A genomic interval at 75C1,2 is required for programmed cell death in Drosophila. We identified a new activator of apoptosis, grim, which maps between two previously identified cell death genes in this region reaper (rpr) and head involution defective (hid). Expression of grim RNA coincided with the onset of programmed cell death at all stages of embryonic development, whereas ectopic induction of grim triggered extensive apoptosis in both transgenic animals and in cell culture. Cell killing by grim was blocked by coexpression of p35, a viral product that inactivates ICE-like proteases, and did not require the functions of rpr or hid. The predicted grim protein shares an amino-terminal motif in common with rpr. However, grim was sufficient to elicit apoptosis in at least one context, where rpr was not. The grim gene product might thus function in a parallel circuit of cell death signaling that ultimately activates a common set of downstream apoptotic effectors.

IL-1 alpha and IL-1 beta bind to receptors termed the type I and type II IL-1 receptors. The type I IL-1 receptor is responsible for specific signaling, while the type II IL-1 receptor functions as a nonsignaling decoy receptor. To determine the effect of a defect in IL-1-mediated signaling, mice have been produced with a genetically disrupted type I IL-1 receptor gene. Mice lacking type I IL-1 receptors are of normal vigor and exhibit no overt phenotype. B cells from type I IL-1R-/- mice activated in vitro with anti-IgM do not proliferate in response to IL-1, but do so in response to IL-4. Injection of murine IL-1 alpha does not induce detectable serum IL-6 levels in type I IL-1R-/- mice, but equivalent levels are produced in response to LPS. Type I IL-1R-/- mice have normal serum Ig levels and generate equivalent primary and secondary Ab responses as wild-type mice. In response to LPS, acute phase protein mRNA induction are equivalent in type I IL-1R-/- and wild-type mice. Type I IL-1R-/- mice do not differ from control mice in susceptibility to either a lethal challenge with D-galactosamine plus LPS or high dose LPS. Interestingly, ICE-/-/type I IL-1R-/- double mutant mice are resistant to high dose LPS. Type I IL-1R-/- mice backcrossed to the C57BL/6 background were as equally resistant as wild-type mice to Listeria monocytogenes.

Signal transducers and activators of transcription (STATs) enhance transcription of specific genes in response to cytokines and growth factors. STAT1 is also required for efficient constitutive expression of the caspases Ice, Cpp32, and Ich-1 in human fibroblasts. As a consequence, STAT1-null cells are resistant to apoptosis by tumor necrosis factor alpha (TNF-alpha). Reintroduction of STAT1alpha restored both TNF-alpha-induced apoptosis and the expression of Ice, Cpp32, and Ich-1. Variant STAT1 proteins carrying point mutations that inactivate domains required for STAT dimer formation nevertheless restored protease expression and sensitivity to apoptosis, indicating that the functions of STAT1 required for these activities are different from those that mediate induced gene expression.

Over the past two years, it has become evident that there is an unexpected link between nuclear pore complex structure and dynamics, nucleocytoplasmic transport and chromosome segregation. In addition, a tomographic three-dimensional reconstruction of native nuclear pore complexes preserved in thick amorphous ice has unveiled a number of new structural features of this supramolecular machine. These data, together with some of the elementary physical principles that underlie nucleocytoplasmic transport, will be discussed in this review.

The etiology of major depression (MDD), a common and complex disorder, remains obscure. Gene expression profiling was conducted on post-mortem brain tissue samples from Brodmann Area 10 (BA10) in the prefrontal cortex from psychotropic drug-free persons with a history of MDD and age, gender, and post-mortem interval-matched normal controls (n=14 pairs of subjects). Microarray analysis was conducted using the Affymetrix Exon 1.0 ST arrays. A set of differential expression changes was determined by dual-fold change-probability criteria (∣average log ratios∣>0.585 [equivalent to a 1.5-fold difference in either direction], P<0.01), whereas molecular pathways of interest were evaluated using Gene Set Enrichment Analysis software. The results strongly implicate increased apoptotic stress in the samples from the MDD group. Three anti-apoptotic factors, Y-box-binding protein 1, caspase-1 dominant-negative inhibitor pseudo-ICE, and the putative apoptosis inhibitor FKGS2, were over-expressed. Gene set analysis suggested up-regulation of a variety of pro- and anti-inflammatory cytokines, including interleukin 1α (IL-1α), IL-2, IL-3, IL-5, IL-8, IL-9, IL-10, IL-12A, IL-13, IL-15, IL-18, interferon gamma (IFNγ), and lymphotoxin α (TNF superfamily member 1). The genes showing reduced expression included metallothionein 1M (MT1M), a zinc-binding protein with a significant function in the modulation of oxidative stress. The results of this study indicate that post-mortem brain tissue samples from BA10, a region that is involved in reward-related behavior, show evidence of local inflammatory, apoptotic, and oxidative stress in MDD.

