or immersed in an RNase inhibitor solution. In addition, when compared to the previously reported in situ ductal perfusion technique, our method does not cause histological artifacts.     

Increase in expression of the homeobox gene, GBX1, in retinol-induced epidermal mucous metaplasia

Using a degenerate RT-PCR-based screening method, we isolated the homeobox gene, Gbx1, from the shank skin of 13-day-old chick embryos. By in situ hybridization analysis we showed that the Gbx1 was expressed in the epidermis of the skin and the mucous epithelium of the intestine, and that among many homeobox genes isolated, expression of the Gbx1 strongly increased in the epidermis when the skin was cultured with 20 microM retinol, which induces epidermal mucous metaplasia. The Gbx1 expression in the epidermis was increased by interaction with the retinol-pretreated dermal fibroblasts, resulting in mucous metaplasia. These results suggest that the Gbx1 regulates the differentiation and transdifferentiation of the epithelium and controls the morphology of the epithelium. We isolated the chick Gbx1 cDNA clones. The amino acid sequences in homeodomain and its downstream encoded by human and chick Gbx1 cDNA were almost the same, but those upstream of the homeodomain were rather different.     

Sensitive innervation of the skin of the concha auriculae

By means of the retrograde transport of horseradish peroxidase (HP) method sensitive innervation of the rabbit concha auriculae skin has been studied. For the investigation the skin area 2 X 2 sm large has been chosen on the internal surface of the right concha auriculae 6 sm below the upper edge of the ear. The HP solution is injected in the skin areas 5-6 mm large with normal electrical permeability and with decreased electrical resistance to the constant electrical current. In the first case innervation is ensured only by ipsilateral sensitive neurons of the spinal nodes (SN) CIII. Small neurons make 75% of the total amount of the labelled cells. In the second case about 98% of the skin areas are innervated by the neurons of the SN CIII and about 2% by the neurons of the SN CII. Small and middle neurons make 60% and 38%, respectively. Single labelled cells are revealed in the SN CIV. The HP-positive neurons, innervating the skin areas with an increased electrical permeability nearly 3 times exceed the neurons, projecting on the skin areas with normal electrical permeability. The large labelled neurons make 2% in both cases. Localization of the HP-positive cells in the SN is diffuse, structural compositions of the neurons are not revealed. In the trigeminal node labelled neurons are not found.     

“Density equilibrium” method for the quantitative and rapid in situ determination of lipid,hydrocarbon, or biopolymer content in microorganisms

The work provides a simple method, based on a direct density equilibrium measurement, for the rapid in situ estimation of total lipid, hydrocarbon or biopolymer content in a variety of prokaryotic and eukaryotic samples. The method can be readily applied to live microalgae and photosynthetic bacteria, single‐celled or colonial microorganisms, as well as cellular fractions and isolated subcellular compartments or components. In this approach, the absolute lipid, hydrocarbon, or biopolymer content of the cells can be readily calculated. This method is especially useful for tracking the oil or polymer content of strains of microalgae and other microorganisms, whose lipid, hydrocarbon or biopolymer content may change with cultivation conditions and/or time, as the case would be in microorganism lipid‐induction industrial processes. The method is also useful for the direct in situ measurement of storage polymer accumulation in live cells, such as starch in microalgae and polyhydroxybutyrate, or other polyhydroxyalkanoates, in photosynthetic and non‐photosynthetic bacteria. Biotechnol. Bioeng. 2009;102: 1406–1415. © 2008 Wiley Periodicals, Inc.     

Urinalysis of 4-hydroxynonenal, a marker of oxidative stress, using stir bar sorptive extraction-thermal desorption-gas chromatography/mass spectrometry

A simple and fast method for the measurement of 4-hydroxynonenal (4HNE), a highly toxic end-product of lipid peroxidation, in urine samples is described. The method combines stir bar sorptive extraction (SBSE) with two derivatization steps, followed by thermal desorption and GC/MS. 4HNE is derivatized in situ with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine and the oxime is extracted from the aqueous phase with SBSE. The 4HNE-oxime is further acylated by headspace derivatization prior to thermal desorption. Derivatization reactions and extraction were optimized in terms of reagent quantities, temperature and time. The method is linear over a concentration range of 0.5-5 ng mL(-1) with a correlation coefficient of 0.997. The limit of detection and limit of quantitation are 22 and 75 pg mL(-1) urine, respectively. The high sensitivity of the method allows the measurement of physiological concentrations of 4HNE in urine samples.     

The nature of transepithelial potential of the liver

The method of continuous monitoring of rat liver transepithelial electrical potential in situ (the potential between electrodes inserted into the common bile duct and blood flow) with simultaneous measurement of liver blood filling by photoelectroplethysmography is described. It was suggested that spontaneous oscillations of potential value with the period 20-30 sec found in the experiment were associated with synchronous oscillations of blood vessels and bile ducts cross section square caused by synchronization of pre- and postcapillary sphincters activity.     

