We have developed a PCR approach to clone new apoptotic Ced-3/Ice-like cysteine protease genes. This approach uses degenerate oligonucleotides encoding the highly conserved pentapeptides QACRG and GSWFI that are present in all known apoptotic cysteine proteases. Using this approach, we have cloned a novel apoptotic gene from human Jurkat T lymphocytes. The new gene encodes a approximately 34-kilodalton protein that is highly homologous to human CPP32, Caenorhabditis elegans cell death protein CED-3, mammalian Ich-1 (Nedd2), and mammalian interleukin-1 beta converting enzyme. Because of its high homology to the C. elegans Ced-3 gene, we named the new gene mammalian Ced-3 homologue Mch2. Two Mch2 transcripts (Mch2 alpha, 1.7 kb; Mch2 beta, 1.4 kb) were detected in Jurkat T lymphocytes and other cell lines. We believe that the Mch2 alpha transcript encodes the full-length Mch2, whereas the Mch2 beta transcript encodes a shorter Mch2 isoform, probably as a result of alternative splicing. Like interleukin-1 beta converting enzyme and CPP32, recombinant Mch2 alpha, but not Mch2 beta, possesses protease activity, as determined by its ability to cleave the fluorogenic peptide DEVD-AMC. CPP32 and Mch2 alpha can also cleave poly(ADP-ribose) polymerase in vitro, suggesting that these enzymes participate in poly(ADP-ribose) polymerase cleavage observed during cellular apoptosis. In addition, overexpression of recombinant Mch2 alpha, but not Mch2 beta, induces apoptosis in Sf9 insect cells. Our data suggest that Mch2 is a Ced-3/interleukin-1 beta converting enzyme-like cysteine protease and could be another important mediator of apoptosis in mammalian cells.

The ribosome is formed by assembly of proteins and nucleic acids, and synthesizes proteins according to genetic instructions in all organisms. Many of the biochemical steps of this fundamental process are known, but a detailed understanding requires a well-defined structural model of the ribosome. Electron microscopy combined with image reconstruction of two-dimensional crystals or single ribosomes has been the most promising technique, but the resolution of the resulting models has been insufficient. Here we report a 25-A reconstruction of the ribosome from Escherichia coli, obtained by combining 4,300 projections of ice-embedded single particles. Our new reconstruction reveals a channel in the small ribosomal subunit and a bifurcating tunnel in the large subunit which may constitute pathways for the incoming message and the nascent polypeptide chain, respectively. Based on these new findings, a three-dimensional model of the basic framework of protein synthesis is presented.

Whole genome duplication (WGD) is often considered to be mechanistically associated with species diversification. Such ideas have been anecdotally attached to a WGD at the stem of the salmonid fish family, but remain untested. Here, we characterized an extensive set of gene paralogues retained from the salmonid WGD, in species covering the major lineages (subfamilies Salmoninae, Thymallinae and Coregoninae). By combining the data in calibrated relaxed molecular clock analyses, we provide the first well-constrained and direct estimate for the timing of the salmonid WGD. Our results suggest that the event occurred no later in time than 88 Ma and that 40-50 Myr passed subsequently until the subfamilies diverged. We also recovered a Thymallinae-Coregoninae sister relationship with maximal support. Comparative phylogenetic tests demonstrated that salmonid diversification patterns are closely allied in time with the continuous climatic cooling that followed the Eocene-Oligocene transition, with the highest diversification rates coinciding with recent ice ages. Further tests revealed considerably higher speciation rates in lineages that evolved anadromy--the physiological capacity to migrate between fresh and seawater--than in sister groups that retained the ancestral state of freshwater residency. Anadromy, which probably evolved in response to climatic cooling, is an established catalyst of genetic isolation, particularly during environmental perturbations (for example, glaciation cycles). We thus conclude that climate-linked ecophysiological factors, rather than WGD, were the primary drivers of salmonid diversification.