The last 3.85 Gyr of Earth history have been divided into five stages. During stage 1 (3.85-2.45 Gyr ago (Ga)) the atmosphere was largely or entirely anoxic, as were the oceans, with the possible exception of oxygen oases in the shallow oceans. During stage 2 (2.45-1.85 Ga) atmospheric oxygen levels rose to values estimated to have been between 0.02 and 0.04 atm. The shallow oceans became mildly oxygenated, while the deep oceans continued anoxic. Stage 3 (1.85-0.85 Ga) was apparently rather 'boring'. Atmospheric oxygen levels did not change significantly. Most of the surface oceans were mildly oxygenated, as were the deep oceans. Stage 4 (0.85-0.54 Ga) saw a rise in atmospheric oxygen to values not much less than 0.2 atm. The shallow oceans followed suit, but the deep oceans were anoxic, at least during the intense Neoproterozoic ice ages. Atmospheric oxygen levels during stage 5 (0.54 Ga-present) probably rose to a maximum value of ca 0.3 atm during the Carboniferous before returning to its present value. The shallow oceans were oxygenated, while the oxygenation of the deep oceans fluctuated considerably, perhaps on rather geologically short time-scales.

Viruses are the most abundant biological entities and can control microbial communities, but their identity in terrestrial and freshwater Antarctic ecosystems is unknown. The genetic structure of an Antarctic lake viral community revealed unexpected genetic richness distributed across the highest number of viral families that have been found to date in aquatic viral metagenomes. In contrast to other known aquatic viromes, which are dominated by bacteriophage sequences, this Antarctic virus assemblage had a large proportion of sequences related to eukaryotic viruses, including phycodnaviruses and single-stranded DNA (ssDNA) viruses not previously identified in aquatic environments. We also observed that the transition from an ice-covered lake in spring to an open-water lake in summer led to a change from a ssDNA- to a double-stranded DNA-virus-dominated assemblage, possibly reflecting a seasonal shift in host organisms.

Electron tomography is a powerful technique capable of giving unique insights into the three-dimensional structural organization of pleomorphic biological objects. However, visualization and interpretation of the resulting volumetric data are hampered by an extremely low signal-to-noise ratio, especially when ice-embedded biological specimens are investigated. Usually, isosurface representation or volume rendering of such data is hindered without any further signal enhancement. We propose a novel technique for noise reduction based on nonlinear anisotropic diffusion. The approach combines efficient noise reduction with excellent signal preservation and is clearly superior to conventional methods (e.g., low-pass and median filtering) and invariant wavelet transform filtering. The gain in the signal-to-noise ratio is verified and demonstrated by means of Fourier shell correlation. Improved visualization performance after processing the 3D images is demonstrated with two examples, tomographic reconstructions of chromatin and of a mitochondrion. Parameter settings and discretization stencils are presented in detail.

