Subepidermal Tissue Research Articles

Auxin-transport-dependent leaf vein formationThis paper is one of a selection published in a Special Issue comprising papers presented at the 50th Annual Meeting of the Canadian Society of Plant Physiologists (CSPP) held at the University of Ottawa, Ontario, in June 2008.

The formation of vein patterns in leaves has captivated biologists, mathematicians, and philosophers. In leaf development, files of vein-forming procambial cells emerge from within a seemingly homogeneous subepidermal tissue through the selection of anatomically inconspicuous preprocambial cells. Although the molecular details underlying the orderly formation of veins in the leaf remain elusive, gradually restricted transport paths of the plant signaling molecule auxin have long been implicated in defining sites of vein differentiation. Several recent advances converge to more precisely define the role of auxin flow at successive stages of vascular development. The picture that emerges is that of vein formation as a self-organizing, reiterative, auxin-transport-dependent process.

The volumetric component of individual leaf expansion: Taking into account sub-epidermal tissues in the description of leaf expansion over time

Most leaf development studies at the cell and organ levels have been limited to the leaf surface, with data referring to the leaf surface area and to the number and surface area of epidermal cells. However, leaf sub-epidermal tissues, the palisade and spongy mesophyll, contain the main actors in photosynthesis. The number and thickness of palisade cell layers and the volume occupied by spongy mesophyll (cells and intercellular spaces) affect the accumulation of photosynthates and, as such, whole plant growth. Studies into the leaf phenotype of growth-affected Arabidopsis thaliana mutants have revealed a higher variability in leaf thickness than in leaf surface area. In general, there is no correlation between these two variables, which means that to describe a leaf phenotype, leaf volume has to be taken into account. A method has been developed for high-resolution imaging of leaves in three dimensions usingmultiphoton laser scanning microscopy, and for the analysis of images, providing data on volumes and volumetric proportions of cells and tissues and cell density. The method has been used in the study of A. thaliana leaf expansion from emergence to the onset of senescence for leaves located at different nodal positions in the rosette, completing our knowledge of individual leaf development processes with their volumetric component. The method will further be applied in the study of leaf plasticity in response to the environment for both A. thaliana and apple tree, a model and an agronomic species, respectively. (Texte integral)

Moxa sticks: Thermal properties and possible implications for clinical trials

The aim of the study was to examine the infrared spectrum and the irradiance of different types of moxa sticks to develop a basis for a moxa stick control therapy. An IR radiometer was used to measure the spectral infrared irradiance of seven glowing moxa sticks of different types in dependence of distance to the heat. All sticks investigated showed a similar spectral distribution of the emitted IR radiation with maxima of about 83.5-87.5% in the range long-wavelength IR radiation (IR-C) and with small contributions of both short wavelength IR radiation (IR-A) between 2.2% and 5.5% and of middle wavelength IR radiation (IR-B) between 9.0% and 12.1% of the total IR emission. Study results showed that only a small proportion of the IR radiation emitted by moxa sticks is capable of affecting subepidermal tissue. This finding indicates that thermal effects of moxa sticks are caused primarily by superficial effects on the skin. Because most heat receptors are located in the superficial skin, it thus appears impossible to separate the effects of moxa sticks from the sensation of heat. These results should be taken into account while developing placebo or sham moxibustion devices.

Expression analysis of ANTHOCYANINLESS2 gene in Arabidopsis

The ANTHOCYANINLESS2 ( ANL2) gene of Arabidopsis thaliana affects the accumulation of anthocyanin in subepidermal cells and the organization of cells in the primary root, suggesting that this homeobox gene determines the nature of epidermis and/or subepidermis. In situ hybridization showed that ANL2 mRNA accumulated in peripheral region of young leaves, flower meristems, anthers and pistils. Promoter activity of ANL2 was also examined by using uidA as a reporter. β-Glucuronidase (GUS) was expressed in the shoot apex, young leaves, root tips, anthers and ovules. In rosette leaves, subepidermal tissues showed strong GUS expression, while epidermal cells including guard cells and trichome had weak GUS expression. In the primary root, GUS was expressed in epidermal and lateral root cap cells. In the rosette leaf, the pattern of GUS expression was correlated with the pattern of anthocyanin accumulation in the subepidermis but not in epidermis. The expression of ANL2 was not affected by irradiation and the defects in TTG1.

