A genetic look at the active site of RNA polymerase III

rpc160-112, a mutant of the RNA polymerase III active site, is corrected in vivo by six second-site mutants obtained by random mutagenesis. These mutants introduce single-site amino acid replacements at the two large subunits of the enzyme. The mutated motifs are conserved in RNA polymerases I and II and, for some of them, in the bacterial enzyme, thus delineating key elements of the active site in eukaryotic RNA polymerases.     

P-selectin,and not E-selectin,negatively regulates murine megakaryocytopoiesis

To assess the role of P-selectin and E-selectin in megakaryocytopoiesis, in vitro assays were performed in animal models deficient in both adhesion receptors. There was a significantly greater number of IL-3-responsive megakaryocyte progenitors CFU (CFU-MK) and an increase in immature megakaryoblasts in response to IL-6 in the P-selectin-null mice compared with the wild-type controls. Furthermore, P-selectin-null mice showed a greater number of CFU-MK colonies derived from CD34(+) cells in response to IL-3 or IL-3 plus stem cell factor. A significant shift in baseline ploidy with a reduction in 8N cells and an increase in 32N cells was also observed in the P-selectin-null mice. Secretion of the inhibitory growth factor TGF-beta1 and not TGF-beta2 was significantly lower in the supernatants of cultures containing bone marrow cells from P-selectin-deficient mice as compared with those from the wild-type control bone marrow cells. No differences in the responsiveness of murine CFU-MK, immature megakaryocytes, or 5-fluorouracil-selected stem cells to cytokines were observed in E-selectin-null mice as compared with the control mice. These studies indicate that the absence of P-selectin, and not E-selectin, resulted in an altered adhesion environment with subsequent expansion of megakaryocyte progenitors and immature megakaryoblasts, enhanced secretion of TGF-beta1, and apparent increased responsiveness to inflammatory cytokines.     

Contribution of molecular modeling and site-directed mutagenesis to the identification of two structural residues,Arg-220 and Asp-227, in aminopeptidase A

Aminopeptidase A is a zinc metalloenzyme involved in the formation of brain angiotensin III, which exerts a tonic stimulatory action on the central control of blood pressure. Thus, central inhibitors of aminopeptidase A constitute putative central antihypertensive agents. Mutagenic studies have been performed to investigate organization of the aminopeptidase A active site, with a view to designing such inhibitors. The structure of one monozinc aminopeptidase (leukotriene A(4) hydrolase) was recently resolved and used to construct a three-dimensional model of the aminopeptidase A ectodomain. This new model, highly consistent with the results of mutagenic studies, showed a critical structural interaction between two conserved residues, Arg-220 and Asp-227. Mutagenic replacement of either of these two residues disrupted maturation and subcellular localization and abolished the enzymatic activity of aminopeptidase A, confirming the critical structural role of these residues. In this study, we generated the first three-dimensional model of a strict aminopeptidase, aminopeptidase A. This model constitutes a new tool to probe further the active site of aminopeptidase A and to design new inhibitors of this enzyme.     

Defective microtubule-dependent podosome organization in osteoclasts leads to increased bone density in Pyk2(-/-) mice

The protein tyrosine kinase Pyk2 is highly expressed in osteoclasts, where it is primarily localized in podosomes. Deletion of Pyk2 in mice leads to mild osteopetrosis due to impairment in osteoclast function. Pyk2-null osteoclasts were unable to transform podosome clusters into a podosome belt at the cell periphery; instead of a sealing zone only small actin rings were formed, resulting in impaired bone resorption. Furthermore, in Pyk2-null osteoclasts, Rho activity was enhanced while microtubule acetylation and stability were significantly reduced. Rescue experiments by ectopic expression of wild-type or a variety of Pyk2 mutants in osteoclasts from Pyk2(-/-) mice have shown that the FAT domain of Pyk2 is essential for podosome belt and sealing zone formation as well as for bone resorption. These experiments underscore an important role of Pyk2 in microtubule-dependent podosome organization, bone resorption, and other osteoclast functions.     

Trace element levels in fish from clean and polluted coastal marine sites in the Mediterranean Sea, Red Sea and North Sea

The bioaccumulation of Hg, Cd, Zn, Cu, Mn and Fe was evaluated in the muscle and liver tissue of four fish species (Siganus rivulatus, Diplodus sargus, Lithognatus mormyrus and Plathychtis flesus) from clean and polluted marine coastal sites in the Red Sea, Mediterranean Sea and North Sea within the framework of the MARS 1 program. Representative liver samples were screened for organic contaminants (DDE, PCBs and PAHs) which exhibited very low concentrations. The levels of Cd, Cu, Zn, Fe and Mn found in the muscle tissue in this study were similar among the four species and within the naturally occurring metal ranges. However, differences were found among the sites. In the Red Sea, Cu was higher in the muscle of S. rivulatus at Ardag and Zn at the Observatory (OBS). Cu, Zn and Mn were higher in the Red Sea than in the specimens from the Mediterranean. The differences were attributed to different diets derived from distinctively different natural environments. D. sargus from Haifa Bay (HB) had higher Cd, Cu and Mn values than specimens from Jaffa (JFA), and L. mormyrus higher Cd, Fe and Mn in HB, corresponding to the polluted environmental status of the Bay. No differences in metal levels were found among the North Sea sites, except for Fe that was lower at the Eider station. Hg was low in all the specimens, but the values varied with species and sites. The lowest Hg values were found in S. rivulatus, the herbivorous species, as expected from its trophic level. Hg in P. flesus was higher than in S. rivulatus but still low. Higher Hg values were found in the muscle tissue of L. mormyrus,with the highest values in D. sargus, both carnivorous species from the same family. Hg in D. sargus was higher in HB than in JFA, as expected, but in the larger specimens of L. mormyrus from JFA values were higher, while in the small specimens there were no differences in Hg values. The levels of all metals were higher in the liver than in the muscle, with enrichment factors ranging from 3 to 104, depending on species and sites. The lowest enrichment values were found for Hg. Based on liver values, the specimens of S. rivulatus from the OBS had the highest levels, as well as D. sargus and L. mormyrus from JFA, contrary to the known relative environmental status of the sites. Received: 25 February 1999 / Received in revised form: 5 June 1999 / Accepted: 7 June 1999     

Characterization of hemin antibacterial action on Staphylococcus aureus

Abstract The mechanism of inactivation of Staphylococcas aureus cells by hemin is described. Protection experiments by sulfhydryl reagents such as cysteine, mercaptoethanol, glutathione or thioglycolate in their reduced form prevent S. aureus bacteria from inactivation by hemin (1.5 × 10⁻⁵ M). The treatment of bacteria by hemin in the presence of one of those reagents (1 × 10⁻² M) showed that the growth rate and viability of the culture remained unchaged. On the other hand sulfhydryl reagents did not prevent the binding of hemin to the bacteria. When cysteine or glutathione were introduced to a culture after exposure to hemin it could neither reverse the damage done to the cells nor shorten the time of the culture's recovery. Another type of protection was obtained by addition of serum albumin which prevented hemin molecules from binding to the bacterial envelopes. Furthermore, when albumin was introduced after the bacteria were treated by hemin it prevented further damage to the survivors and thus shortened the time required for recovery. None of the singlet oxygen quenchers or hydroxyl radical scavengers could protect the bacteria from hemin inactivation. The mechanism by which hemin affects S. aureus is assumed to be by oxidizing a major system within the cell.