The serine protease Omi/HtrA2 regulates apoptosis by binding XIAP through a reaper-like motif

The inhibitor-of-apoptosis proteins (IAPs) play a critical role in the regulation of apoptosis by binding and inhibiting caspases. Reaper family proteins and Smac/DIABLO use a conserved amino-terminal sequence to bind to IAPs in flies and mammals, respectively, blocking their ability to inhibit caspases and thus promoting apoptosis. Here we have identified the serine protease Omi/HtrA2 as a second mammalian XIAP-binding protein with a Reaper-like motif. This protease autoprocesses to form a protein with amino-terminal homology to Smac/DIABLO and Reaper family proteins. Full-length Omi/HtrA2 is localized to mitochondria but fails to interact with XIAP. Mitochondria also contain processed Omi/HtrA2, which, following apoptotic insult, translocates to the cytosol, where it interacts with XIAP. Overexpression of Omi/HtrA2 sensitizes cells to apoptosis, and its removal by RNA interference reduces cell death. Omi/HtrA2 thus extends the set of mammalian proteins with Reaper-like function that are released from the mitochondria during apoptosis.     

Cranial reconstruction with computer-generated hard-tissue replacement patient-matched implants: indications, surgical technique, and long-term follow-up

The aim of this clinical study was to evaluate the effectiveness and safety of using computer-generated alloplastic (hard-tissue replacement) implants for the reconstruction of large defects of the upper craniofacial region. Fourteen patients who had large (> 150 cm2) preexisting defects of the cranium or cranio-orbital region underwent surgical reconstruction. Preoperatively, a three-dimensional computed tomographic scan was obtained from which an anatomic model was fabricated. The defect in the model was then used to create an alloplastic (hard tissue-replacement polymer) implant for reconstruction and surgical placement. At the time of surgery, the implant was secured into position with either metal or resorbable fixation. In cases where the frontal sinus was in proximity to the implant, the frontal sinus was either cranialized and covered with a pericranial flap or obliterated with hydroxyapatite cement. In cases that had been previously irradiated or infected, wide bony debridement and coverage with a vascularized muscle was initially performed, followed by implant reconstruction 6 months later. All implants fit easily into the bone defects, and only four (29 percent) required some minor adjustments to complete the fit. All patients healed uneventfully. With a minimum of 1 year follow-up (average, 3 years) in all cases, excellent contours have been maintained and all patients have remained infection-free. In large cranial defects, custom implants fabricated from porous, hydrophilic hard-tissue replacement polymer provide an exacting anatomic fit and a solid stable reconstruction. This method of reconstruction in these defects is rapid and exact, and significantly reduces operative time. Critical attention must be paid, however, to management of the frontal sinus and preexisting bone infection and the quality of the overlying soft-tissue cover.     

Asaia sp., an Unusual Spoilage Organism of Fruit-Flavored Bottled Water

A gram-negative bacillus was isolated from a batch of fruit-flavored bottled water, which had spoiled as a result of bacterial overgrowth (>10⁶ CFU/ml). The spoilage organism was extremely difficult to identify phenotypically and was poorly identified as Pasturella sp. (78.7% identification profile) employing the API 20NE identification scheme, which gave the profile 5040000. Molecular identification through PCR amplification of a partial region of the 16S rRNA gene followed by direct automated sequencing of the PCR amplicon allowed identification of the organism. Due to the sequence identity (100%) between the spoilage organism and a reference strain in GenBank, the spoilage isolate was considered to be an Asaia sp., a recently described genus and member of the acetic acid bacteria. This is the first report of Asaia sp. causing spoilage of a foodstuff and highlights the benefits of molecular identification techniques based on 16S rRNA gene sequences in the identification of unusual spoilage organisms.     

Aggregation of misfolded proteins can be a selective process dependent upon peptide composition

Intracellular aggregation of misfolded proteins is observed in a number of human diseases, in particular, neurologic disorders in which expanded tracts of polyglutamine residues play a central role. A variety of other proteins are prone to aggregation when mutated, indicating that this process is a common pathologic mechanism for inherited disorders. However, little is known about the relationship between the sequence of aggregating peptides and the specificity of intracellular accumulation. Here we demonstrate that substitution of two residues eliminates aggregation of a 111-amino acid peptide derived from the C-terminal portion of the cystic fibrosis transmembrane conductance regulator (CFTR). We also show that fusion to a reporter protein considerably alters the subcellular distribution of aggregating peptide. When fused to green fluorescent protein, the peptide containing amino acids 1370-1480 of CFTR accumulates in large perinuclear or nuclear aggregates. The same CFTR fragment devoid of green fluorescent protein localizes predominantly to discrete accumulations associated with mitochondria. Importantly, both types of accumulation are dependent on the presence of the same two amino acids within the CFTR sequence. Co-expression studies show that both CFTR-derived proteins can co-localize in large cytoplasmic/nuclear aggregates. However, neither CFTR construct accumulates in intracellular inclusions formed by N-terminal fragment of huntingtin. In addition to unique accumulation patterns, each aggregating peptide shows differences in association with chaperone proteins. Thus, our results indicate that the process of intracellular aggregation can be a selective process determined by the composition of the aggregating peptides.     