Time‐lapse imaging of Arabidopsis leaf development shows dynamic patterns of procambium formation

The principles underlying the formation of leaf veins have long intrigued developmental biologists. In leaves, networks of vascular precursor procambial cells emerge from seemingly homogeneous subepidermal tissue through the selection of anatomically inconspicuous preprocambial cells. Understanding dynamics of procambium formation has been hampered by the difficulty of observing the process in vivo. Here we present a live-imaging technique that allows visual access to complex events occurring in developing leaves. We combined this method with stage-specific fluorescent markers in Arabidopsis (Arabidopsis thaliana) to visualize preprocambial strand formation and procambium differentiation during the undisturbed course of development and upon defined perturbations of vein ontogeny. Under all experimental conditions, we observed extension, termination and fusion of preprocambial strands and simultaneous initiation of procambium differentiation along entire individual veins. Our findings strongly suggest that progressiveness of preprocambial strand formation and simultaneity of procambium differentiation represent inherent properties of the mechanism underlying vein formation.

The Relationship of Langerhans Cells to Melanocytes and Schwann Cells in Mature Cystic Teratomas of the Ovary

Mature cystic teratomas have been widely studied relative to their tissue components derived from all 3 embryonic layers, and immunohistochemical methods have demonstrated a variety of neurohormonal polypeptides. To our knowledge, Langerhans cells (LCs), which are a peculiar component of epidermis, have not been reported in ovarian teratomas. The origin of these cells is still a matter of debate, ranging from bone marrow stem cells to neural elements. Thirty mature teratomas of the ovary were studied by immunohistochemistry using CD1 (specific against dendritic LCs), S100 protein (against LCs and melanocytes), and melan-A and HMB45 (against melanocytes). Furthermore, antibodies for identifying subsets of lymphocytes and monocytes (CD3, CD4, CD8, CD20, and CD68) were used. Histologic examination showed teratomas with the presence of all 3 embryonic layers in variable proportions in 23 cases, while 7 teratomas were composed only of epidermis without appendages or other tissues. Immunohistochemistry identified LCs among the suprabasal layers of epidermis in the same sites at which melanocytes were seen in the basal layer. CD1-positive LCs sometimes appeared to cross the basal membrane and penetrate the subepidermal tissue (related to their known migratory ability), and they were associated there with T-cell line lymphocytes (CD3 positive). These findings were commonly observed in teratomas that included all 3 embryonic layers and neural tissues. Notably, LCs and melanocytes were undetectable in the 7 teratomas composed of epidermis only. Our observations of LCs in ovarian teratomas led us to consider these cells to be derived from neural cells, possibly related to Schwann cells, in accord with the original description by Langerhans. In fact, LCs are always associated with melanocytes, which are universally considered to be derived from the neural crest, as are Schwann cells and peripheral nerves. Finally, we propose that LCs may be part of a cytoimmunologic system related to the T-cell compartment, with a stem cell derived from the neural crest.

Colonization and Nitrogen-Fixing Ability of Herbaspirillum sp. Strain B501 gfp1 and Assessment of Its Growth-Promoting Ability in Cultivated Rice

The endophytic colonization, nitrogen fixation, and plant growth-promoting abilities of Herbaspirillum sp. strain B501 gfp1, which is a diazotrophic endophyte isolated from wild rice, were studied after infection (at 102 and 108 cells ml-1) of seedlings of cultivated rice Oryza sativa cv. Nipponbare. Both doses resulted in colonization of the roots and stem (basal stem and leaf sheath). No colonization of leaves was observed. Higher bacterial populations were observed in the roots than stems. The bacteria colonized the intercellular spaces of the root epidermis and the spaces at the junctions of the lateral roots. They also colonized the epidermis and pericycle of the basal stem and the sub-epidermal tissues of the dermal tissue system of the leaf sheath at later stages. The colonizing bacteria incorporated significant amounts of 15N2 into the infected plants. The inoculated plants also had higher dry weights and fresh weights than the control (uninoculated) plants.

Isolation and study of active ATP-dependent phosphofructokinase from apple fruits Pyrus domestica Borkh

The activity of ATP-dependent phosphofructokinase (PFK) from subepidermal tissue of apple fruits was studied. The enzyme extracted under optimal conditions was stable for 14 h at room temperature. The enzyme was partially purified by ammonium sulfate fractionation and dialysis. PFK from apple fruits was found to be ATP-, UTP-, and CTP-specific. It was inhibited by PEP, Gly-2-P, Gly-1,3-DP, and ADP. The effect of the listed inhibitors was diminished by the presence of phosphate. The activity of PFK was stimulated by magnesium cations. The activity of the enzyme in fruits of an Antonovka cultivar was higher than in the Simirenko rennet cultivar by a factor of 1.3.