The relationship between balance ability and sport injury risk has been established in many cases, but the relationship between balance ability and athletic performance is less clear. This review compares the balance ability of athletes from different sports, determines if there is a difference in balance ability of athletes at different levels of competition within the same sport, determines the relationship of balance ability with performance measures and examines the influence of balance training on sport performance or motor skills. Based on the available data from cross-sectional studies, gymnasts tended to have the best balance ability, followed by soccer players, swimmers, active control subjects and then basketball players. Surprisingly, no studies were found that compared the balance ability of rifle shooters with other athletes. There were some sports, such as rifle shooting, soccer and golf, where elite athletes were found to have superior balance ability compared with their less proficient counterparts, but this was not found to be the case for alpine skiing, surfing and judo. Balance ability was shown to be significantly related to rifle shooting accuracy, archery shooting accuracy, ice hockey maximum skating speed and simulated luge start speed, but not for baseball pitching accuracy or snowboarding ranking points. Prospective studies have shown that the addition of a balance training component to the activities of recreationally active subjects or physical education students has resulted in improvements in vertical jump, agility, shuttle run and downhill slalom skiing. A proposed mechanism for the enhancement in motor skills from balance training is an increase in the rate of force development. There are limited data on the influence of balance training on motor skills of elite athletes. When the effectiveness of balance training was compared with resistance training, it was found that resistance training produced superior performance results for jump height and sprint time. Balance ability was related to competition level for some sports, with the more proficient athletes displaying greater balance ability. There were significant relationships between balance ability and a number of performance measures. Evidence from prospective studies supports the notion that balance training can be a worthwhile adjunct to the usual training of non-elite athletes to enhance certain motor skills, but not in place of other conditioning such as resistance training. More research is required to determine the influence of balance training on the motor skills of elite athletes.

BACKGROUND

About an eighth of the earth's land surface is in protected areas (hereafter "PAs"), most created during the 20(th) century. Natural landscapes are critical for species persistence and PAs can play a major role in conservation and in climate policy. Such contributions may be harder than expected to implement if new PAs are constrained to the same kinds of locations that PAs currently occupy.

RESULTS

Quantitatively extending the perception that PAs occupy "rock and ice", we show that across 147 nations PA networks are biased towards places that are unlikely to face land conversion pressures even in the absence of protection. We test each country's PA network for bias in elevation, slope, distances to roads and cities, and suitability for agriculture. Further, within each country's set of PAs, we also ask if the level of protection is biased in these ways. We find that the significant majority of national PA networks are biased to higher elevations, steeper slopes and greater distances to roads and cities. Also, within a country, PAs with higher protection status are more biased than are the PAs with lower protection statuses.

CONCLUSIONS

In sum, PAs are biased towards where they can least prevent land conversion (even if they offer perfect protection). These globally comprehensive results extend findings from nation-level analyses. They imply that siting rules such as the Convention on Biological Diversity's 2010 Target [to protect 10% of all ecoregions] might raise PA impacts if applied at the country level. In light of the potential for global carbon-based payments for avoided deforestation or REDD, these results suggest that attention to threat could improve outcomes from the creation and management of PAs.

BACKGROUND

Cryopreservation of the ovarian cortex with subsequent autotransplantation has, on an experimental basis, been performed to preserve fertility in women being treated for a malignant disease. The present study reports ovarian activity and pregnancies following autotransplantation of frozen/thawed ovarian tissue.

METHODS

One complete ovary was cryopreserved from each of six patients who were 26-35 years old prior to treatment. Tissue from three of the patients was transported 4-5 h on ice prior to cryopreservation. After a period of 17-32 months, orthotopic autotransplantation was performed replacing 20-60% of the tissue. Two patients received additional heterotopic transplants.

RESULTS

In all cases, the tissue restored menstrual cyclicity 14-20 weeks following transplantation. Four of the six women conceived following assisted reproduction: two women (who had the tissue transported 4-5 h prior to cryopreservation) each, based on the orthotopic transplanted tissue, delivered one healthy child (February 2007 and January 2008); one woman miscarriaged in gestational Week 7; and the other had a positive hCG test but no clinical pregnancy. The remaining two women did not become pregnant.

CONCLUSIONS

Two additional healthy children have been born as a result of the ovarian cryopreservation procedure. In both cases, the ovarian tissue was transported 4-5 h prior to freezing demonstrating that hospitals may offer cryopreservation without having the necessary expertise locally.

