Co-localization of xenopsin and gastrin immunoreactivity in gastric antral G-cells

Studies indicating evidence for the presence of the amphibian octapeptide xenopsin in gastric mucosa of mammals prompted us to investigate the cellular localization of this peptide. Using the peroxidase-antiperoxidase method and a specific antiserum to xenopsin (Xen-7) on paraffin and adjacent semithin sections of gastric antral mucosa from man, dog, and Tupaia belangeri, we found numerous epithelial cells showing a specific positive immunoreaction. These cells were of typical pyramidal shape and could be classified as of the "open" type. Cell quantification in serial sections processed for xenopsin and gastrin immunoreactivity, respectively, revealed an identical number of cells per section and an identical distribution of these cells in the middle zone of the antral mucosa. Furthermore, adjacent semithin sections demonstrated the colocalization of xenopsin and gastrin immunoreactivity within the same G-cell. The xenopsin antiserum could be completely absorbed with synthetic xenopsin but not with gastrin. Preabsorption tests with neurotensin, a xenopsin related peptide, or with somatostatin, glucagon, and enkephalins gave no evidence for crossreactivity of the xenopsin antiserum with these peptides. It is concluded that gastric antral G-cells in addition to gastrin also contain the amphibian peptide xenopsin.     

Co-localization of xenopsin and gastrin immunoreactivity in gastric antral G-cells

Summary Studies indicating evidence for the presence of the amphibian octapeptide xenopsin in gastric mucosa of mammals prompted us to investigate the cellular localization of this peptide. Using the peroxidase-antiperoxidase method and a specific antiserum to xenopsin (Xen-7) on paraffin and adjacent semithin sections of gastric antral mucosa from man, dog, and Tupaia belangeri, we found numerous epithelial cells showing a specific positive immunoreaction. These cells were of a typical pyramidal shape and could be classified as of the open type. Cell quantification in serial sections processed for xenopsin and gastrin immunoreactivity, respectively, revealed an identical number of cells per section and an identical distribution of these cells in the middle zone of the antral mucosa. Furthermore, adjacent semithin sections demonstrated the colocalization of xenopsin and gastrin immunoreactivity within the same G-cell. The xenopsin antiserum could be completely absorbed with synthetic xenopsin but not with gastrin. Preabsorption tests with neurotensin, a xenopsin related peptide, or with somatostatin, glucagon, and enkephalins gave no evidence for crossreactivity of the xenopsin antiserum with these peptides.It is concluded that gastric antral G-cells in addition to gastrin also contain the amphibian peptide xenopsin.     

A possible alternative mechanism of kinin generation in vivo by cathepsin L

We investigated the ability of cathepsin L to induce a hypotensive effect after intravenous injection in rats and correlated this decrease in blood pressure with kinin generation. Simultaneously with blood pressure decrease, we detected plasma kininogen depletion in the treated rats. The effect observed in vivo was abolished by pre-incubation of cathepsin L with the cysteine peptidase-specific inhibitor E-64 (1 microM) or by previous administration of the bradykinin B2 receptor antagonist JE049 (4 mg/kg). A potentiation of the hypotensive effect caused by cathepsin L was observed by previous administration of the angiotensin I-converting enzyme inhibitor captopril (5 mg/kg). In vitro studies indicated that cathepsin L excised bradykinin from the synthetic fluorogenic peptide Abz-MTSVIRRPPGFSPFRAPRV-NH2, based on the Met375-Val393 sequence of rat kininogen (Abz = o-aminobenzoic acid). In conclusion, our data indicate that in vivo cathepsin L releases a kinin-related peptide, and in vitro experiments suggest that the kinin generated is bradykinin. Although it is well known that cysteine proteases are strongly inhibited by kininogen, cathepsin L could represent an alternative pathway for kinin production in pathological processes.     

