Bcl-2 on the endoplasmic reticulum regulates Bax activity by binding to BH3-only proteins

Bcl-2 family members have been shown to be key mediators of apoptosis as either pro- or anti-apoptotic factors. It is thought that both classes of Bcl-2 family members act at the level of the mitochondria to regulate apoptosis, although the founding anti-apoptotic family member, Bcl-2 is localized to the endoplasmic reticulum (ER), mitochondrial, and nuclear membranes. In order to better understand the effect of Bcl-2 localization on its activity, we have utilized a Bcl-2 mutant that localizes only to the ER membrane, designated Bcl-2Cb5. Bcl-2Cb5 was expressed in MDA-MB-468 cells, which protected against apoptosis induced by the kinase inhibitor, staurosporine. Data presented here show that Bcl-2Cb5 inhibits this process by blocking Bax activation and cytochrome c release. Furthermore, we show that Bcl-2Cb5 can inhibit the activation of a constitutively mitochondrial mutant of Bax, indicating that an intermediate between Bcl-2 on the ER and Bax on the mitochondria must exist. We demonstrate that this intermediate is likely a BH3-only subfamily member. Data presented here show that Bcl-2Cb5 can sequester a constitutively active form of Bad (Bad3A) from the mitochondria and prevent it from activating Bax. These data suggest that Bcl-2 indirectly protects mitochondrial membranes from Bax, via BH3-only proteins.     

Evaluating the effects of enhanced processivity and metal ions on translesion DNA replication catalyzed by the bacteriophage T4 DNA polymerase

The fidelity of DNA replication is achieved in a multiplicative process encompassing nucleobase selection and insertion, removal of misinserted nucleotides by exonuclease activity, and enzyme dissociation from primer/templates that are misaligned due to mispairing. In this study, we have evaluated the effect of altering these kinetic processes on the dynamics of translesion DNA replication using the bacteriophage T4 replication apparatus as a model system. The effect of enhancing the processivity of the T4 DNA polymerase, gp43, on translesion DNA replication was evaluated using a defined in vitro assay system. While the T4 replicase (gp43 in complex with gp45) can perform efficient, processive replication using unmodified DNA, the T4 replicase cannot extend beyond an abasic site. This indicates that enhancing the processivity of gp43 does not increase unambiguously its ability to perform translesion DNA replication. Surprisingly, the replicase composed of an exonuclease-deficient mutant of gp43 was unable to extend beyond the abasic DNA lesion, thus indicating that molecular processes involved in DNA polymerization activity play the predominant role in preventing extension beyond the non-coding DNA lesion. Although neither T4 replicase complex could extend beyond the lesion, there were measurable differences in the stability of each complex at the DNA lesion. Specifically, the exonuclease-deficient replicase dissociates at a rate constant, k(off), of 1.1s(-1) while the wild-type replicase remains more stably associated at the site of DNA damage by virtue of a slower measured rate constant (k(off) 0.009s(-1)). The increased lifetime of the wild-type replicase suggests that idle turnover, the partitioning of the replicase from its polymerase to its exonuclease active site, may play an important role in maintaining fidelity. Further attempts to perturb the fidelity of the T4 replicase by substituting Mn(2+) for Mg(2+) did not significantly enhance DNA synthesis beyond the abasic DNA lesion. The results of these studies are interpreted with respect to current structural information of gp43 alone and complexed with gp45.     

A biochemical and pharmacological comparison of enzyme replacement therapies for the glycolipid storage disorder Fabry disease

Fabry disease is a lysosomal storage disease arising from deficiency of the enzyme alpha-galactosidase A. Two recombinant protein therapeutics, Fabrazyme (agalsidase beta) and Replagal (agalsidase alfa), have been approved in Europe as enzyme replacement therapies for Fabry disease. Both contain the same human enzyme, alpha-galactosidase A, but they are produced using different protein expression systems and have been approved for administration at different doses. To determine if there is recognizable biochemical basis for the different doses, we performed a comparison of the two drugs, focusing on factors that are likely to influence biological activity and availability. The two drugs have similar glycosylation, both in the type and location of the oligosaccharide structures present. Differences in glycosylation were mainly limited to the levels of sialic acid and mannose-6-phosphate present, with Fabrazyme having a higher percentage of fully sialylated oligosaccharides and a higher level of phosphorylation. The higher levels of phosphorylated oligomannose residues correlated with increased binding to mannose-6-phosphate receptors and uptake into Fabry fibroblasts in vitro. Biodistribution studies in a mouse model of Fabry disease showed similar organ uptake. Likewise, antigenicity studies using antisera from Fabry patients demonstrated that both drugs were indistinguishable in terms of antibody cross-reactivity. Based on these studies and present knowledge regarding the influence of glycosylation on protein biodistribution and cellular uptake, the two protein preparations appear to be functionally indistinguishable. Therefore, the data from these studies provide no rationale for the use of these proteins at different therapeutic doses.     

CD134 as target for specific drug delivery to auto-aggressive CD4<Superscript>+</Superscript>T cells in adjuvant arthritis

T cells have an important role during the development of autoimmune diseases. In adjuvant arthritis, a model for rheumatoid arthritis, we found that the percentage of CD4⁺ T cells expressing the activation marker CD134 (OX40 antigen) was elevated before disease onset. Moreover, these CD134⁺ T cells showed a specific proliferative response to the disease-associated epitope of mycobacterial heat shock protein 60, indicating that this subset contains auto-aggressive T cells. We studied the usefulness of CD134 as a molecular target for immune intervention in arthritis by using liposomes coated with a CD134-directed monoclonal antibody as a drug targeting system. Injection of anti-CD134 liposomes subcutaneously in the hind paws of pre-arthritic rats resulted in targeting of the majority of CD4⁺CD134⁺ T cells in the popliteal lymph nodes. Furthermore, we showed that anti-CD134 liposomes bound to activated T cells were not internalized. However, drug delivery by these liposomes could be established by loading anti-CD134 liposomes with the dipalmitate-derivatized cytostatic agent 5'-fluorodeoxyuridine. These liposomes specifically inhibited the proliferation of activated CD134⁺ T cells in vitro, and treatment with anti-CD134 liposomes containing 5'-fluorodeoxyuridine resulted in the amelioration of adjuvant arthritis. Thus, CD134 can be used as a marker for auto-aggressive CD4⁺ T cells early in arthritis, and specific liposomal targeting of drugs to these cells via CD134 can be employed to downregulate disease development.     

Benzyl ether structure-activity relationships in a series of ketopiperazine-based renin inhibitors

Inhibition of renin enzymatic activity by a series of ketopiperazine-based compounds containing a C6 benzyloxymethyl substituent correlated with a +(pi+sigma) effect. A 3-pyridinyloxymethyl substituent was also found to be equipotent as higher molecular weight analogs, and exhibited decreased CYP3A4 inhibition levels and improved pharmacokinetic properties.     

Blue Light Overrides Repression of Asexual Sporulation by Mating Type Genes in the Basidiomycete Coprinus cinereus

Monokaryotic mycelia of the homobasidiomycete Coprinus cinereus form asexual spores (oidia) constitutively in abundant numbers. Mycelia with mutations in both mating type loci (Amut Bmut homokaryons) also produce copious oidia but only when exposed to blue light. We used such an Amut Bmut homokaryon to define environmental and inherent factors that influence the light-induced oidiation process. We show that the Amut function causes repression of oidiation in the dark and that light overrides this effect. Similarly, compatible genes from different haplotypes of the A mating type locus repress sporulation in the dark and not in the light. Compatible products of the B mating type locus reduce the outcome of light on A-mediated repression but the mutated B function present in the Amut Bmut homokaryons is not effective. In dikaryons, the coordinated regulation of asexual sporulation by compatible A and B mating type genes results in moderate oidia production in light. Copyright 1998 Academic Press.     

'Navius' kissing stents for coronary bifurcation stenosis,recreating a new metallic carina: an IVUS-assessed case report

We report a case of implantation of a new design of stent which allows creation of a double-hemispheric lumen for the treatment of a bifurcational stenosis. The unfavourable outcome following the implantation of this stent is described.     

A role for brain size and cognition in food webs

Predators tend to be large and mobile, enabling them to forage in spatially distinct food web compartments (e.g. littoral and pelagic aquatic macrohabitats). This feature can stabilise ecosystems when predators are capable of rapid behavioural response to changing resource conditions in distinct habitat compartments. However, what provides this ability to respond behaviourally has not been quantified. We hypothesised that predators require increased cognitive abilities to occupy their position in a food web, which puts pressure to increase brain size. Consistent with food web theory, we found that fish relative brain size increased with increased ability to forage across macrohabitats and increased relative trophic positions in a lacustrine food web, indicating that larger brains may afford the cognitive capacity to exploit various habitats flexibly, thus contributing to the stability of whole food webs.     

Short pulse of 1080 improves the survival of brown kiwi chicks in an area subjected to long-term stoat trapping

Many mainland populations of kiwi are declining because stoats (Mustela erminea) kill most of their chicks. Stoats are often trapped during conservation programmes, but the long-term effectiveness of trapping has not been measured. During continuous trapping of mammalian predators in the 9800?ha Whangarei Kiwi Sanctuary, the survival of brown kiwi (Apteryx mantelli) chicks declined over time. Following the use of sodium fluoroacetate (1080) to kill rats (Rattus spp.) and possums (Trichosurus vulpecula) and likely secondary poisoning of stoats, chick survival at Riponui increased from 5% to 56%, and the 62% chick survival at Rarewarewa was better than the 20% recorded in a trapped-only area nearby. We suggest that untrappable stoats accumulate in areas subjected to continuous predator trapping. Conservation managers should build into their long-term pest control programmes a periodic pulse of an alternative tool to kill pests that, for whatever reason, actively avoid the primary control tool.