Flavin-containing polyamine oxidase is a hydrogen peroxide source in the oxidative response to the protein phosphatase inhibitor cantharidin in Zea mays L.

In this study, the specific contribution of polyamine oxidase (PAO), a hydrogen peroxide (H2O2)-producing enzyme, to the oxidative burst induced in maize mesocotyl by the phosphatase inhibitor cantharidin was examined. For this purpose, a pharmacological approach was applied using, either in vitro or in vivo, two strong inhibitors of maize PAO (MPAO), N-prenylagmatine (G3) and its structural analogue Ro5, as well as diphenyleneiodonium (DPI), an inhibitor of the phagocyte NAD(P)H oxidase. DPI was shown to be a good MPAO inhibitor in vitro. G3, Ro5, and DPI were very effective in inhibiting in vivo the extracellular accumulation of H2O2 that is released by mesocotyl segments upon spermidine supply. G3 and Ro5 did not show any inhibition in vitro of either horseradish peroxidase or barley oxalate oxidase. Moreover, G3 and Ro5 did not inhibit the extracellular accumulation of superoxide radical that is released in vivo upon NADH supply. G3, Ro5, and DPI strongly affected H2O2 production induced in maize mesocotyl by cantharidin. Histochemical localization of H2O2 in cantharidin-treated mesocotyl cross-sections revealed an increase of H2O2-specific staining in the epidermal and subepidermal tissues. The effect was also inhibited by G3 and DPI. Moreover, an increase in MPAO activity was observed in the same tissues upon cantharidin treatment. All these data suggest that G3 and Ro5 behave as powerful and selective inhibitors of MPAO activity either in vitro or in vivo and that MPAO activity contributes to a major part of the cantharidin-induced H2O2 synthesis in the apoplastic milieu of maize mesocotyl.

Control of leaf vascular patterning by polar auxin transport.

The formation of the leaf vascular pattern has fascinated biologists for centuries. In the early leaf primordium, complex networks of procambial cells emerge from homogeneous subepidermal tissue. The molecular nature of the underlying positional information is unknown, but various lines of evidence implicate gradually restricted transport routes of the plant hormone auxin in defining sites of procambium formation. Here we show that a crucial member of the AtPIN family of auxin-efflux-associated proteins, AtPIN1, is expressed prior to pre-procambial and procambial cell fate markers in domains that become restricted toward sites of procambium formation. Subcellular AtPIN1 polarity indicates that auxin is directed to distinct "convergence points" in the epidermis, from where it defines the positions of major veins. Integrated polarities in all emerging veins indicate auxin drainage toward pre-existing veins, but veins display divergent polarities as they become connected at both ends. Auxin application and transport inhibition reveal that convergence point positioning and AtPIN1 expression domain dynamics are self-organizing, auxin-transport-dependent processes. We derive a model for self-regulated, reiterative patterning of all vein orders and postulate at its onset a common epidermal auxin-focusing mechanism for major-vein positioning and phyllotactic patterning.

Vertical differences in Phragmites australis culm anatomy along a water depth gradient

We reported on quantitative anatomical comparisons of reed culms grown under a wide range of water depth (from −10 to +230 cm). The study focused mainly on the above water parts of ramets, but also provided an insight into the submerged internodes. Investigated anatomical features were: radial thickness of the internode wall and that of each tissue constituting it (epidermis and subepidermal tissues, aerenchyma channels, cortical sclerenchyma and parenchyma); areas of aerenchyma channels and the summed transversal area of them; areas of the innermost vascular bundles involving the bundle sheath sclerenchyma, phloem and metaxylem vessels; the cross-sectional area of the pith cavity and that of the internode wall. After a 2–3% decrease below the uppermost internode, the thickness of internode tissues, except for cortical sclerenchyma, continuously increased downwards. Quantitative differences between the ramets grown at different water depths were observed only from the lower aerial internodes. Parenchyma taking part considerably in the mechanical stability of culms with its large extension and thick cell walls was more-layered and thicker (with the maximum thicknesses of approximately 1200 and 1000 μm) in ramets from 180–200 than from 20–30 cm water depth. Areas of the innermost vascular bundles increased downwards and decreased after a maximum value appearing in lower internodes of ramets grown in deeper than shallower water. Aerenchyma channels appeared farther up from the water surface in culms in shallower than deeper water: the top ends were found about 50 cm higher in culms from 20–30 than from 180–200 cm water depth. Along the transect at right angles to the shore, the radial thickness and area of aerenchyma channels were higher in ramets grown in medium water depth—the summed transversal area of them was greater than 4 mm 2 in internodes at the water surface, while plants from the two ends of the water depth gradient had smaller aerenchyma channels (with approximately 1 mm 2 total areas). In contrast, pith cavity area at the water surface continuously increased – up to 60 mm 2 – from shallower to deeper water. Therefore, oxygen transport in ramets grown at the open water fringe of stands may occur mostly in the pith cavity.

