Packing of ribosomes in crystals from the lizard Lacerta sicula

The packing of ribosomes in the large crystalline sheets found in the lizard Lacerta sicula has been investigated by electron microscopy. The ribosomes in each of the two layers composing a sheet are organised as tetramers on a P4 space group lattice. The two layers face in opposite directions and tend to be related to one another crystallographically, generating a family of P422 crystals of different unit cell dimensions. The projected structure of one layer was determined from negatively stained, isolated sheets by separating the contributions from each layer in Fourier transforms computed from electron micrographs. Comparison of the projection map with other, low resolution, analyses of images of isolated eukaryotic ribosomes indicates that the large subunit- small subunit axis lies approximately parallel to the plane of the sheet.     

Acylphosphatase increases the rate of ethanol production from glucose in cell-free extracts of Saccharomyces cerevisiae

Addition of acylphosphatase exerted a stimulating effect on the alcoholic fermentation of glucose by Saccharomyces cerevisiae. The rates of glucose degradation and ethanol production by cell-free extracts of the S-288C strain were measured in the absence and in the presence of various levels of this enzyme. Two acylphosphatase isoenzymes were used; one was purified from horse skeletal muscle and the other from human erythrocytes. Both increased the rate of alcoholic fermentation, but that from erythrocytes proved to be the more efficient. This stimulating action is probably due to an "uncoupling effect" of acylphosphatase on the fermentative process, through hydrolysis of 3-phosphoglyceroyl phosphate. This was demonstrated by the fact that alcoholic fermentation was stimulated considerably by a mixture of ADP and inorganic phosphate and by arsenate as well. The possibility of improving the fermentative capacity of microorganisms may have important biotechnological applications.     

Routine colposcopic survey of patients with squamous atypia. A method for identifying cases with false-negative smears

Routine colposcopy was performed on 376 women with cervical squamous atypia (originally reported as "inflammatory atypia"). Colposcopy showed no abnormalities in 240 cases and a lesion in 136 cases; the latter were sampled by colposcopy-guided biopsy. The biopsy samples showed evidence of human papillomavirus (HPV) infection and/or grade I cervical intraepithelial neoplasia (CIN I) in 42 cases (11.1%), CIN II in 4 cases (1.1%) and CIN III in 5 cases (1.3%); the other 85 biopsied cases were histologically negative. Most cases of HPV/CIN I (35 of 42) and all of the cases of CIN II-III occurred in women under the age of 40. The detection rates were 4.4% for CIN II-III in women under the age of 40, 4.0% for HPV/CIN I in women 40 and older and 17.2% for HPV/CIN I in women under the age of 40 (P less than .001). It thus appears that women under the age of 40 who show cytologic evidence of squamous atypia would benefit from colposcopic examination.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Nuclear localization of TRK-A in liver cells

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.     

Effect of copper on callus growth and gene expression of in vitro-cultured pith explants of Nicotiana glauca

Abstract

Copper is a vital component of electron transfer reactions mediated by proteins such as superoxide dismutase, cytochrome c oxidase and plastocyanin, but its concentrations in the cells needs to be maintained at low levels. In fact, the same ability of this essential metal ion to transfer electrons can also make it toxic to cells when present in excess. In vitro cultured explants of Nicotiana have been extensively used as a model to analyse metal-DNA interactions. In this report, we examined the effect of copper (1, 10 and 100 μM CuSO₄) on callus growth and protein synthesis of in vitro-cultured pith explants of Nicotiana glauca. In addition, a N. glauca cDNA library from Cu-treated (100 μM CuSO₄) pith explants cultured in vitro for 24 h was analysed by mRNA differential screening. The copper treatments inhibited callus growth of pith explants. The extent of inhibition was directly correlated to metal concentration. One and 10 μM CuSO₄ induced a notable increase of proteins synthesis relative to control explants. By contrast, 100 μM CuSO₄ inhibited protein synthesis relative to control extracts. The SDS-PAGE fluorography of pith proteins revealed, in Cu-treated extracts qualitative and/or quantitative differences in the synthesis of some polypeptides compared with control explants. Copper-modulated patterns of gene expression were also analysed by mRNA differential screening. The N. glauca genes isolated from Cu-treated pith explants shared common identities with other genes known to be elicited by diverse stresses, including pathogenesis and abiotic stress. In particular, the cDNAs were homologues to genes encoding cell wall proteins (i.e., extensin, and arabinogalactan-protein) and pathogenesis-related proteins (i.e., osmotin, endochitinase and a member of the Systemic Acquired Resistance gene family). In addition, an MD-2-related lipid-recognition (ML) domain protein and the enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase appeared involved in the response to copper stress. In animal cells, AdoHcy hydrolase is a copper binding protein in vivo, which suggests that, also in plant tissues, this enzyme may play an important role in regulating the levels and intracellular distribution of copper.     

In vitro activity of novel in silico‐developed antimicrobial peptides against a panel of bacterial pathogens

Antimicrobial‐peptide‐based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β‐sheet structure in 10‐mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram‐negative and 0.94 to 20.65 μM (1.72‐46.5 µg/ml) for Gram‐positive bacteria. Interestingly, in high‐salt environment, the antibacterial activity was generally maintained for Gram‐negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram‐negative bacteria. A linear time‐dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg²⁺ and Ca²⁺, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti‐infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram‐negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg²⁺ and Ca²⁺, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.