The three-dimensional structures of double and single-shelled simian rotavirus have been determined to a resolution of 40 A by image processing electron micrographs of unstained, unfixed virus particles embedded in vitreous ice. This study demonstrates that the icosahedral surface lattices in these structures have a triangulation number of 13 in a left-handed configuration. The double-shelled virion has a smooth outer surface with 60 slender spikes. The single-shelled virion, in contrast, exhibits a bristly surface. On the basis of these structures, the locations and number of copies of outer and inner shell proteins have been deduced. The spikes likely correspond to VP3, a hemagglutinin, while the rest of the mass density in the outer shell represents 780 molecules of VP7, a neutralization antigen. The 260 morphological units, located on all the local and strict 3-fold axes of the single-shelled virion are proposed to represent 260 trimers of VP6, which is a subgroup antigen. The regions of closed contact between the outer and the inner shells are located mainly near the local and strict 3-fold axes. A distinctive feature in the rotavirus structure is the presence of 132 large channels spanning across both the shells at all 5 and 6-co-ordinated positions linking the outermost surface with the inner core. In the transcriptionally active single-shelled virion, these channels may provide pathways for importing the metabolites required for the viral RNA transcription and exporting the newly synthesized RNA molecules for subsequent viral replication processes.

BACKGROUND

Short-term studies suggest that intravenous immunoglobulin might reduce disability caused by chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) but long-term effects have not been shown. We aimed to establish whether 10% caprylate-chromatography purified immune globulin intravenous (IGIV-C) has short-term and long-term benefit in patients with CIDP.

METHODS

117 patients with CIDP who met specific neurophysiological inflammatory neuropathy cause and treatment (INCAT) criteria participated in a randomised, double-blind, placebo-controlled, response-conditional crossover trial. IGIV-C (Gamunex) or placebo was given every 3 weeks for up to 24 weeks in an initial treatment period, and patients who did not show an improvement in INCAT disability score of 1 point or more received the alternate treatment in a crossover period. The primary outcome was the percentage of patients who had maintained an improvement from baseline in adjusted INCAT disability score of 1 point or more through to week 24. Patients who showed an improvement and completed 24 weeks of treatment were eligible to be randomly re-assigned in a blinded 24-week extension phase. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00220740.

RESULTS

During the first period, 32 of 59 (54%) patients treated with IGIV-C and 12 of 58 (21%) patients who received placebo had an improvement in adjusted INCAT disability score that was maintained through to week 24 (treatment difference 33.5%, 95% CI 15.4-51.7; p=0.0002). Improvements from baseline to endpoint were also recorded for grip strength in the dominant hand (treatment difference 10.9 kPa, 4.6-17.2; p=0.0008) and the non-dominant hand (8.6 kPa, 2.6-14.6; p=0.005). Results were similar during the crossover period. During the extension phase, participants who continued to receive IGIV-C had a longer time to relapse than did patients treated with placebo (p=0.011). The incidence of serious adverse events per infusion was 0.8% (9/1096) with IGIV-C versus 1.9% (11/575) with placebo. The most common adverse events with IGIV-C were headache, pyrexia, and hypertension.

CONCLUSIONS

This study, the largest reported trial of any CIDP treatment, shows the short-term and long-term efficacy and safety of IGIV-C and supports use of IGIV-C as a therapy for CIDP.

The Sino-Japanese Floristic Region (SJFR) of East Asia harbors the most diverse of the world's temperate flora, and was the most important glacial refuge for its Tertiary representatives ('relics') throughout Quaternary ice-age cycles. A steadily increasing number of phylogeographic studies in the SJFR of mainland China and adjacent areas, including the Qinghai-Tibetan-Plateau (QTP) and Sino-Himalayan region, have documented the population histories of temperate plant species in these regions. Here we review this current literature that challenges the oft-stated view of the SJFR as a glacial sanctuary for temperate plants, instead revealing profound effects of Quaternary changes in climate, topography, and/or sea level on the current genetic structure of such organisms. There are three recurrent phylogeographic scenarios identified by different case studies that broadly agree with longstanding biogeographic or palaeo-ecological hypotheses: (i) postglacial re-colonization of the QTP from (south-)eastern glacial refugia; (ii) population isolation and endemic species formation in Southwest China due to tectonic shifts and river course dynamics; and (iii) long-term isolation and species survival in multiple localized refugia of (warm-)temperate deciduous forest habitats in subtropical (Central/East/South) China. However, in four additional instances, phylogeographic findings seem to conflict with a priori predictions raised by palaeo-data, suggesting instead: (iv) glacial in situ survival of some hardy alpine herbs and forest trees on the QTP platform itself; (v) long-term refugial isolation of (warm-)temperate evergreen taxa in subtropical China; (vi) 'cryptic' glacial survival of (cool-)temperate deciduous forest trees in North China; and (vii) unexpectedly deep (Late Tertiary/early-to-mid Pleistocene) allopatric-vicariant differentiation of disjunct lineages in the East China-Japan-Korea region due to past sea transgressions. We discuss these and other consequences of the main phylogeographic findings in light of palaeo-environmental evidence, emphasize notable gaps in our knowledge, and outline future research prospects for disentangling the evolution and biogeographic history of the region's extremely diverse temperate flora.