Loss of albumin and megalin binding to renal cubilin in rats results in albuminuria after total body irradiation

The role of the renal apical brush-border membrane (BBM) endocytic receptors cubilin and megalin in the onset of albuminuria in rats exposed to a single dose of total body irradiation (TBI) has been investigated. Albuminuria was evident as immunoblot (IB) analysis of the urine samples from TBI rats revealed excretion of large amounts of albumin. IB analysis of the BBM proteins did not reveal any significant changes in cubilin or megalin levels, but (125)I-albumin binding to BBM from TBI rats declined by 80% with a fivefold decrease (from 0.5 to 2.5 microM) in the affinity for albumin. IB analysis of cubilin from the BBM demonstrated a 75% loss when purified using albumin, but not intrinsic factor (IF)-cobalamin (Cbl) ligand affinity chromatography. Immunoprecipitation (IP) of Triton X-100 extract of the BBM with antiserum to cubilin followed by IB of the immune complex with an antiserum to megalin revealed a 75% loss of association between megalin and cubilin. IP studies with antiserum to cubilin or megalin and IB with antiserum to the cation-independent mannose 6-phosphate/insulin-like growth factor II-receptor (CIMPR) revealed that CIMPR interacted with both cubilin and megalin. In addition, TBI did not disrupt the association of CIMPR with either cubilin or megalin in BBM. These results suggest that albuminuria noted in TBI rats is due to selective loss of albumin and megalin, but not CIMPR or IF-Cbl binding by cubilin. Furthermore, these results also suggest that albumin and IF-Cbl binding to cubilin occur at distinct sites and that in the rat renal BBM, CIMPR interacts with both cubilin and megalin.     

Extent of invasion of Tasmanian native vegetation by the exotic bumblebee Bombus terrestris (Apoidea: Apidae)

Abstract Observations of the large earth bumblebee, Bombus terrestris (L.), in native vegetation were collated to determine the extent to which this exotic species has invaded Tasmanian native vegetation during the first 9 years after its introduction. The range of B. terrestris now encompasses all of Tasmania's major vegetation types, altitudes from sea level to 1260m a.s.L, and the entire breadth of annual precipitation in the state from more than 3200 mm to less than 600 mm. Observations of workers carrying pollen, together with the presence of large numbers of bumblebees at many localities across this range indicate that colonies are frequently established in native vegetation. Evidence that colonies are often successful was obtained from repeated observations of the species during more than 1 year at particular sites. Unequivocal evidence of colonies was obtained from six National Parks, including four of the five in the Tasmanian Wilderness World Heritage Area (WHA). Indeed, the species has been present in the WHA for at least as long as it has in the city of Hobart, where it was first recorded. In southwestern Tasmania, evidence of colonies was obtained up to 40km from gardens, 61 km from small towns and 93 km from large towns. Hence, contrary to previous suggestions, the species is established in the most remote parts of Tasmania and is not dependent on introduced garden plants. Given their strong record of invasion, it is likely that B. terrestris will form feral populations on the mainland of Australia and in many other parts of the world if introduced. Because of their likely negative impacts on native animals and plants, and potential to enhance seed production in weeds, the spread of bumblebees should be avoided.     

The cytogenetic systems of grasshoppers and locusts

The three endemic species of the Australian genusTolgadia are all distributed in the northern tropical and subtropical areas. They are distinguishable both on morphological and cytological grounds. All three have a derived neo XY, neo XX sex chromosome system involving distinct elements of a basically 11-membered autosome set, namely (spacies-1) (bivittata) and (infirma). Additionallybivittata is homozygous for two autosomal fusions involving and giving 2n=18 as compared to the complement of 2n=22 present in the other two species. Two of the species,bivittata and species-1, have a comparable mean cell chiasma frequency despite their difference in chromosome number. That ofinfirma is significantly higher. Coupled with this, some of the populations ofinfirma are polymorphic for supernumerary heterochromatic segments, principally on the smallest autosome. Such segments increase the mean cell chiasma frequency still further. Thusinfirma, which is the least habitat restricted despite its brachypterous nature, not only has the highest mean cell chiasma frequency but, in addition, has at its disposal a polymorphism capable of magnifying this difference.