GTPγS restores nucleophosmin (NPM) localization to nucleoli of GTP-depleted HeLa cells

Previous studies showed that localization of nucleophosmin/B23 (NPM) to nucleoli requires adequate cellular GTP levels (Finchet al., J Biol Chem 268, 5823–5827, 1993). In order to study whether hydrolysis of GTP plays a role in NPM localization, we introduced a nonhydrolyzable GTP analog into HeLa cells. Cells were first depleted of GTP with the IMP dehydrogenase inhibitor, mycophenolic acid (MA), to induce translocation of NPM from the nucleoli to the nucleoplasm. Non-hydrolyzable GTP analogs were then introduced into cells by electroporation. We found that introduction of the non-hydrolyzable analog, GTPS, was effective in restoring NPM localization to nucleoli. Cells incubated in medium containing G-nucleotides without electroporation showed no effect. To reduce the possibility that cells use guanine from degraded nucleotide to supplement GTP pools via salvage pathways, experiments were also performed in the presence of (6-mercaptopurine) 6MP, a competitive inhibitor of the salvage enzyme, HGPRT (hypoxanthine guanine phosphoribosyl transferase), in addition to MA. Under these conditions, introduction of GTPS still effectively restored the localization of NPM into nucleoli. This study demonstrates that electroporation can be used effectively to introduce nucleotides into cultured cells without excessive loss of viability. Our results also indicate that the GTP dependent localization of NPM to the nucleoli may not require GTP hydrolysis.     

The use of luciferase as a reporter for response of plant cells to the fireblight pathogen Erwinia amylovora

Summary We have introduced the gene encoding luciferase from Photinus pyralis into pear and tobacco cells in order to judge the reaction of plant tissue to damaging conditions such as incubation at high temperature or inoculation with a pathogen. The constitutive expression of the luciferase gene via a strong promoter slowly decreased during propagation of the transformed pear cell line. After various stress treatments the resulting luciferase activity and the ATP content of the plant cells were determined by bioluminescence and found to correspond to each other. Inoculation of transformed pear cells with Erwinia amylovora resulted in a continuous decrease of luciferase activity in contrast to tobacco cells, where the enzyme activity was significantly higher in the first period after inoculation with bacteria compared to the untreated control cells. The pattern of the luciferase activity reflected the slow damage of the host-plant cells by E. amylovora and the elevated metabolism of the non-host cells after inoculation with the pathogen.Abbreviations 2,4-D 2,4-dichloro-phenoxyacetic acid - CaMV Cauliflower mosaic virus - DTT dithiothreitol - EDTA ethylenediaminetetraacetate - FDA fluorescein diacetate - HEPES (hydroxyethyl)piperazine(ethanesulfonic acid) - HR hypersensitive reaction - Tris tris (hydroxymethyl)amino-methane     

Mass spectra of underivatized peptide amides related to substance P

Mass spectra of underivatized hexa- and heptapeptide amides related to Substance P have been obtained with a conventional electron ionization mass spectrometer using sample vaporization from a tungsten wire by the technique of rapid heating, proton transfer ionization using ammonia, and photoplate recording of spectra. These spectra exhibit little evidence of sample pyrolysis and are readily interpreted to yield amino acid sequences.     

Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.     

Stimulation by prostaglandin F2α of acidic glycosaminoglycan production in cultured fibroblasts

The effect of PGF2α on the synthesis of hexosamine-containing substances (acidic glycosaminoglycans and glycoproteins) was studied in cultured fibroblasts derived from a rat carrageenin granuloma. Treatment with PGF2α ranging from 0.01 μg/ml to 20 μg/ml resulted in a significant increase of the production of these macromolecules by the cells. The stimulatory effect was found significant even at the low concentration of 10 ng/ml, and could be seen as early as 3h after exposure to PGF2α. The hexosamine-containing substances increased by PGF2α revealed that 80% of the increase was due to acidic glycosaminoglycans and the rest was due to glycoproteins.