Entamoeba histolytica: intracellular distribution of the sec61alpha subunit of the secretory pathway and down-regulation by antisense peptide nucleic acids

The Sec61alpha protein is defined as a highly conserved essential integral component of the endoplasmic reticulum in eukaryotic cells. We report a detailed immunolocalization of the Entamoeba histolytica homologue of the Sec61alpha subunit (EhSec61alpha), which shows an irregular pattern throughout the cell and is also found on the cell surface, its effective down-regulation by means of antisense peptide nucleic acids and its effects on cell proliferation, subcellular distribution of two virulence factors, and the ability of the trophozoites to cause liver abscess in hamsters. Although Sec61alpha levels are specifically decreased in antisense PNA-treated trophozoites, which proliferate more slowly than the controls, mobilization of the cysteine protease 5 and amoebapore to the cell surface is not significantly impeded and the capacity to induce liver abscess in hamsters is largely unaffected. The implications of these findings are discussed in the context of the peculiar cell biology of E. histolytica.     

Zizimin2: a novel, DOCK180-related Cdc42 guanine nucleotide exchange factor expressed predominantly in lymphocytes

A novel superfamily of guanine nucleotide exchange factors for Rho GTPases includes DOCK180 and zizimin1. The zizimin subfamily includes three genes of which only zizimin1 has been cloned. We report here the cloning of zizimin2, identified in a screen for genes enriched in germinal center B cells. Zizimin2 and zizimin1 have similar primary structures and both proteins bound and activated Cdc42 but not the Cdc42-related proteins TC10 or TCL. Their tissue distributions are distinct, however, with zizimin2 expressed predominantly in lymphocytes and an opposite pattern for zizimin1. Zizimin3 was also analyzed and showed distinct GTPase specificity and tissue distribution.     

Synthesis of peptide nucleic acid-peptide chimeras carrying the c-myc tag-sequence

Summary The preparation of peptide nucleic acids (PNA) carrying a c-myc tag-peptide sequence is described. These PNA-peptide chimeras have higher affinity to complementary DNA than unmodified oligonucleotides. Moreover, they can be used as nonradioactive probes with sensitivity similar to other nonradioactive methods.     

Evidence for non-coordinated synthesis of 5 S RNA in the thermophilic fungus,Thermomyces lanuginosus

Analysis of ribosomes and the post ribosomal supernatant fraction of actively growing cells ofThermomyces lanuginosus showed the presence of free 5 S RNA in the supernatant fraction. This 5 S RNA was identical to the ribosomal 5 S RNA in its electrophoretic mobility on 10% Polyacrylamide gel and in its base composition. 5 S RNA from both the sources gave evidence for the presence of diphosphate at the 5’ end. Most of the 5 S RNA that appeared in the cytoplasm was that transported from the nucleus during the isolation. This could be prevented by the use of a hexylene glycol-HEPES buffer.     

Inhibition of Gene Expression by Peptide Nucleic Acids in Cultured Cells

Abstract

We have tested in cultured cells the capacity of antisense and antigene PNAs to inhibit, in a sequence specific manner, the expression of oncogenes in leukaemia and pancreatic carcinoma cells. The results observed appeared promising and suggest that PNA may play in the future an important role in targeting disease-related genes.     

Structural Studies on Porphyrin–PNA Conjugates in Parallel PNA:PNA Duplexes: Effect of Stacking Interactions on Helicity

Parallel PNA:PNA duplexes were synthesized and conjugated with meso‐tris(pyridyl)phenylporphyrin carboxylic acid at the N‐terminus. The introduction of one porphyrin unit was shown to affect slightly the stability of the PNA:PNA parallel duplex, whereas the presence of two porphyrin units at the same end resulted in a dramatic increase of the melting temperature, accompanied by hysteresis between melting and cooling curves. The circular dichroism (CD) profile of the Soret band and fluorescence quenching strongly support the occurrence of a face‐to‐face interaction between the two porphyrin units. Introduction of a L‐lysine residue at the C‐terminal of one strand of the parallel duplex induced a left‐handed helical structure in the PNA:PNA duplex if the latter contains only one or no porphyrin moiety. The left‐handed helicity was revealed by nucleobase CD profile at 240–280 nm and by the induced‐CD observed in the presence of the DiSC₂(5) cyanine dye at ~500–550 nm. Surprisingly, the presence of two porphyrin units led to the disappearance of the nucleobase CD signal and the absence of CD exciton coupling within the Soret band region. In addition, a dramatic decrease of induced CD of DiSC₂(5) was observed. These results are in agreement with a model where the porphyrin–porphyrin interactions cause partial loss of chirality of the PNA:PNA parallel duplex, forcing it to adopt a ladder‐like conformation. Chirality 27:864–874, 2015. © 2015 Wiley Periodicals, Inc.     

PNA-DNA Chimeras Forming Quadruplex Structures

Abstract

¹H-NMR, CD, and UV spectroscopy have been used to investigate the structure of PNA/DNA chimeras forming quadruplex structures. In particular, we synthesized ^5′TGGG^3′-t (1) and ^5′TGG^3′-gt (2), where lower and upper case letters indicate PNA and DNA residues, respectively. CD spectrum and all NMR data of (1) are typical of quadruplexes involving four parallel strands. UV melting profile of (1) indicates that its thermal stability is quite similar to that observed for the reference structure [d(TGGGT)]₄. ¹H-NMR spectrum for ^5′TGG^3′-gt (2) shows that this oligonucleotide is not able to fold into a single, well-defined species.