The site of vitellogenin synthesis in Chinese mitten-handed crab Eriocheir sinensis

The objective of this study was to investigate the synthesis site of vitellogenin in the Chinese mitten-handed crab, Eriocheir sinensis. Using the RT-PCR techniques, the cDNA fragments isolated from the ovaries of vitellogenic female crab, we found that its deduced amino acid sequence had a high identity with that from other decapods crustacean vitellogenin. This cDNA fragments were used as probes to examine the transcription of mRNAs encoding the Vg. The mRNA expression was observed in vitellogenic female hepatopancreas, which was not detected in any other tissues including muscle, heart, and subepidermal tissues. The positive immunocytological staining with antibody against vitellin were found in ovaries and hepatopancreas of vitellogenic female, which was determined by immunological and immunohistochemical techniques. These results suggest that both ovaries and hepatopancreas are capable of synthesizing vitellogenin. Therefore, it was concluded that hepatopancreas is the extraovarian site of vitellogenin synthesis in E. sinensis.

Behavioural differences during host selection between alate virginoparae of generalist and tobacco‐specialist Myzus persicae

AbstractHost plant selection and acceptance by aphids involves four consecutive steps: (1) prealighting behaviour, (2) leaf surface exploration and probing of subepidermal tissues, (3) deep probing of plant tissues, and (4) evaluation of the phloem sap. Host specialisation in aphids may involve not only different performances on potential hosts, but also different strategies for host selection and acceptance. Myzus persicae s.s. (Sulzer) (Homoptera: Aphididae) is one of the most polyphagous aphid species, although a tobacco‐adapted subspecies, M. persicae nicotianae, has been described. These two taxa constitute a good system for studying the effect of host range on host selection strategies. We studied the first two steps in the host selection process by alate virginoparae of M. persicae s.s. and M. persicae nicotianae on host and non‐host plants, using three types of behavioural assays: wind tunnel, olfactometry, and video‐recording. Alate virginoparae of M. persicae nicotianae recognised and chose their host plant more efficiently than M. persicae s.s., on the basis of olfactory and visual cues, and factors residing at cuticular and subcuticular levels. Host recognition was evident before phloem tissues were contacted. Olfactory cues were apparently not involved in host selection by M. persicae s.s.

Morphological Changes in the Temporal Bone and the Expression of Fas Ligand in Patients with Cholesteatoma

Objectives The unique feature of cholesteatoma is the hyperproliferation and accumulation of keratin debris within the middle ear and mastoid cavity, a process that leads to destruction of the surrounding structures in the temporal bone. The proliferation of keratin and the resulting bony destruction in the cholesteatoma matrix are associated with apoptosis. FasL, when conjugated with Fas, is known to trigger apoptosis. This study aimed to analyze the temporal bone patterns in patients with cholesteatoma and the expression of FasL in the cholesteatoma matrix. Methods. From July 1999 to July 2001, all patients with cholesteatoma who received ear operations at the China Medical University Hospital were enrolled in this study. Surgical specimens from all patients were shown histopathologically to have cholesteatoma. The temporal bone patterns in these patients were reviewed by high resolution temporal bone computed tomography. Immunoperoxidase stain with a monoclonal antibody to FasL evaluated the expression of FasL in the cholesteatoma matrix. Postauricular skin, which was harvested during the same surgical procedure served as the control. Result& The temporal bone patterns in the patients were classified as blunted scutum (58%), ossicular chain erosion (58%), erosion of tegmen mastoid-tympanicum (30%), erosion of the otic capsule (25%) and marked sclerosis (50%). Expression of FasL was not detected in the postauricular skin, cholesteatoma matrix or subepidermal granulation tissue. Conclusions. The temporal bone patterns in the patients with cholesteatoma included bony destruction and new bone formation. No expression of FasL was detected in the cholesteatoma matrix or subepidermal granulation tissue. This indicates that apoptosis in the cholesteatoma matrix may not be through the FasL pathway. Therefore, the destruction of the temporal bone in patients with cholesteatoma may not be associated with FasL.

