Biophysical Instrumentation Research Articles

1P312 細胞解析の為のトラップ及びリリース機構を有するマイクロ流路デバイス(バイオエンジニアリング、計測,口頭発表,第45回日本生物物理学会年会)

1P312 細胞解析の為のトラップ及びリリース機構を有するマイクロ流路デバイス(バイオエンジニアリング、計測,口頭発表,第45回日本生物物理学会年会)

1P325 昆虫ウイルス由来タンパク質ポリヘドリンを用いた外来タンパク質固定化技術の開発(バイオエンジニアリング、計測,口頭発表,第45回日本生物物理学会年会)

1P325 昆虫ウイルス由来タンパク質ポリヘドリンを用いた外来タンパク質固定化技術の開発(バイオエンジニアリング、計測,口頭発表,第45回日本生物物理学会年会)

1P357 原子間力顕微鏡による生細胞の応力緩和・熱振動測定(計測,ポスター発表,第45回日本生物物理学会年会)

1P357 原子間力顕微鏡による生細胞の応力緩和・熱振動測定(計測,ポスター発表,第45回日本生物物理学会年会)

1P340 TOF-SIMSによる電極固定化リゾチウムの表面構造測定(計測,ポスター発表,第45回日本生物物理学会年会)

1P340 TOF-SIMSによる電極固定化リゾチウムの表面構造測定(計測,ポスター発表,第45回日本生物物理学会年会)

1P333 一細胞時系列を用いたin silico細胞周期同調法の開発(バイオエンジニアリング、計測,口頭発表,第45回日本生物物理学会年会)

1P333 一細胞時系列を用いたin silico細胞周期同調法の開発(バイオエンジニアリング、計測,口頭発表,第45回日本生物物理学会年会)

1P322 セロトニン受容体に特異的なリガンド検出用バイオセンサー(バイオエンジニアリング、計測,口頭発表,第45回日本生物物理学会年会)

1P322 セロトニン受容体に特異的なリガンド検出用バイオセンサー(バイオエンジニアリング、計測,口頭発表,第45回日本生物物理学会年会)

1P349 研究室系X線蛋白質溶液散乱回折装置の立ち上げ(計測,ポスター発表,第45回日本生物物理学会年会)

1P349 研究室系X線蛋白質溶液散乱回折装置の立ち上げ(計測,ポスター発表,第45回日本生物物理学会年会)

Atopic xerosis: employment of noninvasive biophysical instrumentation for the functional analyses of the mildly abnormal stratum corneum and for the efficacy assessment of skin care products

The subtle dryness of the skin surrounding the lesions of atopic dermatitis (AD) is called atopic dry skin or atopic xerosis (AX). AX is more susceptible to the development of AD skin lesions under various environmental stimuli than the clinically normal skin of the people who have or have had or will have AD, which might be called normal atopic skin (NAS) that shows no functional differences as compared to the skin of normal individuals. Routine histopathologic studies of AX that involve the invasive procedures of biopsy are not so helpful in clarifying the underlying pathogenesis. Modern, noninvasive biophysical instrumentation provides rich and quantitative information about various functional aspects of skin. The stratum corneum (SC) of AX reveals not only decreased hydration but also mildly impaired barrier function demonstrable as an increase in transepidermal water loss, elevated pH values, and an increased turnover rate of the SC consisting of thick layers of smaller-sized corneocytes. These data suggest that AX is related to mildly increased epidermal proliferation as a result of the presence of subclinical cutaneous inflammation. Although AX skin does not display any impairment in the recovery of barrier function after physical skin irritation by tape-stripping, it produces a much more severe, long-lasting inflammatory response together with a delay in barrier repair after chemical irritation such as that induced by sodium lauryl sulphate. The SC of AX is biochemically characterized by reduction in the amounts of ceramides, especially ceramide I, sebum lipids, and water-soluble amino acids. None of these changes in SC functions are seen in NAS, which includes not only the normal-looking skin of AD patients long after regression of all active lesions but also of latent atopic skin such as neonates who later develop AD. This suggests that all of the observed functional as well as biochemical abnormalities of AX are a reflection of subclinical inflammation. The presence of the underlying inflammation in AX also differentiates it from senile xerosis. The mildly impaired SC functions of AX can be improved by daily repeated applications of effective moisturizers, i.e., corneotherapy, which is effective in preventing the exacerbating progression of AX to AD resulting from inadvertent scratching of the skin that facilitates the penetration of environmental allergens into the skin. The biophysical confirmation of such efficacy of moisturizers, including cosmetic bases on the mildly impaired barrier function and decreased water-holding capacity of the SC of AX, definitely substantiates the importance of skin care for the cosmetic skin problems that affect every individual in the cold and dry season ranging from late autumn to early spring.