Specific sites of fatty acid sterol synthesis in isolated skin components

Metabolic studies on isolated mouse skin components were undertaken to determine the specific sites of fatty acid and sterol synthesis. The concentrations of long-chain fatty acids and sterols and the incorporation of radioactivity from acetate-1-(14)C into these lipids are reported for various skin components and intact whole skin. Only fatty acids having chain lengths of 18 carbons or less were produced by the connective tissue cells of the dermis, while fatty acids containing 20 carbons or more, as well as the acids of 18 carbons or less, were synthesized in the upper dermis (papillary reticulum). The upper dermis also produced significant quantities of eicosenoic acid and of an octadecadienoic acid (not linoleic acid), and incorporated labeled acetate into fatty acids containing an odd number of carbons. Removal of the epidermis and adnexa diminished sterol synthesis. However, the upper region of the dermis was capable of synthesizing, from acetate, large quantities of unidentified nonsaponifiable lipids which were neither sterols nor squalene.     

Newly explored electrical properties of normal skin and special skin sites.

Skin impedance measurements at various skin sites yield different impedance loci for normal skin and special skin sites. The results of skin impedance measurements taken at such sites with a two-electrode measurement system are presented herein. Some of these sites can be identified as acupuncture points. Data from 4 volunteers were acquired by means of a data acquisition board and a measuring system consisting of the measurement circuit, including several electrode types, and a power supply. The Cole model is a model for an equivalent electrical circuit of the skin-electrode system. The system was used to derive skin-typical parameters from the Bode plot of the whole system. These parameters are the fractional power a, the pseudo-capacity K, the parallel resistance Rp, and the serial resistance Rs of the equivalent electrical circuit. The results show that the measured parameters differ between normal skin and special skin sites. These effects have not previously been discovered by other authors, since there has been no systematic investigation of many acupuncture points to date, and there has been no apparent need for such an investigation. A number of necessary criteria for acupuncture point detection can be derived from the results obtained.     

Biomedical Implementation of Liquid Metal Ink as Drawable ECG Electrode and Skin Circuit

Background

Conventional ways of making bio-electrodes are generally complicated, expensive and unconformable. Here we describe for the first time the method of applying Ga-based liquid metal ink as drawable electrocardiogram (ECG) electrodes. Such material owns unique merits in both liquid phase conformability and high electrical conductivity, which provides flexible ways for making electrical circuits on skin surface and a prospective substitution of conventional rigid printed circuit boards (PCBs).

Methods

Fundamental measurements of impedance and polarization voltage of the liquid metal ink were carried out to evaluate its basic electrical properties. Conceptual experiments were performed to draw the alloy as bio-electrodes to acquire ECG signals from both rabbit and human via a wireless module developed on the mobile phone. Further, a typical electrical circuit was drawn in the palm with the ink to demonstrate its potential of implementing more sophisticated skin circuits.

Results

With an oxide concentration of 0.34%, the resistivity of the liquid metal ink was measured as 44.1 µΩ·cm with quite low reactance in the form of straight line. Its peak polarization voltage with the physiological saline was detected as −0.73 V. The quality of ECG wave detected from the liquid metal electrodes was found as good as that of conventional electrodes, from both rabbit and human experiments. In addition, the circuit drawn with the liquid metal ink in the palm also runs efficiently. When the loop was switched on, all the light emitting diodes (LEDs) were lit and emitted colorful lights.

Conclusions

The liquid metal ink promises unique printable electrical properties as both bio-electrodes and electrical wires. The implemented ECG measurement on biological surface and the successfully run skin circuit demonstrated the conformability and attachment of the liquid metal. The present method is expected to innovate future physiological measurement and biological circuit manufacturing technique in a large extent.     

The Hyperpolarizing Region of the Current-Voltage Curve in Frog Skin

Excitability (action potential and refractory period) has been described by A. Finkelstein in the depolarizing region of the current-voltage (I-V) curve of the isolated frog skin. Recently Fishman and Macey interpreted this phenomenon as a consequence of a region with negative resistance that confers to the I-V curve an N shape. We have studied the I-V relation of the isolated frog skin in the hyperpolarizing region with a current-ramp system. It was found that in Na₂SO₄ Ringer's, the resistance continuously increases in the hyperpolarizing direction. When hyperpolarization reaches 300 mv an electrical breakdown occurs, occasionally followed by a region of negative resistance. In NaCl Ringer's the breakdown was also found although the I-V relation was reasonably linear. Unidirectional Na⁺ outflux was measured at different levels of voltage clamping across the skin and with different Na⁺ concentrations in the solutions. The Na⁺ outflux was found to be relatively independent of these parameters. Based on these results a Na⁺ rectifying structure is postulated. An electrical model for active Na⁺ transport including a diode and an oscillator is proposed. The effects of CO₂, nitrogen, amiloride, and ouabain on the I-V relation are described.     