Tissue eosinophilia in pemphigoid gestationis: association with eotaxin and upregulated activation markers on transmigrated eosinophils.

We studied chemotactic cytokines and activation of skin-homed eosinophils in pemphigoid gestationis, a rare autoimmune bullous disease of late pregnancy. Eotaxin and interleukin (IL)-5 were analysed in patients' blister fluid and serum by enzyme-linked immunosorbent assay. Lesional tissue was investigated for eotaxin deposition by immunohistochemistry. Adhesion molecules and activation markers on tissue eosinophils were analysed by immunofluorescence staining. Eotaxin was elevated in serum of two pemphigoid gestationis patients compared with nine healthy pregnant women. Blister fluid contained even higher amounts of eotaxin and in addition IL-5. Eotaxin was mainly expressed in subepidermal tissue in close proximity to eosinophils that expressed high levels of adhesion molecules (CD11b, CD11c, CD18 and CD49d) and the activation marker HLA-DR. High local levels of eotaxin and IL-5 as present in the blister fluid correspond to the high state of activation of the infiltrated eosinophils.

Comparative Proteomic Profiling of Murine Skin

Mammalian skin is regularly exposed to different environmental stresses, each of which results in specific compensatory changes in protein expression that can be assessed by proteomic analysis. We have established a reference proteome map of BALB/c murine skin allowing the resolution of greater than 500 protein spots in a single two-dimensional polyacrylamide gel. Forty-four protein spots, corresponding to 28 different cutaneous proteins, were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. Twenty-five proteins were expressed at higher levels in the epidermis, whereas only nine were found predominantly in the subepidermal tissues. A subset of protein spots exhibited strain-specific expression. Proteins of diverse function were identified, including those involved in stress response, apoptosis, growth inhibition, the maintenance of structural integrity, translational control, energy metabolism, calcium binding, cholesterol transport, and the scavenging of free radicals. Prohibitin expression was detected cutaneously, with more abundant protein and mRNA levels in the epidermis. Five molecular chaperones including protein di-sulfide isomerase, 78 kDa glucose-regulated protein precursor, heat shock protein 60 (HSP60), HSP70, and HSP27 were also identified. Of these, HSP27 expression was confined mainly to the epidermis, and expression of protein disulfide isomerase was found primarily in the subepidermal tissues. Proteomic analysis of skin following heat or cold shock resulted in increased levels of HSP27, HSP60, and HSP70 suggesting involvement of these chaperones in the cutaneous response mechanism to temperature stress. These data establish numerous reference markers within the proteome map of murine skin and provide an important framework for future efforts aimed at characterization of the epidermal and subepidermal responses to environmental changes.

Pepper (capsicum annuum L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot

Three auxin-type herbicides, namely 2.4-dichlorophenoxyacetic acid (2,4-D), (4-chlorophenoxy)acetic acid 2-(dimethylamino)ethyl ester (centrophenoxine), and quinolinecarboxylic acid (quinclorac) induced direct somatic embryogenesis in seed-derived zygotic embryo explants of sweet pepper (Capsicum annuum L.) when added to Murashige and Skoog medium with 200 mM sucrose. Optimum concentrations for embryogenesis induction were 0.40–0.45 mM and 1.15–1.30 μM for 2.4-D and centrophenoxine, respectively (in the presence of 5.0 gl−1 activated charcoal), or 40 μM for quinclorac (in medium without activated charcoal). Somatic embryos emerged from the epidermal and subepidermal tissues and developed on the surface of the explant. Centrophenoxine- or 2.4-D-mediated embryogenesis was accomplished from 95% of the explants in about 3 wk and, on average, six embryos were formed per explant. Induction efficieney was lower for quinelorac. Centrophenoxine-mediated embryognesis was possible in 10 pepper cultivars, the extent of the reponse-being genotype-dependent. embryos detached from the explant and transplanted onto a growth regulator-free medium germinated; however, the recovered regenerants were without a shoot, and some of them bore a single deformed cotyledon while others had no cotyledons. Regenerants lacking a shoot were generated irrespective of the auxin type applied and across all responsive genotypes investigated. Absence of a shoot, resulting from a failure in the establishment of a normal functioning apical shoot meristem, was the principal developmental disorder that precluded regeneration of normal plants via direct somatic embryogenesis. Since stem cells of the shoot meristem become established in globular and heart-stage embryos, we deduce that the absence of a shoot in germinating embryos could orginate from deviant differentiation at these early stages of embryogeny.