Objective evaluation of the efficacy of daily topical applications of cosmetics bases using the hairless mouse model of atopic dermatitis

Dry skin (xerosis) is a characteristic change associated with atopic dermatitis (AD) and has often been treated with topical petrolatum applications despite its unfavorable feel. Recently, various therapeutically effective skin-care products with better feel have been introduced. To elucidate the mechanisms underlying the clinical effectiveness of these newer treatments, we used our recently established hairless mouse model of AD. We produced AD-like skin lesions in hairless mice with repeated applications of 2,4,6-trinitro-1-chrolobenzene (TNCB) in acetone for 36 days as reported previously. Groups of five mice with AD-like skin were treated once daily with an emollient-type cream containing petrolatum, a moisturizer-type cream containing 10% glycerin, a solution of 0.01% dexamethasone in acetone, or were left untreated. Over the duration of these treatments, we conducted non-invasive measurements of skin surface condition with biophysical instruments and electron microscopic evaluation of the surface area size and density of rear surface villi of superficial corneocytes. We also obtained skin biopsy samples and blood samples at each time point for histopathological evaluation and to assess serum IgE levels, respectively. After cessation of topical TNCB applications, AD-like skin underwent spontaneous resolution with normalization of skin appearance. A similar reduction in skin fold thickness was observed in the cream-treated mice and in the untreated mouse group, whereas a significant decrease in skin thickness was observed in the dexamethasone-treated mice. Transepidermal water loss, a measure of stratum corneum barrier function, rapidly normalized in all groups, without any statistical differences noted among groups. In comparison with untreated skin, skin surface hydration markedly improved after repeated applications of the moisturizer-type cream, whereas it consistently remained low in dexamethasone-treated skin. The skin treated with emollient-type cream appeared similar to skin that received no treatment. Reduced corneocyte surface area size resulting from repeated applications of TNCB returned to control size with cream treatments, while the corneocyte surface area size became much larger following dexamethasone treatment. In addition, the density of villi on the rear surface of corneocytes decreased with application of the creams or dexamethasone. By contrast, no changes were observed in the number of leukocytes in the epidermis or in serum IgE levels among the different treatment groups. In all treatment groups, even after 32 days of treatment, reapplication of TNCB resulted in early-stage skin swelling, but only in the steroid-treated animals did this swelling show a remarkably prolonged time course. Our present results indicate that the efficacy of skin-care products containing no active ingredients in treating atopic xerosis can be objectively evaluated using the hairless mouse model of AD.

Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding

Thermodynamic stability is fundamental to the biology of proteins. Information on protein stability is essential for studying protein structure and folding and can also be used indirectly to monitor protein-ligand or protein-protein interactions. While clearly valuable, the experimental determination of a protein's stability typically requires biophysical instrumentation and substantial quantities of purified protein, which has limited the use of this technique as a general laboratory method. We report here a simple new method for determining protein stability by using pulse proteolysis with varying concentrations of denaturant. Pulse proteolysis is designed to digest only the unfolded proteins in an equilibrium mixture of folded and unfolded proteins that relaxes on a time scale longer than the proteolytic pulse. We used this method to study the stabilities of Escherichia coli ribonuclease H and its variants, both in purified form and directly from cell lysates. The DeltaG(unf) degrees values obtained by this technique were in agreement with those determined by traditional methods. We also successfully used this method to monitor the binding of maltose-binding protein to maltose, as well as to rapidly screen cognate ligands for this protein. The simplicity of pulse proteolysis suggests that it is an excellent strategy for the high-throughput determination of protein stability in protein engineering and drug discovery applications.

BIONANOMECHANICAL SYSTEMS

▪ Abstract Over the past two decades, advances in biophysical instrumentation have enabled the study of molecular motors at the single molecule level. These studies have inspired the creation of biological/inorganic systems powered by such motors in an attempt to exploit their unique sizes, speeds, functions, and energy utilization capabilities. We give a brief overview of the state-of-the-art of biological and synthetic molecular motors and discuss some initial efforts to exploit their function in engineered structures. We also briefly discuss the construction of devices powered by organized and coordinated arrays of millions of motors in which the growth of cardiac muscle tissue over a microfabricated silicon “skeleton” is directed and controlled.