Characterisation of bombesin-like immunoreactivity in human fetal lung

A sensitive radioimmunoassay for bombesin-like immunoreactivity (BLI) was developed and utilised in conjunction with G50 gel chromatography and reverse-phase HPLC, to study the content and molecular characteristics of bombesin-like peptides in acid extracts of human fetal lung. The antiserum, (B5), is directed towards the C-terminal region of the bombesin molecule and cross-reacts 70% with synthetic porcine GRP and the synthetic GRP fragment, GRP (14-27). Specimens of lung were collected from fetuses of gestational ages 15-22 weeks, following prostaglandin termination of pregnancy. The tissue was extracted into 0.1 N HCl at 90 degrees C. The mean BLI content was 50.2 pg/mg wet weight of tissue (range 15.5-136 pg/mg; n = 13). No correlation between gestational age and BLI content could be established. G50 gel chromatography of acid extracts, under dissociating conditions, revealed two peaks of BLI, one in the position of synthetic porcine GRP and the second, constituting greater than 90% of the immunoreactivity, eluting with synthetic amphibian bombesin. Reverse-phase ODS silica HPLC of this major G50 peak, utilising a methanol/trifluoroacetic acid gradient, indicated that this peptide was similar to the GRP C-terminal fragment, GRP (14-27). We have therefore (1) confirmed the presence and heterogeneity of BLI in human fetal lung, and (2) shown, for the first time, that the majority of this BLI more closely resembles a fragment of GRP than amphibian bombesin itself.     

Localization of xenopsin and xenopsin precursor fragment immunoreactivities in the skin and gastrointestinal tract of Xenopus laevis

Summary Xenopsin (Xp) and xenopsin precursor fragment (XPF) are bioactive peptides derived from a single precursor molecule; both were isolated previously from extracts of Xenopus laevis skin. The present immunohistochemical study was undertaken to determine the specific cellular localization of these two peptides in the skin and also in the gastrointestinal tract of adult Xenopus. We report here that Xp-like and XPF-like immuno-reactivities co-exist in the granular glands of the skin and specific granular cells in the lower esophagus and stomach. However, only Xp-like immunoreactivity, not XPF-like immunoreactivity, was detected in tall, thin cells of the duodenum and in club-shaped cells of the large intestine. The immunochemical co-localization of the two peptides in specific cells of the skin, lower esophagus and stomach suggests that the same gene is expressed in each of these cells, and that the precursor molecule undergoes similar post-translational processing. In contrast, the observation that certain cells of the duodenum and large intestine display only one peptide immunoreactivity suggests an alternative phenomenon, possibly involving selective peptide accumulation or expression of a different gene.     

In vivo characterization of the mechanical properties of human skin derived from MRI and indentation techniques

The human skin is an exceedingly complex and multi-layered material. This paper aims to introduce the application of the finite element analysis (FEA) to the in vivo characterization of the non-linear mechanical behaviour of three human skin layers. Indentation tests combined with magnetic resonance imaging (MRI) technique have been performed on the left dorsal forearm of a young man in order to reveal the mechanical behaviour of all skin layers. Using MRI images processing and a pre and post processor allows to make numerically individualized 2D model which consists of three skin layers and the muscles. FEA has been applied to simulate indentation tests. Neo-Hookean slightly compressible material model of two material constants (C(10), K) has been used to model the mechanical behaviour of the three skin layers and the muscles. The identification of material model parameters was done by applying Levenberg-Marquardt algorithm (LMA). Our methodology of identification provides a range of values for each constant. Range of values of different material properties of epidermis, dermis, hypodermis are respectively, C10(E)=0.12+/-0.06 MPa, C10(D)=1.11+/-0.09 MPa, C10(H)=0.42+/-0.05 KPa, K(E)=5.45+/-1.7 MPa, K(D)=29.6+/-1,28 MPa, K(H)=36.0+/-0.9 KPa.     