Two morphological types of pineal window in catfish in relation to photophase and scotophase activity: a morphological and experimental study.

The pineal window is a transparent/translucent pineal covering on the dorsal surface of the cranium of certain fishes and is associated with light reactions of fish. In the present study, catfish species Clarias batrachus, Heteropneustes fossilis, Mystus vittatus, M. seenghala, and M. cavassius were examined for the type of pineal window present. Two morphologically different types of pineal window were found: an opaque-looking pineal window in C. batrachus and H. fossilis and a translucent type of pineal window in M. vittatus, M. seenghala, and M. cavassius. The distributional pattern of pigments in the melanophores at the pineal window were studied in terms of Melanophore Index (MI). In all of the species studied, a pineal foramen, a subepidermal lens-like tissue, and pineal end vesicle were present. Experiments were carried out on catfish having the opaque pineal window, as it is uncommon in catfish. Catfish with normal and shielded pineal window were exposed to conditions of artificial constant illumination (LL) and darkness (DD) to evaluate the effects of altered photoperiods on the state of pigmentation of melanophores at the pineal window. Recordings of diel activity patterns, which are light dependent in catfish, were carried out under both natural and artificial photoperiods in fish with a normal or shielded window in order to assess its functional nature. The existence of two morphologically and functionally different types of pineal window in a relatively closely related group of catfish has been demonstrated in this study. The nature of the opaque type of pineal window has been reconsidered based on new experimental evidence.

Silicon-Induced Cell Wall Fortification of Rice Leaves: A Possible Cellular Mechanism of Enhanced Host Resistance to Blast

ABSTRACT Locations of silicon accumulation in rice leaves and its possible association with resistance to rice blast were investigated by electron microscopy and X-ray microanalysis. A blast-susceptible cultivar, Jinmi, and a partially resistant cultivar, Hwaseong, were grown under a hydroponic culture system with modified Yoshida's nutrient solution containing 0, 50, 100, and 200 ppm of silicon. Electron-dense silicon layers were frequently found beneath the cuticle in epidermal cell walls of silicon-treated plants. Increasing levels of silicon were detected in the outer regions of epidermal cell walls. Silicon was present mainly in epidermal cell walls, middle lamellae, and intercellular spaces within subepidermal tissues. Furthermore, silicon was prevalent throughout the leaf surface, with relatively small deposition on stomatal guard cells in silicon-treated plants. Silicon accumulation and epidermal cell wall thickness in leaves were greater in cv. Jinmi than in cv. Hwaseong. However, the thickness ratios of the silicon layers to epidermal cell walls were greater in cv. Hwaseong (53.25 to 93.28%) than in cv. Jinmi (36.58 to 66.54%). Leaf blast severity was lower in cv. Hwaseong than in cv. Jinmi and was significantly reduced in silicon-treated plants of both cultivars. These results suggest that silicon-induced cell wall fortification of rice leaves may be closely associated with enhanced host resistance to blast.

Application of the minimal trauma tissue biopsy to transdermal clinical pharmacokinetic studies

Although the transdermal administration of drugs has gained considerable importance, reliable methods for the quantitative assessment of transdermal drug penetration are scarce. The aim of the present study was therefore to evaluate the scopes and limits of the minimal trauma tissue biopsy (MTTB) technique for the in vivo characterization of the transdermal penetration process and the assessment of dermal drug kinetics in humans following topical drug application. Nicotine TTS (21 mg/24 h) was administered transdermally to 13 healthy volunteers. Repeated minimally invasive dermal and subdermal tissue biopsies were obtained at defined time points from defined skin layers directly underlying the TTS. The position of the biopsy needle and depth of biopsate were determined by 2D ultrasound scanning. The biopsy procedure was well tolerated by all volunteers and up to six biopsies within a period of 10 h were easily accepted. Dermal pharmacokinetic profiles for nicotine were obtained in all experiments and corresponded well to the values measured in previous studies on transdermal nicotine penetration. Mean area under the nicotine concentration time curve ( AUC) in subepidermal layers underneath the application site of the TTS was 70.0±55.1 μg/g per h. There was a correlation between the depth of biopsy sampling and dermal nicotine concentrations at steady state ( r=0.7). The MTTB is a suitable, well tolerated technique for the detection of transdermally applied compounds in defined subepidermal tissue layers and could therefore become a valuable tool in the development and assessment of transdermal dosage forms.