Identification of dynorphin 1-8 in human placenta by mass spectrometry

The human placental villus tissue contains opioid receptors and peptides. The opioid peptides extracted from the villus tissue were fractionated using reverse-phase high performance liquid chromatography and a radio-receptor assay. The presence of dynorphin 1-8 was corroborated by mass spectrometric production of (M + H) ion in the fast atom bombardment mode. This octa-peptide could be the natural ligand of the kappa opioid receptors present in the human placental villus tissue.     

Response of tissue diamine oxidase activity to polyamine administration.

The administration to rats of putrescine (750 mumol/kg body wt.) caused in liver, kidney and heart an increase in putrescine at 1 h and in diamine oxidase (EC 1.4.3.6) activity within 3-6 h. An increase in spermidine was observed at 9 h in liver and at 6 h in kidney, whereas in heart there was no change. The increase in diamine oxidase activity by exogenous putrescine was prevented by the administration of actinomycin D and cycloheximide, suggesting that syntheses of mRNA and protein are involved. Equimolar doses of 1,3-diaminopropane, 1,5-diaminopentane and monoacetylputrescine stimulated, similarly to putrescine, hepatic, renal and cardiac diamine oxidase activity. After the injection of a non-toxic dose of spermidine (750 mumol/kg body wt.), the increase in diamine oxidase activity occurred at 9 h in all the tissues studied, when a substantial putrescine formation from spermidine occurred. sym-Norspermidine, which is unable to form putrescine, did not cause an increase in enzyme activity. The possibility that the tissue contents of putrescine might regulate diamine oxidase activity is discussed.     

Polyamine levels and diamine oxidase activity in hypertrophic heart of spontaneously hypertensive rats and of rats treated with isoproterenol

Polyamine levels and diamine oxidase (EC 1.4.3.6) activity were studied in hypertrophic heart of spontaneously hypertensive rats as well as in the heart of Wistar rats during the development and regression of cardiac hypertrophy induced by isoproterenol administration. In spontaneously hypertensive rats, putrescine content and diamine oxidase activity were higher than those found in normotensive Kyoto-Wistar control rats. During the development of cardiac hypertrophy induced by isoproterenol, there was an increase in polyamine content and diamine oxidase activity. The administration of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity during the first 24 h after isoproterenol administration, demonstrating that the rise in diamine oxidase activity was due to synthesis of new enzyme. Following the cessation of isoproterenol treatment, cardiac hypertrophy regressed and polyamine levels and diamine oxidase activity diminished toward control values. The administration of aminoguanidine to isoproterenol-treated rats caused in the heart an inhibition of diamine oxidase activity that led to an increase in putrescine level beyond the values found in animals given isoproterenol alone. The results suggest that the enhancement of diamine oxidase activity plays a role in the regulation of putrescine level in hypertrophic heart.     

The amino acid sequence of hemoglobin II from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin II from the gill of the clamLucina pectinata consists of 150 amino acid residues, has a calculatedM _m of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J.,J. Biol. Chem. 265, 16054–16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of theLucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 Å of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 inLucina Hb II.     

The complete amino acid sequence of porcine gastrotropin, an ileal protein which stimulates gastric acid and pepsinogen secretion

The stomach is stimulated by an enterooxyntin factor in a delayed response to feeding, resulting in an increase in both gastric acid and pepsinogen secretion. We have previously reported on the identity of such a factor from the porcine ileum (Wider, M. D., Vinik, A. I., and Heldsinger, A. (1984) Endocrinology 115, 1484-1491). This protein, termed gastrotropin, is localized to the distal region of the ileum where it constitutes less than 0.1% of the cytosolic protein. We have completed the primary structure of porcine gastrotropin by Edman degradation and mass spectrometry. Gastrotropin (Mr = 14,054) contains 127 amino acid residues and has a blocked (acetylated) alanine at its NH2 terminus. The sequence of porcine gastrotropin is similar to rat liver fatty acid-binding protein (FABP), with 44 of 127 residues being identical (35%). Homology with other members of the FABP family is significantly less apparent, with the order of similarity being liver FABP greater than heart FABP greater than retinol-binding protein greater than intestine FABP. The sequences of the NH2-terminal regions of these proteins account for virtually all of the homology; there are 9 conserved residues common to all five proteins. Gastrotropin represents the first member of the FABP family which has an extracellular function.     

Immunoreactive evidence of beta-endorphin and methionine-enkephalin-Arg-Gly-Leu in human tooth pulp

For the first time, beta-endorphin-like immunoreactivity (BE-LI) has been measured in human tooth pulp. Separation of peptides from pulp tissue was achieved by acid extraction followed by chromatographic separation through a Sep-Pak disposable cartridge. Reversed phase high performance liquid chromatographic (RP-HPLC) was performed on the peptide-rich fractions for further peptide separation. Radioreceptor assay (RRA) data of the HPLC fractions was used to construct a profile of opioid-receptor active peptides. Radioimmunoassay (RIA) data provided further information. Following acute mechanical stress, a monotonic decrease in BE-LI concentrations was evident according to a four bicuspid extraction order.     