In vivo characterization of the mechanical properties of human skin derived from MRI and indentation techniques

The human skin is an exceedingly complex and multi-layered material. This paper aims to introduce the application of the finite element analysis (FEA) to the in vivo characterization of the non-linear mechanical behaviour of three human skin layers. Indentation tests combined with magnetic resonance imaging (MRI) technique have been performed on the left dorsal forearm of a young man in order to reveal the mechanical behaviour of all skin layers. Using MRI images processing and a pre and post processor allows to make numerically individualized 2D model which consists of three skin layers and the muscles. FEA has been applied to simulate indentation tests. Neo-Hookean slightly compressible material model of two material constants (C₁₀, K) has been used to model the mechanical behaviour of the three skin layers and the muscles. The identification of material model parameters was done by applying Levenberg–Marquardt algorithm (LMA). Our methodology of identification provides a range of values for each constant. Range of values of different material properties of epidermis, dermis, hypodermis are respectively, C10_E = 0.12 ± 0.06 MPa, C10_D = 1.11 ± 0.09 MPa, C10_H = 0.42 ± 0.05 KPa, K _E = 5.45 ± 1.7 MPa, K _D = 29.6 ± 1,28 MPa, K _H = 36.0 ± O.9 KPa.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin

We previously reported that fetal porcine skin-derived stem cells were capable of differentiation into oocyte-like cells (OLCs). Here we report that newborn mice skin-derived stem cells are also capable of differentiating into early OLCs. Using stem cells from mice that are transgenic for Oct4 germline distal enhancer-GFP, germ cells resulting from their differentiation are expected to be GFP(+). After differentiation, some GFP(+) OLCs reached 40-45 μM and expressed oocyte markers. Flow cytometric analysis revealed that ～ 0.3% of the freshly isolated skin cells were GFP(+). The GFP-positive cells increased to ～ 7% after differentiation, suggesting that the GFP(+) cells could be of in vivo origin, but are more likely induced upon being cultured in vitro. To study the in vivo germ cell potential of skin-derived cells, they were aggregated with newborn ovarian cells, and transplanted under the kidney capsule of ovariectomized mice. GFP(+) oocytes were identified within a subpopulation of follicles in the resulting growth. Our finding that early oocytes can be differentiated from mice skin-derived cells in defined medium may offer a new in vitro model to study germ cell formation and oogenesis.     

A possible mechanism for the generation and motion of blobs in tokamaks

A possible mechanism for the generation and motion of so-called blobs—peculiar perturbations that are observed in a tokamak edge plasma—is proposed. It is suggested that blobs are self-contracting plasma filaments generated either by the thermal-radiative instability of a plasma with impurities or by the nonradiative resonant charge-exchange instability resulting from the presence of neutral hydrogen atoms near the tokamak wall. Instability occurs in a narrow temperature range in which pressure is a decreasing function of density. Under these conditions, the most typical perturbations are the local ones that originate spontaneously in the form of separate growing hills and wells in the density. The temperature at the centers of the hills is lower than that in the surrounding plasma, but they are denser and, consequently, brighter than the background. The (denser) hills should move (“sink”) toward the separatrix, while the (less dense) wells should “rise” in the opposite direction, as is observed in experiments. It may even be said that they behave in accordance with a peculiar Archimedes' principle.     

Transfer RNA-like structure of the human Alu family: Implications of its generation mechanism and possible functions

Summary Structural resemblance of the human Alu family with a subset of vertebrate tRNAs was detected. Of four tRNAs, tRNA^Lys, tRNA^Ile, tRNA^Thr, and tRNA^Tyr, which comprise a structurally related family, tRNA^Lys is the most similar to the human Alu family. Of the 76 nucleotides in lysine tRNA (including the CCA tail), 47 are similar to the human Alu family (60% identity). The secondary structure of the human Alu family corresponding to the D-stem and anticodon stem regions of the tRNA appears to be very stable. The 7SL RNA, which is a progenitor of the human Alu family, is less similar to lysine tRNA (55% identity), and the secondary structure of the 7SL RNA folded like a tRNA is less stable than that of the human Alu family folded likewise. Insertion of the tetranucleotide GAGA, which is an important region of the second promoter for RNA polymerase III in the Alu sequence, occurred during the deletion and ligation process to generate the Alu sequence from the parental 7SL RNA. These results suggest that the human Alu family was generated from the 7SL RNA by deletion, insertion, and mutations, which thus modified the ancestral 7SL sequence so that it could form a structure more closely resembling lysine tRNA. The similarities of several short interspersed sequences to the lysine tRNA were also examined. TheGalago type 2 family, which was reported to be derived from a methionine initiator tRNA, was also found to be similar to the lysine tRNA. Thus lysine tRNA-like structures are widespread in genomes in the animal kingdom. The implications of these findings in relation to the mechanism of generation of the human Alu family and its possible functions are discussed.     