A portable device using a closed chamber system for measuring transepidermal water loss: comparison with the conventional method

The measurement of transepidermal water loss (TEWL) is frequently used to evaluate the barrier function of the stratum corneum (SC) of diseased skin in vivo, as well as normal skin damaged by external injurious agents. For this purpose, most people use biophysical instruments, based on an open chamber system, measuring the water vapor pressure gradient produced above the skin surface by vapor passing through the SC barrier. Recently, we tested a new portable device (Model H4300 NIKKISO-YSI CO., LTD., Tokyo, Japan) based on a closed chamber system. Its measuring principle is based on a computed TEWL estimation, from the progressive increase in relative humidity inside a closed chamber, due to continuous water evaporation from the skin surface on which the chamber is placed. We conducted an in vivo comparative study between the new portable device and a conventional method, using the DermaLab (Cortex Technology, Hadsund, Denmark), on both healthy and diseased skin surfaces, under well-controlled, standardized, experimental conditions (21 degrees C and 50% relative humidity). We found that the sensitivity of the new device was similar to that of the DermaLab. For example, the poor SC barrier function of normal facial skin was clearly demonstrated by significantly higher TEWL values than those of the extremities (P<0.0001). The compromised barrier function of the lesional skin of atopic dermatitis and psoriasis was also demonstrated in a severity-dependent fashion. However, the TEWL values measured with the new device were much lower than those obtained with the DermaLab, probably reflecting differences in the methodological principles. Nevertheless, they correlated well with those of the latter method (P<0.001). For the instruments compared in the present study, we found the following equation useful for conversion: [corresponding value obtained with the DermaLab]= 1.42 x [the recorded value with Nikkiso-YSI Model H4300] + 1.892. We consider this closed chamber system practical because it can be carried easily and its portability enables TEWL measurements to be taken in any location, without ensuring a special environment against air turbulence. Moreover, it is far less expensive than other devices currently available.

Effects of silicone gel sheet on the stratum corneum hydration

Various groups have reported the efficacy of treatment with topical silicone gel sheet (SGS) for keloids and hypertrophic scars. Because its hydrating effect on the stratum corneum (SC) has been suggested as a mechanism underlying its therapeutic effectiveness, we evaluated it by comparing it with simple plastic film occlusion. With biophysical instruments we assessed the water content of the skin surface as well as its water evaporation on the flexor aspects of bilateral forearms of 10 healthy volunteers for 30min after removal of dressings of SGS or a plastic film that were applied either for 1 day or for 7 days. Occlusion with SGS or plastic film induced hydration of the skin surface, which was followed by an initial quick and later slow process of dehydration when the skin was exposed to the ambient atmosphere. The magnitude of the increase in hydration induced by SGS was always smaller than that of the plastic film occlusion and, unlike the latter treatment, hydration became less with repetition of SGS treatment. On day 7, the SC hydration quickly reduced to the level of non-treated control skin after removal of the dressings. An in vivo test demonstrated that the water-holding capacity of the SC normalised after 7 days of SGS treatment. SGS probably produces a favourable condition for the skin by protecting it from various environmental stimuli, while keeping the SC in an adequately but not over-hydrated condition.

Cultured Skin Substitutes: A Review

Skin substitutes composed of cultured cells and biopolymers provide alternative materials for study of skin biology and pathology, treatment of skin wounds, safety testing of consumer products, and therapeutic delivery of gene products. Most frequently, substitutes for epidermis consist of cultured keratinocytes and dermal substitutes consist of resorbable biopolymers populated with cultured fibroblasts. Preclinical models characterize cellular morphogenesis, antigen expression, and barrier properties in vitro, and recovery of tissue function after grafting. Clinical considerations include time required to prepare cultured autografts, time required for graft vascularization, management of microbial contamination in wounds, mechanical fragility of cultured grafts, and high cost. Safety in graft preparation generally requires the use of materials and procedures that comply with standards for quality assurance. Efficacy of engineered skin substitutes has been evaluated predominantly by subjective criteria, but evaluation may become more objective and quantitative by application of noninvasive biophysical instrumentation. Future directions with engineered skin substitutes are expected to include gene therapy by addition or deletion of selected gene products and establishment of international standards for fabrication and assessment of engineered skin.

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.

Tanner symposium on biophysical measurements and instrumentation

Tanner symposium on biophysical measurements and instrumentation

The first 100 years of “voiceprinting.”

