Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.     

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.     

GABA and Homovanillic Acid in the Plasma of Schizophrenic and Bipolar I Patients

We have determined the plasma (p) concentration of gamma-aminobutyric acid (GABA) and the dopamine metabolite homovanillic acid (HVA), and the pHVA/pGABA ratio in schizophrenic and bipolar patients. The research was undertaken in a geographic area with an ethnically homogeneous population. The HVA plasma concentrations were significantly elevated in the schizophrenic patients compared to the bipolar patients. The levels of pGABA was significantly lower in the two groups of patients compared to the control group, while the pHVA/pGABA ratio was significantly greater in the both groups of patients compared to the controls. As the levels of pHVA and pGABA are partially under genetic control it is better to compare their concentrations within an homogeneous population. The values of the ratio pHVA/pGABA are compatible with the idea of an abnormal dopamine-GABA interaction in schizophrenic and bipolar patients. The pHVA/pGABA ratio may be a good peripheral marker in psychiatric research.     

In vitro Culture of Medicago arborea L. Anthers: Initial Response

High production of viable somatic embryos was obtained from cultured anthers in the second phase of meiosis, using microscopic level observations of tetrads. The medium with the greatest embryogenic efficiency was H6, composed of Murashige and Skoog (MS) medium with 2 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l⁻¹ of kinetin. All (100%) of the somatic embryos obtained germinated and produced 63% green and 37% albino seedlings. In general, embryogenic calli had a higher ion concentration than non-embryogenic calli, with the exception of calcium whose concentration was higher in non-embryogenic calli. The calli induced in the different media differed in their sucrose and starch compositions. The most embryogenic medium H6-induced calli with the highest sucrose concentration and the lowest starch concentration, before visible embryos were observed. In the leaves of the albino seedlings, sucrose concentrations were very high while those of starch were very low. Ion concentrations were also lower in albino plants than in the leaves of green seedlings, with the exception of calcium, whose concentration was higher. Most of the albino individuals were homozygous, even when their progenitors were heterozygous, thereby confirming their haploid nature.     

Interleukin-1 beta-induced anorexia is reversed by ghrelin

Interleukins, in particular interleukin-1β (IL-1β), reduce food intake after peripheral and central administration, which suggests that they contribute to anorexia during various infectious, neoplastic, and autoimmune diseases. On the other hand, ghrelin stimulates food intake by acting on the central nervous system (CNS) and is considered an important regulator of food intake in both rodents and humans. In the present study, we investigated if ghrelin could reverse IL-1β-induced anorexia. Intracerebroventricular (i.c.v.) injection of 15, 30 or 45 ng/μl of IL-1β caused significant suppression of food intake in 20 h fasting animals. This effect lasted for a 24 h period. Ghrelin (0.15 nmol or 1.5 nmol/μl) produced a significant increase in cumulative food intake in normally fed animals. However, it did not alter food intake in 20 h fasting animals. Central administration of ghrelin reduced the anorexic effect of IL-1β (15 ng/μl). The effect was observed 30 min after injection and lasted for the next 24 h. This study provides evidence that ghrelin is an orexigenic peptide capable of antagonizing IL-1β-induced anorexia.     

Rat organic anion transporting protein 1A1 (Oatp1a1): purification and phosphopeptide assignment

Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated or singly or doubly phosphorylated forms and sensitive to alkaline phosphatase treatment. The beta-elimination reaction resulted in a mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosphorylated peptide, while phosphorylation at S634 and S635 accounted for all doubly phosphorylated peptide. These findings identify the site of oatp1a1 phosphorylation and demonstrate that it is an ordered process, in which phosphorylation at S634 precedes that at S635. The mechanism by which phosphorylation results in loss of transport activity in hepatocytes remains to be established. Whether phosphorylation near the C-terminus inhibits C-terminal oligomerization of oatp1a1, required for normal transport function, can be speculated upon but is as yet unknown.     

Impact of arbuscular mycorrhizal fungi (AMF) and atmospheric CO2 concentration on the biomass production and partitioning in the forage legume alfalfa

The influence of mycorrhizal symbiosis, atmospheric CO₂ concentration and the interaction between both factors on biomass production and partitioning were assessed in nodulated alfalfa (Medicago sativa L.) associated or not with arbuscular mycorrhizal fungi (AMF) and grown in greenhouse at either ambient (392 μmol?mol^?1) or elevated (700 μmol?mol^?1) CO₂ air concentrations. Measurements were performed at three stages of the vegetative period of plants. Shoot and root biomass achieved by plants at the end of their vegetative period were highly correlated to the photosynthetic rates reached at earlier stages, and there was a significant relationship between CO₂ exchange rates and total nodule biomass per plant. In non-mycorrhizal alfalfa, the production of leaves, stems and nodules biomass significantly increased when plants had been exposed to elevated CO₂ concentration in the atmosphere for 4 weeks. Regardless CO₂ concentration at which alfalfa were cultivated, mycorrhizal symbiosis improved photosynthetic rates and growth of alfalfa at early stages of the vegetative period and then photosynthesis decreased, which suggests that AMF shortened the vegetative period of the host plants. At final stages of the vegetative period, AMF enhanced both area and biomass of leaves as well as the leaves to stems ratio when alfalfa plants were cultivated at ambient CO₂. The interaction of AMF with elevated CO₂ improved root biomass and slightly increased the leaves to stems ratio at the end of the vegetative growth. Therefore, AMF may favor both the forage quality of alfalfa when grown at ambient CO₂ and its perennity for next cutting regrowth cycle when grown under elevated CO₂. Nevertheless, this hypothesis needs to be checked under natural conditions in field.     

Hyaluronan and Layilin Mediate Loss of Airway Epithelial Barrier Function Induced by Cigarette Smoke by Decreasing E-cadherin

Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that the HA receptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS.