XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP.     

Oil pollution and climate have wide-scale impacts on seabird demographics

Oil spills often spell disaster for marine birds caught in slicks. However, the impact of oil pollution on seabird population parameters is poorly known because oil spills usually occur in wintering areas remote from breeding colonies where birds may be distributed over a wide area, and because it is difficult to separate the effects of oil pollution from the effect of natural environmental variation on seabird populations. Using a long-term data set we show that over-winter survival of adult common guillemots (Uria aalge) is negatively affected by both the incidence of four major oil-spills in their wintering grounds and high values of the North Atlantic Oscillation (NAO) index. After controlling for the effect of the NAO index, we show that winter mortality of adult guillemots is doubled by major oil pollution incidents. Our results demonstrate that oil pollution can have wide-scale impacts on marine ecosystems that can be quantified using populations of marked individuals to estimate survival.     

Modification of gibberellin biosynthesis in the grafted apple scion allows control of tree height independent of the rootstock

The availability of short stature apple scions that required minimal applications of chemical growth retardants and could be used with a range of rootstocks would be of considerable benefit to fruit growers. We have suppressed the expression of a gene encoding the gibberellin (GA) biosynthetic enzyme GA 20-oxidase to reduce the levels of bioactive GAs in a scion variety, resulting in significant reductions in stem height. Application of GA3 reversed the effect. The scion remained dwarfed after grafting on to normally invigorating rootstocks, whilst control plants of the same cultivar displayed the expected vigour when grafted on to these rootstocks. This approach could be applicable to any perennial crop variety, allowing dwarf trees to be obtained on any available rootstock or on their own roots without the need for chemical growth retardant application. In effect, seedlings that are well suited to local conditions (drought, salinity) could be employed as tree rootstocks, as could existing rootstocks valued for characters other than vigour control, such as pest and disease resistance.     

The unusual mitochondrial compartment of Cryptosporidium parvum

Recent studies, including the Cryptosporidium parvum Genome Project, have provided evidence for a mitochondrial-derived compartment in this parasite. This organelle appears to lack a genome, and thus must be entirely dependent on nuclear-encoded proteins. Here, we review the evidence for such an organelle in C. parvum and its probable function. There is no adequate treatment for infection by this parasite and so the elucidation of the role of this organelle and the effective targeting of its functions by antimicrobial agents might provide new treatments for infection by C. parvum.     

Evaluation of specificity and effects of monoclonal antibodies submitted to the Eighth Human Leucocyte Differentiation Antigen Workshop on rotavirus-cell attachment and entry

Rotavirus infection of permissive cells is a multi-step process that requires interaction with several cell surface receptors. Integrins alpha2beta1, alpha4beta1, alphaXbeta2, and alphavbeta3 are involved in the attachment and entry into permissive cells for many rotavirus strains. However, possible roles of known partners of these integrins in this process have not been studied. Here, the specificities of new monoclonal antibodies directed to beta1 and beta2 integrins were determined using integrin-transfected cells. The ability of monoclonal antibodies to integrin partners CD82, CD151, CD321, and CD322 to bind rotavirus-permissive cell lines (MA104, Caco-2, and RD) and K562 cells expressing or lacking alpha4beta1 also was investigated. CD82 and CD151 were expressed on K562, alpha4-K562, and RD cells. CD321-specific antibodies bound K562, alpha4-K562, MA104, and Caco-2 cells. CD322 expression was detected on MA104 but not Caco-2 cells. Antibodies to CD82, CD151, CD321, and CD322 that bound these cells were investigated for their ability to inhibit cellular attachment and entry by rotaviruses. Antibody blockade of these integrin-associated proteins did not affect cell attachment or entry of the integrin-using rhesus rotavirus RRV or porcine rotavirus CRW-8, which uses alpha4beta1 integrin for infection. Antibody blockade of CD322 did not alter cell attachment or infectivity by human rotavirus strain RV-3, so RV-3 infection was independent of CD322. Overall, these studies indicate that CD82, CD151, CD321, and CD322 are unlikely to play a role in rotavirus-cell binding or entry.     

Testosterone and autumn territorial behavior in male red grouse Lagopus lagopus scoticus

In many bird species, males exhibit territorial aggression outside the breeding season, when testosterone concentrations are low and may not regulate territorial behaviors. The hormonal regulation of aggression at this time of year has only been studied in passerine birds. Here, we investigated the role of testosterone in the regulation of aggression in a non-passerine bird, the red grouse Lagopus lagopus scoticus. Male red grouse are aggressive in early spring when breeding starts, in autumn when they establish territories, and sporadically through much of the winter. We first describe seasonal variations in plasma testosterone concentrations and in the size of males' sexual ornaments, their red combs, which relates to aggressiveness. Testosterone concentrations and comb size were correlated. Both increased in autumn to a peak in October, and then increased again in spring, to a greater peak in early April. Secondly, we experimentally investigated the effects of testosterone, and of an anti-androgen (flutamide) used in combination with an aromatase inhibitor (ATD), on autumn territorial behavior. Males were treated with either empty implants, as controls (C-males), testosterone implants (T-males), or with flutamide and ATD implants (FA-males). One month after implanting, both T- and FA-males had higher concentrations of testosterone than C-males. Comb size, aggressive call rate, and response to playbacks of territorial call all significantly increased in T-males. However, the increase in testosterone in FA-males did not increase comb size or aggressive behavior. In the following spring, after the content of implants was used, FA-males had significantly lower testosterone than C-males, and had a reduced seasonal increase in comb size. The results suggest that testosterone plays a significant role in regulating red grouse aggressive behavior in autumn. However, the observation that flutamide and ATD treatment did not reduce territorial behavior, suggests that estradiol may also be involved in the regulation of non-breeding aggression.     

