Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization

The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.

Dissociation of the Tubulin-sequestering and Microtubule Catastrophe-promoting Activities of Oncoprotein 18/Stathmin

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100–147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5–25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminal–truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (α, β)-methylene diphosphonate–tubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis.

Detection and selective dissociation of intact ribosomes in a mass spectrometer

Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg2+ concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 ± 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components.

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase, promotes dissociation of αs from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of αs from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant αs prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated αs (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound αs, but not αs· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of αs and that dissociation of αs·GTP from βγ is likely to mediate receptor-induced αs depalmitoylation and translocation of the protein to cytosol in intact cells.

A fission yeast homolog of Int-6, the mammalian oncoprotein and eIF3 subunit, induces drug resistance when overexpressed

Through a screen to identify genes that induce multi-drug resistance when overexpressed, we have identified a fission yeast homolog of Int-6, a component of the human translation initiation factor eIF3. Disruption of the murine Int-6 gene by mouse mammary tumor virus (MMTV) has been implicated previously in tumorigenesis, although the underlying mechanism is not yet understood. Fission yeast Int6 was shown to interact with other presumptive components of eIF3 in vivo, and was present in size fractions consistent with its incorporation into a 43S translation preinitiation complex. Drug resistance induced by Int6 overexpression was dependent on the AP-1 transcription factor Pap1, and was associated with increased abundance of Pap1-responsive mRNAs, but not with Pap1 relocalization. Fission yeast cells lacking the int6 gene grew slowly. This growth retardation could be corrected by the expression of full length Int6 of fission yeast or human origin, or by a C-terminal fragment of the fission yeast protein that also conferred drug resistance, but not by truncated human Int-6 proteins corresponding to the predicted products of MMTV-disrupted murine alleles. Studies in fission yeast may therefore help to explain the ways in which Int-6 function can be perturbed during MMTV-induced mammary tumorigenesis.

Vaccination of healthy and diseased koalas (Phascolarctos cinereus) with a Chlamydia pecorum multi-subunit vaccine : evaluation of immunity and pathology

Chlamydial infections represent a major threat to the long-term survival of the koala and a successful vaccine would provide a valuable management tool. Vaccination however has the potential to enhance inflammatory disease in animals exposed to a natural infection prior to vaccination, a finding in early human and primate trials of whole cell vaccines to prevent trachoma. In the present study, we vaccinated both healthy koalas as well as clinically diseased koalas with a multi-subunit vaccine consisting of Chlamydia pecorum MOMP and NrdB mixed with immune stimulating complex as adjuvant. Following vaccination, there was no increase in inflammatory pathological changes in animals previously infected with Chlamydia. Strong antibody (including neutralizing antibodies) and lymphocyte proliferation responses were recorded in all vaccinated koalas, both healthy and clinically diseased. Vaccine induced antibodies specific for both vaccine antigens were observed not only in plasma but also in ocular secretions. Our data shows that an experimental chlamydial vaccine is safe to use in previously infected koalas, in that it does not worsen infection-associated lesions. Furthermore, the prototype vaccine is effective, as demonstrated by strong levels of neutralizing antibody and lymphocyte proliferation responses in both healthy and clinically diseased koalas. Collectively, this work illustrates the feasibility of developing a safe and effective Chlamydia vaccine as a tool for management of disease in wild koalas.

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.

Chemically modified ribbon edge stimulated H2 dissociation: A first-principles computational study

First-principles computational studies indicate that (B, N, or O)-doped graphene ribbon edges can substantially reduce the energy barrier for H2 dissociative adsorption. The low barrier is competitive with many widely used metal or metal oxide catalysts. This suggests that suitably functionalized graphene architectures are promising metal-free alternatives for low-cost catalytic processes.

Adsorption and dissociation of ammonia borane outside and inside single-walled carbon nanotubes : A density functional theory study

Amonia borane (AB) has been identified as a potential candidate highcapacity hydrogen storage material. This work probes the adsorption and dissociation of AB inside and outside single-walled carbon nanotubes (SWCNTs) within the framework of density functional theory. The dissociation barriers of AB have been calculated and compared with that of the isolated AB molecule. On the basis of the present calculations, no notable improvement results from SWCNT confinement; on the contrary, the dissociation barrier slightly increases with respect to isolated AB.

The role of Ti as a catalyst for the dissociation of hydrogen on a Mg(0001) surface

In this paper, the dissociative chemisorption of hydrogen on both pure and Ti-incorporated Mg(0001) surfaces are studied by ab initio density functional theory (DFT) calculations. The calculated dissociation barrier of hydrogen molecule on a pure Mg(0001) surface (1.05 eV) is in good agreement with comparable theoretical studies. For the Ti-incorporated Mg(0001) surface, the activated barrier decreases to 0.103 eV due to the strong interaction between the molecular orbital of hydrogen and the d metal state of Ti. This could explain the experimentally observed improvement in absorption kinetics of hydrogen when transition metals have been introduced into the magnesium materials.

G-protein β3 subunit gene (GNB3) variant in causation of essential hypertension

Essential hypertensives display enhanced signal transduction through pertussis toxin-sensitive G proteins. The T allele of a C825T variant in exon 10 of the G protein β3 subunit gene (GNB3) induces formation of a splice variant (Gβ3-s) with enhanced activity. The T allele of GNB3 was shown recently to be associated with hypertension in unselected German patients (frequency=0.31 versus 0.25 in control). To confirm and extend this finding in a different setting, we performed an association study in Australian white hypertensives. This involved an extensively examined cohort of 110 hypertensives, each of whom were the offspring of 2 hypertensive parents, and 189 normotensives whose parents were both normotensive beyond age 50 years. Genotyping was performed by polymerase chain reaction and digestion with BseDI, which either cut (C allele) or did not cut (T allele) the 268-bp polymerase chain reaction product. T allele frequency in the hypertensive group was 0.43 compared with 0.25 in the normotensive group (χ2=22; P=0.00002; odds ratio=2.3; 95% CI=1.7 to 3.3). The T allele tracked with higher pretreatment blood pressure: diastolic=105±7, 109±16, and 128±28 mm Hg (mean±SD) for CC, CT, and 7T, respectively (P=0.001 by 1-way ANOVA). Blood pressures were higher in female hypertensives with a T allele (P=0.006 for systolic and 0.0003 for diastolic by ANOVA) than they were in male hypertensives. In conclusion, the present study of a group with strong family history supports a role for a genetically determined, physiologically active splice variant of the G protein β3 subunit gene in the causation of essential hypertension.

The B subunit of coagulation factor XIII is linked to renin and the Duffy blood group to α-spectrin on human chromosome 1

Family linkage studies were used to detect two linkage relationships on human chromosome 1. The B subunit of coagulation factor XIII showed significant linkage to renin with a maximum lod score of 5.071 at a distance of 10 cM. Significant linkage was also shown between the Duffy blood group and α-spectrin with linkage results giving a combined lod score of 3.194 at 5 cM.