Identification of a novel fibroblast growth factor, FGF-22, preferentially expressed in the inner root sheath of the hair follicle

We isolated cDNA encoding a novel fibroblast growth factor (FGF-22) (170 amino acids) from human placenta. Of the FGF family members, FGF-22, which appears to be a secreted protein, is most similar to FGF-10 and FGF-7 (approximately 46% and approximately 40% amino acid identities, respectively). The human FGF-22 gene was localized on chromosome 19p13.3. We also isolated mouse cDNA encoding FGF-22 (162 amino acids) from the skin. Mouse FGF-22 shows high homology (87% amino acid identity) to human FGF-22. Mouse FGF-22 mRNA was found to be preferentially expressed in the skin among the mouse adult tissues examined by Northern blotting analysis. By in situ hybridization, FGF-22 mRNA in the skin was found to be preferentially expressed in the inner root sheath of the hair follicle. Therefore, FGF-22 is expected to be a unique FGF that plays a role in hair development.     

Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers

We have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controlled scanner. The median lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single strand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determine pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling.     

Numerical sensitivity modeling for the detection of skin tumors by using tetrapolar probe

The measurement of electrical impedance of skin using surface electrodes permits the assessment of changes in local properties of the skin and can be used in the detection of tumors. The sensitivity of this technique depends mainly on the geometry of the probe and the size of the tumor. In this article, the impedance method was used to estimate the sensitivity of a tetrapolar probe in detecting small regions of increased conductivity in a stratified model of human skin. The impedance method was used to model the potential distribution using fasorial analysis to solve the node equations of the equivalent circuit. Interpolation was applied to reduce discretization error. The skin was modeled as a three-layer structure with different conductivity and permittivity obtained from the literature. A tumor was modeled as a small volume with admittivity four times higher than the normal tissue. Sensitivity calculation was made as a function of electrode diameter and separation, tumor size, and excitation frequency. The simulations indicated that by inserting a one square millimeter tumor in the epidermis, the load impedance to the current source varies about 1% while the transfer impedance varied 8%. The sensitivity also increases nonlinearly with increasing tumor area and thickness. Additionally, it was found that the sensitivity of the transfer impedance has a maximum value when the electrodes are separated by 1.8 mm. The results show that transfer impedance measurements of the skin may detect small skin tumors with a reasonable sensitivity by using an appropriate tetrapolar probe.     

Fiberoptic measurement of tendon forces is influenced by skin movement artifact

Fiberoptic cables have previously been used for tendon force measurements in vivo. To measure forces in the Achilles tendon, a cable is passed mediolaterally through the skin and tendon, transverse to the loading axis. As the tendon is loaded, its fibers compress the cable and modulate the intensity of transmitted light, which can be related to tendon force by an in situ calibration. The relative movement between skin and tendon at the cable entry and exit sites may cause error by bending the cable and thus altering transducer output. Cadaver simulations of walking were conducted to compare fiberoptic measurements of Achilles tendon forces to known loads applied to the tendon by actuators attached in series. Force measurement errors, which were high when the skin was intact (RMS errors 24-81% peak forces), decreased considerably after skin removal (RMS errors 10-33% peak forces). The fiberoptic transducer is a useful tool for measurement of tendon forces in situ under natural loading conditions when skin can be removed, but caution should be exercised during in vivo use of this technique or under circumstances where skin is in contact with the fiberoptic cable at the insertion and exit sites.     

Permeability parameters of the toad isolated stratum corneum.

A technique for isolating the stratum corneum from the subjacent layers of the epithelium was developed which permits studying the stratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50 degrees C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1 +/- 1.3 omega cm2 (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (Lp) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8 X 10(-5) +/- 1.5 X 10(-5) cm sec-1 atm-1 (n=10) in distilled water and 9.2 X 10(-5) +/- 1.4 X 10(-5) cm sec-1 atm-1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skin Lp. The stratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH'S. The membrane presents an isoelectric pH of 4.6 +/- 0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca++ or Cu++) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

Numerical sensitivity modeling for the detection of skin tumors by using tetrapolar probe

The measurement of electrical impedance of skin using surface electrodes permits the assessment of changes in local properties of the skin and can be used in the detection of tumors. The sensitivity of this technique depends mainly on the geometry of the probe and the size of the tumor. In this article, the impedance method was used to estimate the sensitivity of a tetrapolar probe in detecting small regions of increased conductivity in a stratified model of human skin. The impedance method was used to model the potential distribution using fasorial analysis to solve the node equations of the equivalent circuit. Interpolation was applied to reduce discretization error. The skin was modeled as a three-layer structure with different conductivity and permittivity obtained from the literature. A tumor was modeled as a small volume with admittivity four times higher than the normal tissue. Sensitivity calculation was made as a function of electrode diameter and separation, tumor size, and excitation frequency. The simulations indicated that by inserting a one square millimeter tumor in the epidermis, the load impedance to the current source varies about 1% while the transfer impedance varied 8%. The sensitivity also increases nonlinearly with increasing tumor area and thickness. Additionally, it was found that the sensitivity of the transfer impedance has a maximum value when the electrodes are separated by 1.8?mm. The results show that transfer impedance measurements of the skin may detect small skin tumors with a reasonable sensitivity by using an appropriate tetrapolar probe.