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin

Summary Calcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin generelated immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.     

Estimating material parameters of human skin in vivo

An accurate mathematical representation of the mechanical behaviour of human skin is essential when simulating deformations occurring in the skin during body movements or clinical procedures. In this study constitutive stress–strain relationships based on experimental data from human skin in vivo were obtained. A series of multiaxial loading experiments were performed on the forearms of four age- and gender matched subjects. The tissue geometry, together with recorded displacements and boundary forces, were combined in an analysis using finite element modelling. A non-linear optimization technique was developed to estimate values for the material parameters of a previously published constitutive law, describing the stress–strain relationship as a non-linear anisotropic membrane. Ten sets of material parameters where estimated from the experiments, showing considerable differences in mechanical behaviour both between individual subjects as well as mirrored body locations on a single subject. The accuracy of applications that simulate large deformations of human skin could be improved by using the parameters found from the in vivo experiments as described in this study.     

The kinetic properties and reaction mechanism of histamine methyltransferase from human skin.

The substrate kinetic properties of histamine methyltransferase from human skin were studied at limiting concentrations of both histamine and S-adenosylmethionine. Substrate inhibition by histamine was observed at concentrations above 10 microM. Primary plots showed evidence of a sequential reaction mechanism. The Michaelis constants were derived from secondary plots of slopes from the primary plots ([S]/v versus [S]) versus reciprocal of the second substrate concentration. The mean Km values for histamine and S-adenosylmethionine were 4.2 and 1.8 microM respectively. Histamine in concentrations of 25-100 microM inhibited enzyme activity uncompetitively with respect to S-adenosylmethionine. No substrate inhibition was observed with S-adenosylmethionine. To elucidate the reaction mechanism further, inhibition by the two products, S-adenosylhomocysteine and 1-methylhistamine, was studied. S-Adenosylhomocysteine inhibited non-competitively with respect to histamine and competitively with respect to S-adenosylmethionine. 1-Methylhistamine inhibited non-competitively with respect to histamine and to S-adenosylmethionine. These results are interpreted as providing evidence for an ordered sequential Bi Bi reaction mechanism, with the methyl-group donor S-adenosylmethionine as the first substrate that adds to the enzyme and histamine as the second substrate. 1-Methylhistamine is the first product to leave the enzyme and S-adenosylhomocysteine is the second. The results are discussed in terms of the possible role that this enzyme could play in the modulation of histamine-mediated reactions in skin.     

Finite deformation theory for in vivo human skin

A finite deformation mathematical model of in vivo human skin has been developed for the normal physiological load range. Uniaxial load-deformation measurements were carried out with a non-invasive extensometer and utilized in formulating the model. The in vivo strain energy function was found to be a linear function of the first two strain invariants and a quadratic function of the third strain invariant. Only three independent constants were necessary to specify the strain energy function completely for the upper extremities of human volunteers.     

Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo

We describe the differentiation of human embryonic stem (hES) cells into endothelial cells using a scalable two-dimensional method that avoids an embryoid-body intermediate. After transplantation into severe combined immunodeficient (SCID) mice, the differentiated cells contributed to arborized blood vessels that integrated into the host circulatory system and served as blood conduits for 150 d.