Genetic analyses of Caenorhabditis elegans has identified three genes that function in the regulation of nematode cell death. Mammalian homologs of two of these genes, ced-9 and ced-3, have been identified and comprise proteins belonging to the Bcl-2 and ICE families, respectively. To date, it is unclear where the negative regulators, ced-9 and bcl-2, function relative to the death effectors, ced-3 and the mammalian ced-3 homologs, respectively. Here, the molecular order of the cell death pathway is defined. Our results establish that Bcl-2 and Bcl-xL function upstream of two members of the ICE/CED-3 family of cysteine proteases, Yama (CPP32/apopain) and ICE-LAP3 (Mch3).

Polar fishes are known to have serum proteins and glycoproteins that protect them from freezing, by a noncolligative process. Measurements of antifreeze concentrations in ice and scanning electron micrographs of freeze-dried antifreeze solutions indicate that the antifreezes are incorporated in ice during freezing. The antifreezes also have a pronounced effect on the crystal habit of ice grown in their presence. Each of four antifreezes investigated caused ice to grow in long needles whose axes were parallel to the ice c axis. Together these results indicate the antifreezes adsorb to ice surfaces and inhibit their growth. A model in which adsorbed antifreezes raise the curvature of growth steps on the ice surface is proposed to account for the observed depression of the temperature at which freezing occurs and agrees well with experimental observations. The model is similar to one previously proposed for other cases of crystal growth inhibition.

The Fas/APO-1 receptor is one of the major regulators of apoptosis. We report here that Fas/APO-1-mediated apoptosis requires the activation of a new class of cysteine proteases, including interleukin-1 beta-converting enzyme (ICE), which are homologous to the product of the Caenorhabditis elegans cell-death gene ced-3 (refs 11, 12). Triggering of Fas/APO-1 rapidly stimulated the proteolytic activity of ICE. Overexpression of ICE, achieved by electroporation and microinjection, strongly potentiated Fas/APO-1-mediated cell death. In addition, inhibition of ICE activity by protease inhibitors, as well as by transient expression of the pox virus-derived serpin inhibitor CrmA or an antisense ICE construct, substantially suppressed Fas/APO-1-triggered cell death. We conclude that activation of ICE or an ICE-related protease is a critical event in Fas/APO-1-mediated cell death.

The origins and development of the study of speciation, hybrid zones and phylogeography are outlined using evolutionary iconography. This traces the ideas in this field from Lamarck and Darwin through to the present as represented in diagrams and figures. A 'tree of trees' summarizes this growth and current vitality. The new facility to use various DNA sequences from nuclear, mitochondrial and chloroplast genomes to determine genetic variation throughout a species range is examined particularly. There is great genomic subdivision across species distributions, which can be interpreted in the light of the recent demonstrations of severe palaeoclimatic oscillations. Refugia and postglacial colonization routes are proposed for several organisms across Europe. The role of geography in speciation through the Pleistocene is considered. These emerging principles and analyses are applied to data available on a variety of organisms in other regions of the world, such as the Arctic, North America and the Tropics, and including the progress of Homo sapiens through the last ice age. Some suggestions are made for future research directions.

The notion of mimicking natural structures in the synthesis of new structural materials has generated enormous interest but has yielded few practical advances. Natural composites achieve strength and toughness through complex hierarchical designs that are extremely difficult to replicate synthetically. We emulate nature's toughening mechanisms by combining two ordinary compounds, aluminum oxide and polymethyl methacrylate, into ice-templated structures whose toughness can be more than 300 times (in energy terms) that of their constituents. The final product is a bulk hybrid ceramic-based material whose high yield strength and fracture toughness [ approximately 200 megapascals (MPa) and approximately 30 MPa.m(1/2)] represent specific properties comparable to those of aluminum alloys. These model materials can be used to identify the key microstructural features that should guide the synthesis of bio-inspired ceramic-based composites with unique strength and toughness.