A comprehensive assessment of bacterial diversity and community composition in arctic and antarctic pack ice was conducted through cultivation and cultivation-independent molecular techniques. We sequenced 16S rRNA genes from 115 and 87 pure cultures of bacteria isolated from arctic and antarctic pack ice, respectively. Most of the 33 arctic phylotypes were >97% identical to previously described antarctic species or to our own antarctic isolates. At both poles, the alpha- and gamma-proteobacteria and the Cytophaga-Flavobacterium group were the dominant taxonomic bacterial groups identified by cultivation as well as by molecular methods. The analysis of 16S rRNA gene clone libraries from multiple arctic and antarctic pack ice samples revealed a high incidence of closely overlapping 16S rRNA gene clone and isolate sequences. Simultaneous analysis of environmental samples with fluorescence in situ hybridization (FISH) showed that approximately 95% of 4',6'-diamidino-2-phenylindole (DAPI)-stained cells hybridized with the general bacterial probe EUB338. More than 90% of those were further assignable. Approximately 50 and 36% were identified as gamma-proteobacteria in arctic and antarctic samples,respectively. Approximately 25% were identified as alpha-proteobacteria, and 25% were identified as belonging to the Cytophaga-Flavobacterium group. For the quantification of specific members of the sea ice community, new oligonucleotide probes were developed which target the genera Octadecabacter, Glaciecola, Psychrobacter, Marinobacter, Shewanella, and Polaribacter: High FISH detection rates of these groups as well as high viable counts corroborated the overlap of clone and isolate sequences. A terrestrial influence on the arctic pack ice community was suggested by the presence of limnic phylotypes.

Proinflammatory cytokines, including IL-1beta and tumor necrosis factor-alpha (TNF-alpha), promote cancer cell adhesion and liver metastases by up-regulating the expression of vascular cell adhesion molecule-1 (VCAM-1) on hepatic sinusoidal endothelium (HSE). In this study, hepatic metastasis after intrasplenically injected mouse B16 melanoma (B16M) cells was reduced 84-95% in mice with null mutations for either IL-1beta or the IL-1beta-converting enzyme (ICE, caspase-1) compared with wild-type mice. On day 12, 47% of wild-type mice were dead compared with 19% of either IL-1beta or ICE-deficient mice. In vitro, conditioned medium from B16M cells (B16M-CM) induced the release of TNF-alpha and IL-1beta from cultures of primary murine HSE. The effect of B16M-CM on HSE resulted in increased numbers of B16M cells adhering to HSE, which was completely abrogated by a specific inhibitor of ICE, anti-IL-18 or IL-18-binding protein. Exogenous IL-18 added to HSE also increased the number of adhering melanoma cells; however, this was not affected by IL-1 receptor blockade or TNF neutralization but rather by anti-VCAM-1. These results demonstrate a role for IL-1beta and IL-18 in the development of hepatic metastases of B16M in vivo. In vitro, soluble products from B16M cells stimulate HSE to sequentially release TNF-alpha, IL-1beta, and IL-18. The IL-18 cytokine increases expression of VCAM-1 and the adherence of melanoma cells.

Freezing avoidance conferred by different types of antifreeze proteins in various polar and subpolar fishes represents a remarkable example of cold adaptation, but how these unique proteins arose is unknown. We have found that the antifreeze glycoproteins (AFGPs) of the predominant Antarctic fish taxon, the notothenioids, evolved from a pancreatic trypsinogen. We have determined the likely evolutionary process by which this occurred through characterization and analyses of notothenioid AFGP and trypsinogen genes. The primordial AFGP gene apparently arose through recruitment of the 5' and 3' ends of an ancestral trypsinogen gene, which provided the secretory signal and the 3' untranslated region, respectively, plus de novo amplification of a 9-nt Thr-Ala-Ala coding element from the trypsinogen progenitor to create a new protein coding region for the repetitive tripeptide backbone of the antifreeze protein. The small sequence divergence (4-7%) between notothenioid AFGP and trypsinogen genes indicates that the transformation of the proteinase gene into the novel ice-binding protein gene occurred quite recently, about 5-14 million years ago (mya), which is highly consistent with the estimated times of the freezing of the Antarctic Ocean at 10-14 mya, and of the main phyletic divergence of the AFGP-bearing notothenioid families at 7-15 mya. The notothenioid trypsinogen to AFGP conversion is the first clear example of how an old protein gene spawned a new gene for an entirely new protein with a new function. It also represents a rare instance in which protein evolution, organismal adaptation, and environmental conditions can be linked directly.

OBJECTIVE

Lack of objective biomarkers for brain damage hampers acute diagnosis and clinical decision making about return to play after sports-related concussion.

OBJECTIVE

To determine whether sports-related concussion is associated with elevated levels of blood biochemical markers of injury to the central nervous system and to assess whether plasma levels of these biomarkers predict return to play in professional ice hockey players with sports-related concussion.