“Voiceprinting,” appropriately defined, is the sound-spectro-graphic technology aspect of speech and voice individuation. A speechsound spectrogram is at once a voicesound spectrogram, since voice is the biophysical instrument of speech. Speaker individuation, known widely today as voice identification, has been with us throughout history. Technological implications of speech/voice idiosyncrasy have been sighted, and/or explored instrumentally, for at least 100 years. Here listed is a partially annotated chronology of authors and publication dates significant for this author's 30-yr commitment to the development of an instrumental speaker-individuation technology: Alexander Melville Bell (Visible Speech: The Science of … Physiological Letters, etc.): 1867; Galton (“fingerprinting”): 1892 (cf. Neumueller GREW—1684); Scripture!: 1902 (653 pp.); Liddell! (The Physical Characteristics of Speech Sound): 1924–1925–1927; Paget: 1930; Scripture (“vowel track analysis”): 1933; Lacerda: 1932–1934; Steinberg!: 1934; Liddell: 1940; Gray/Kopp! (“Voice Print Identification,” 41 pp., explicit): 1944; Potter: 1945–1946; Steinberg/French!: 1946; Koenig/Dunn/Lacy: 1946; Kopp/Green!: 1946; Potter/Kopp/Green!: 1947; Joos! (Acoustic Phonetics): 1948; Truby: 1957-1959!-1960 (“cryprinting”); Fant: 1960; Cummins/Midlo (“Dermatoglyphics”): 1961; Truby: 1962; Kersta (“Voiceprint Identification”): 1962; Garvin/Ladefoged (“Speaker Identification … Speaker Recognition”): 1963 (variously: 1950–1970: Peterson, Stevens, Flanagan, Fant, Lehiste, Truby); Tosi et al.: 1971; Smrkovski: 1974; plus the continuing, practical, day-by-day, field-laboratory investigations of Hall, Smrkovski, Chiari, Richardson, Tosi, Truby, and other Certified Voice Identification Examiners: esp., 1972–1977. And whom, after all, does the relevant “scientific community” comprise … in controversial “Voiceprint” affairs? [Supported by International Association of Voice Identification.]

Analytical techniques for cell fractions: VIII. Analytical differential centrifugation in angle-head rotors

A simple method for studying sedimentation in angle-head centrifuges has been developed. This method (made possible by the use of a titanium rotor, polycarbonate tubes, and an electronic ω 2 t integrator) is applicable to the study of particles having sedimentation coefficients ranging from 4 to 10 6 S. The range can be extended by including studies of 1 g. Sheep red blood cells, polystyrene latex spheres, and BSA were employed as test particles. A 9 ml sample was used in each tube. At the end of a centrifuge cycle, the tubes were rotated in situ 180°, and then 7 ml of supernatant was carefully withdrawn. The remaining 2 ml was arbitrarily considered to be the pellet. When the concentration of particles remaining in the supernatant was plotted against the ω 2 t (on a log scale), very similar curves were obtained with each test particle. The transport of BSA to the pellet volume followed closely that predicted from the separation cell equation. It is proposed that the Pickels theory of angle-head centrifugation must be revised to include the effect of Coriolis forces on fluid elements moving radially in the centrifuge tube. The results obtained suggest that the angle-head centrifuge may be used as a precision biophysical instrument.

Cytochemical evaluation of metabolic inhibitors in cell culture

A method for the cytochemical evaluation of the mechanism of action of potential metabolic inhibitors in cell culture, utilizing the rapid-scanning, high-resolution, automatically integrating biophysical instrumentation developed for cell population analyses is described. This method permits the in situ determination of the amount of DNA, RNA, and protein in individual cells in populations of heterogeneous cells, and the effects of inhibitory agents on these biosynthetic processes. The utility of the method has been demonstrated by the cytochemical evaluation of a series of inhibitory agents selected on the basis of what is known of their mechanisms of action with respect to inhibition of mitosis, synthesis of nucleic acids, or protein. The results of cytochemical analyses of populations of cells exposed to these agents agreed in general with what is known of their mechanisms of action from other studies. In addition to the advantages provided by a method based upon the evaluation of the effects of an inhibitory agent on single cells in heterogeneous populations, cytochemical analyses allow determination of the phase of the cell growth cycle in which inhibition occurs; information which is not readily obtained from other methods of assay. The advantages of such a method for the selection of candidate inhibitory agents for sequential or combination therapy of neoplastic disease, as well as detailed studies in cell physiology, are evident.