Regional phylogeographical studies involving co-distributed animal and plant species have been conducted for several areas, most notably for Europe and the Pacific Northwest of North America. Until recently, phylogeographical studies in unglaciated eastern North America have been largely limited to animals. As more studies emerge for diverse lineages (including plants), it seems timely to assess the phylogeography across this region: (i) comparing and contrasting the patterns seen in plants and animals; (ii) assessing the extent of pseudocongruence; and (iii) discussing the potential applications of regional phylogeography to issues in ecology, such as response to climatic change. Unglaciated eastern North America is a large, geologically and topographically complex area with the species examined having diverse distributions. Nonetheless, some recurrent patterns emerge: (i) maritime - Atlantic vs. Gulf Coast; (ii) Apalachicola River discontinuity; (iii) Tombigbee River discontinuity; (iv) the Appalachian Mountain discontinuity; (v) the Mississippi River discontinuity; and (vi) the Apalachicola River and Mississippi River discontinuities. Although initially documented in animals, most of these patterns are also apparent in plants, providing support for phylogeographical generalizations. These patterns may generally be attributable to isolation and differentiation during Pleistocene glaciation, but in some cases may be older (Pliocene). Molecular studies sometimes agree with longstanding hypotheses of glacial refugia, but also suggest additional possible refugia, such as the southern Appalachian Mountains and areas close to the Laurentide Ice Sheet. Many species exhibit distinct patterns that reflect the unique, rather than the shared, aspects of species' phylogeographical histories. Furthermore, similar modern phylogeographical patterns can result from different underlying causal factors operating at different times (i.e. pseudocongruence). One underemphasized component of pseudocongruence may result from the efforts of researchers to categorize patterns visually - similar patterns may, in fact, not fully coincide, and inferring agreement may obscure the actual patterns and lead to erroneous conclusions. Our modelling analyses indicate no clear spatial patterning and support the hypothesis that phylogeographical structure in diverse temperate taxa is complex and was not shaped by just a few barriers.

The reaper gene (rpr) is important for the activation of apoptosis in Drosophila. To investigate whether rpr expression is sufficient to induce apoptosis, transgenic flies were generated that express rpr complementary DNA or the rpr open reading frame in cells that normally live. Transcription of rpr from a heat-inducible promoter rapidly caused wide-spread ectopic apoptosis and organismal death. Ectopic overexpression of rpr in the developing retina resulted in eye ablation. The occurrence of cell death was highly sensitive to the dosage of the transgene. Because cell death induced by the protein encoded by rpr (RPR) could be blocked by the baculovirus p35 protein, RPR appears to activate a death program mediated by a ced-3/ICE (interleukin-1 converting enzyme)-like protease.

Water molecules inside cavities in proteins constitute integral parts of the structure. We have sought a quantitative measure of the hydrophilicity of the cavities by calculating energies and free energies of introducing a water molecule into these cavities. A threshold value of the water-protein interaction energy at -12 kcal/mol was found to be able to distinguish hydrated from empty cavities. It follows that buried waters have entropy comparable to that of liquid water or ice. A simple consistent picture of the energetics of the buried waters provided by this study enabled us to address the reliability of buried waters assigned in experiments.

This special article deals with the role of processing enzymes in the generation of bioactive cytokines, particularly IL-1 beta and the novel cytokine IL-18, which was formerly called IFN gamma-inducing factor (IGIF). The "classical" pathways of cytokine processing are described, as well as the importance of alternative cleavage enzymes. The topic of this review also concerns the biology of IL-18. The regulation of IL-18 production, the IL-18 receptor complex, and the biological effects of this novel cytokine are described.

Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 1beta converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAF1 and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.