Human CD34+/CD90+ ASCs Are Capable of Growing as Sphere Clusters,Producing High Levels of VEGF and Forming Capillaries

Background

Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells.

Methodology/Principal Findings

In this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers – including CD34, CD90, CD29, CD44, CD105, and CD117 – and endothelial-progenitor-cell markers – including CD34, CD90, CD44, and CD54. Interestingly, CD34⁺/CD90⁺ cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34⁺/CD90⁺ quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31⁺/VEGF⁺/Flk-1⁺) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests.

Conclusions/Significance

Our results demonstrate, for the first time, that CD34⁺/CD90⁺ cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.     

ADAM2 interactions with mouse eggs and cell lines expressing α4/α9 (ITGA4/ITGA9) integrins: implications for integrin-based adhesion and fertilization

Background

Integrins are heterodimeric cell adhesion molecules, with 18 α (ITGA) and eight β (ITGB) subunits forming 24 heterodimers classified into five families. Certain integrins, especially the α₄/α₉ (ITGA4/ITGA9) family, interact with members of the ADAM (a disintegrin and metalloprotease) family. ADAM2 is among the better characterized and also of interest because of its role in sperm function. Having shown that ITGA9 on mouse eggs participates in mouse sperm-egg interactions, we sought to characterize ITGA4/ITGA9-ADAM2 interactions.

Methodology/Principal Findings

An anti-β₁/ITGB1 function-blocking antibody that reduces sperm-egg binding significantly inhibited ADAM2 binding to mouse eggs. Analysis of integrin subunit expression indicates that mouse eggs could express at least ten different integrins, five in the RGD-binding family, two in the laminin-binding family, two in the collagen-binding family, and ITGA9-ITGB1. Adhesion assays to characterize ADAM2 interactions with ITGA4/ITGA9 family members produced the surprising result that RPMI 8866 cell adhesion to ADAM2 was inhibited by an anti-ITGA9 antibody, noteworthy because ITGA9 has only been reported to dimerize with ITGB1, and RPMI 8866 cells lack detectable ITGB1. Antibody and siRNA studies demonstrate that ITGB7 is the β subunit contributing to RPMI 8866 adhesion to ADAM2.

Conclusions/Significance

These data indicate that a novel integrin α-β combination, ITGA9-ITGB7 (α₉β₇), in RPMI 8866 cells functions as a binding partner for ADAM2. ITGA9 had previously only been reported to dimerize with ITGB1. Although ITGA9-ITGB7 is unlikely to be a widely expressed integrin and appears to be the result of “compensatory dimerization” occurring in the context of little/no ITGB1 expression, the data indicate that ITGA9-ITGB7 functions as an ADAM binding partner in certain cellular contexts, with implications for mammalian fertilization and integrin function.     

Hepatocyte growth factor signaling regulates transactivation of genes belonging to the plasminogen activation system via hypoxia inducible factor-1

Hepatocyte growth factor (HGF) plays an important role in tumor growth and progression also by regulating invasive/metastatic phenotype and angiogenesis. Here we report that a molecular mechanism possibly contributing to these functions of HGF may be hypoxia inducible factor-1 (HIF-1)-dependent expression of genes of the plasminogen activation system. The following findings support this conclusion: (1) HGF enhanced the activity of a luciferase reporter construct under the control of multiple HIF-1 responsive elements (HRE) in HepG2 cells, and the cotransfection of the dominant negative for the beta-subunit (ARNT) prevented this increase; (2) HGF activated uPA and PAI-1 promoters through HIF-1 activity regulated by PI3K/JNK1 transducers, as demonstrated by cotransfection with the reporter gene promoters and the dominant negative for ARNT, p85 subunit of PI3K or JNK1; (3) hypoxia was additive to HGF in increasing reporter vector activities, but probably through different transduction pathways; (4) JNK1 wild-type expression vector increased HIF-1alpha protein expression probably in a phosphorylated state and, thus, functional for transactivating activity; and (5) c-Jun did not seem to be involved in the activation of the luciferase construct containing multiple HREs because it was not prevented by expression of TAM-67, which is the dominant negative mutant form for c-Jun.     

Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation

Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha (RPTPalpha) regulates SRC family kinases, potassium channels and NMDA receptors. Here, we report that absence of RPTPalpha compromises correct positioning of pyramidal neurons during development of mouse hippocampus. Thus, RPTPalpha is a novel member of the functional class of genes that control radial neuronal migration. The migratory abnormality likely results from a radial glial dysfunction rather than from a neuron-autonomous defect. In spite of this aberrant development, basic synaptic transmission from the Schaffer collateral pathway to CA1 pyramidal neurons remains intact in Ptpra(-/-) mice. However, these synapses are unable to undergo long-term potentiation. Mice lacking RPTPalpha also underperform in the radial-arm water-maze test. These studies identify RPTPalpha as a key mediator of neuronal migration and synaptic plasticity.