Genetic factors influencing bystander signaling in murine bladder epithelium after low-dose irradiation in vivo

Radiation-induced bystander effects occur in cells that are not directly hit by radiation tracks but that receive signals from hit cells. They are well-documented in vitro consequences of low-dose exposure, but their relevance to in vivo radiobiology is not established. To investigate the in vivo production of bystander signals, bladder explants were established from two strains of mice known to differ significantly in both short-term and long-term radiation responses. These were investigated for the ability of 0.5 Gy total-body irradiation in vivo to induce production of bystander signals in bladder epithelium. The studies demonstrate that irradiated C57BL/6 mice, but not CBA/Ca mice, produce bystander signals that induce apoptosis and reduce clonogenic survival in reporter HPV-G-transfected keratinocytes. Transfer of medium from explants established from irradiated animals to explants established from unirradiated animals confirmed these differences in bladder epithelium. The responses to the in vivo-generated bystander signal exhibit genotypic differences in calcium signaling and also in signaling pathways indicative of a major role for the balance of pro-apoptosis and anti-apoptosis proteins in determining the overall response. The results clearly demonstrate the in vivo induction of bystander signals that are strongly influenced by genetic factors and have implications for radiation protection, medical imaging, and radiotherapy.     

Identification of an intestinal heme transporter

Dietary heme iron is an important nutritional source of iron in carnivores and omnivores that is more readily absorbed than non-heme iron derived from vegetables and grain. Most heme is absorbed in the proximal intestine, with absorptive capacity decreasing distally. We utilized a subtractive hybridization approach to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. Here we show a membrane protein named HCP 1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature-dependent and saturable manner. HCP 1 mRNA was highly expressed in duodenum and regulated by hypoxia. HCP 1 protein was iron regulated and localized to the brush-border membrane of duodenal enterocytes in iron deficiency. Our data indicate that HCP 1 is the long-sought intestinal heme transporter.     

The effect of gamete co-incubation time during in vitro fertilization with frozen-thawed unsorted and sex-sorted ram spermatozoa on the development of in vitro matured adult and prepubertal ewe oocytes

In vitro matured adult (Experiment 1) and prepubertal (Experiment 2) ewe oocytes were co-incubated with unsorted or sex-sorted frozen-thawed spermatozoa for 2 to 3 h (short) or 18 to 20 h (long) to determine the effects of reducing the gamete co-incubation time during IVF on subsequent embryonic development in vitro. For oocytes derived from adult ewes, there were no differences in oocyte fertilization and cleavage at 24 h post insemination (hpi) between types of spermatozoa or co-incubation times (P > 0.05). By 48 hpi, oocyte cleavage was higher after a short (390/602, 64.8%) compared with a long (381/617, 61.7%) co-incubation (P < 0.05), and was not significantly different for unsorted (266/372, 71.5%) and sex-sorted (505/849, 59.9%) spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (150/266, 56.4%) and sex-sorted (295/505, 58.4%) spermatozoa, but was higher after a short (240/390, 61.5%) than long (205/381, 53.8%) co-incubation (P < 0.05). Oocyte development to the blastocyst stage was not different for unsorted (150/372; 40.3%) and sex-sorted (295/847; 34.8%) spermatozoa but was significantly increased by a short (240/602, 39.9%) compared with a long (205/617, 33.2%) co-incubation. Fertilization of oocytes from prepubertal ewes was similar for types of spermatozoa and for duration of co-incubation. Oocyte cleavage (48 hpi) was similar for a short (241/377, 63.9%) and long (226/349, 64.8%) co-incubation with unsorted spermatozoa, but was increased (P < 0.05) by a long co-incubation (286/500, 57.2% versus 163/517, 31.5%) with sex-sorted spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (230/467, 49.3%) and sex-sorted (186/449, 41.4%) spermatozoa, and a short (200/404, 49.5%) or long (216/512, 42.1%) co-incubation. However, oocyte development to the blastocyst stage was higher (P < 0.05) after IVF with unsorted (230/726, 37.1%) than sex-sorted (186/1017, 18.3%) spermatozoa. Reducing the duration of gamete co-incubation did not deleteriously affect the in vitro development of adult and prepubertal ewe derived oocytes after IVF with unsorted and sex-sorted spermatozoa. In general, sex-sorting had no substantial influence on fertilization and embryo development rates.     

Mineralization of the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine by Gordonia and Williamsia spp

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as Williamsia sp. KTR4 and Gordonia sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-14C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 microM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 microg/ml, respectively. Mineralization of [U-14C]RDX to 14CO2 was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH4)2SO4- greatly inhibited KTR9's degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.