Single particles embedded in ice pose new challenges for image processing because of the intrinsically low signal-to-noise ratio of such particles in electron micrographs. We have developed new techniques that address some of these problems and have applied these techniques to electron micrographs of the Escherichia coli ribosome. Data collection and reconstruction follow the protocol of the random-conical technique of Radermacher et al. [J. Microscopy 146 (1987) 113]. A reference-free alignment algorithm has been developed to overcome the propensity of reference-based algorithms to reinforce the reference motif in very noisy situations. In addition, an iterative 3D reconstruction method based on a chi-square minimization constraint has been developed and tested. This algorithm tends to reduce the effects of the missing angular range on the reconstruction, thereby facilitating the merging of random-conical data sets obtained from differently oriented particles.

Expression of the adenovirus E1A oncogene stimulates both cell proliferation and p53-dependent apoptosis in rodent cells. p53 implements apoptosis in all or in part through transcriptional activation of bax, the product of which promotes cell death. The adenovirus E1B 19K product is homologous in sequence and in function to Bcl-2, both of which bind to and inhibit the activity of Bax and thereby suppress apoptosis. The E1B 19K protein also interacts with the nuclear lamins, but the role of this interaction in the regulation of apoptosis is not known. Lamins are, however, substrates for members of the interleukin-1 beta-converting enzyme (ICE) family of cysteine proteases that are activated during apoptosis and function downstream of Bcl-2 in the cell death pathway. lamins are degraded during E1A-induced p53-dependent apoptosis. Lamin A and C are cleaved into 47- and 37-kD fragments, respectively, and the site of proteolysis is mapped to a conserved aspartic acid residue at position 230. The cleavage of lamins during apoptosis is consistent with the activation of an ICE-related cysteine protease down-stream of p53. No lamin protease activity was detected in cells expressing the E1B 19K protein, indicating that 19K functions upstream of protease activation in inhibiting apoptosis. Substitution of the aspartic acid at the cleavage site produced a mutant lamin protein that was resistant to proteolysis both in vitro and in vivo. Expression of uncleavable mutant lamin A or B attenuated apoptosis, delaying cell death and the associated DNA fragmentation by 12 h. Mutant lamin expressing cells failed to show the signs of chromatin condensation and nuclear shrinkage typical of cell death by apoptosis. Instead, the nuclear envelope collapsed and the nuclear lamina remained intact. However, the late stage of apoptosis was morphologically unaltered and formation of apoptotic bodies was evident. Thus, lamin breakdown by proteolytic degradation facilitates the nuclear events of apoptosis perhaps by facilitating nuclear breakdown.

Horizontal gene transfer shapes the genomes of prokaryotes by allowing rapid acquisition of novel adaptive functions. Conjugation allows the broadest range and the highest gene transfer input per transfer event. While conjugative plasmids have been studied for decades, the number and diversity of integrative conjugative elements (ICE) in prokaryotes remained unknown. We defined a large set of protein profiles of the conjugation machinery to scan over 1,000 genomes of prokaryotes. We found 682 putative conjugative systems among all major phylogenetic clades and showed that ICEs are the most abundant conjugative elements in prokaryotes. Nearly half of the genomes contain a type IV secretion system (T4SS), with larger genomes encoding more conjugative systems. Surprisingly, almost half of the chromosomal T4SS lack co-localized relaxases and, consequently, might be devoted to protein transport instead of conjugation. This class of elements is preponderant among small genomes, is less commonly associated with integrases, and is rarer in plasmids. ICEs and conjugative plasmids in proteobacteria have different preferences for each type of T4SS, but all types exist in both chromosomes and plasmids. Mobilizable elements outnumber self-conjugative elements in both ICEs and plasmids, which suggests an extensive use of T4SS in trans. Our evolutionary analysis indicates that switch of plasmids to and from ICEs were frequent and that extant elements began to differentiate only relatively recently. According to the present results, ICEs are the most abundant conjugative elements in practically all prokaryotic clades and might be far more frequently domesticated into non-conjugative protein transport systems than previously thought. While conjugative plasmids and ICEs have different means of genomic stabilization, their mechanisms of mobility by conjugation show strikingly conserved patterns, arguing for a unitary view of conjugation in shaping the genomes of prokaryotes by horizontal gene transfer.