METHODS

Multicenter prospective cohort study involving all 12 teams of the top professional ice hockey league in Sweden, the Swedish Hockey League. Two hundred eighty-eight professional ice hockey players from 12 teams contesting during the 2012-2013 season consented to participate. All players underwent clinical preseason baseline testing regarding concussion assessment measures. Forty-seven players from 2 of the 12 ice hockey teams underwent blood sampling prior to the start of the season. Thirty-five players had a concussion from September 13, 2012, to January 31, 2013; of these players, 28 underwent repeated blood sampling at 1, 12, 36, and 144 hours and when the players returned to play.

METHODS

Total tau, S-100 calcium-binding protein B, and neuron-specific enolase concentrations in plasma and serum were measured.

RESULTS

Concussed players had increased levels of the axonal injury biomarker total tau(median, 10.0 pg/mL; range, 2.0-102 pg/mL) compared with preseason values (median, 4.5pg/mL; range, 0.06-22.7 pg/mL) (P < .001). The levels of the astroglial injury biomarker S-100 calcium-binding protein B were also increased in players with sports-related concussion(median, 0.075 μg/L; range, 0.037-0.24 μg/L) compared with preseason values (median,0.045 μg/L; range, 0.005-0.45 μg/L) (P < .001). The highest biomarker concentrations of total tau and S-100 calcium-binding protein B were measured immediately after a concussion, and they decreased during rehabilitation. No significant changes were detected in the levels of neuron-specific enolase from preseason values (median, 6.5 μg/L; range,3.45-18.0 μg/L) to postconcussion values (median, 6.1 μg/L; range, 3.6-12.8 μg/L) (P = .10).

CONCLUSIONS

Sports-related concussion in professional ice hockey players is associated with acute axonal and astroglial injury. This can be monitored using blood biomarkers, which may be developed into clinical tools to guide sport physicians in the medical counseling of athletes in return-to-play decisions.

Terrestrial arthropods survive subzero temperatures by becoming either freeze tolerant (survive body fluid freezing) or freeze avoiding (prevent body fluid freezing). Protein ice nucleators (PINs), which limit supercooling and induce freezing, and antifreeze proteins (AFPs), which function to prevent freezing, can have roles in both freeze tolerance and avoidance. Many freeze-tolerant insects produce hemolymph PINs, which induce freezing at high subzero temperatures thereby inhibiting lethal intracellular freezing. Some freeze-tolerant species have AFPs that function as cryoprotectants to prevent freeze damage. Although the mechanism of this cryoprotection is not known, it may involve recrystallization inhibition and perhaps stabilization of the cell membrane. Freeze-avoiding species must prevent inoculative freezing initiated by external ice across the cuticle and extend supercooling abilities. Some insects remove PINs in the winter to promote supercooling, whereas others have selected against surfaces with ice-nucleating abilities on an evolutionary time scale. However, many freeze-avoiding species do have proteins with ice-nucleating activity, and these proteins must be masked in winter. In the beetle Dendroides canadensis, AFPs in the hemolymph and gut inhibit ice nucleators. Also, hemolymph AFPs and those associated with the layer of epidermal cells under the cuticle inhibit inoculative freezing. Two different insect AFPs have been characterized. One type from the beetles D. canadensis and Tenebrio molitor consists of 12- and 13-mer repeating units with disulfide bridges occurring at least every six residues. The spruce budworm AFP lacks regular repeat units. Both have much higher activities than any known AFPs.

Insect antifreeze proteins (AFP) are considerably more active at inhibiting ice crystal growth than AFP from fish or plants. Several insect AFPs, also known as thermal hysteresis proteins, have been cloned and expressed. Their maximum activity is 3-4 times that of fish AFPs and they are 10-100 times more effective at micromolar concentrations. Here we report the solution structure of spruce budworm (Choristoneura fumiferana) AFP and characterize its ice-binding properties. The 9-kDa AFP is a beta-helix with a triangular cross-section and rectangular sides that form stacked parallel beta-sheets; a fold which is distinct from the three known fish AFP structures. The ice-binding side contains 9 of the 14 surface-accessible threonines organized in a regular array of TXT motifs that match the ice lattice on both prism and basal planes. In support of this model, ice crystal morphology and ice-etching experiments are consistent with AFP binding to both of these planes and thus may explain the greater activity of the spruce budworm antifreeze.

SXT is an integrative and conjugative element (ICE) that confers resistance to multiple antibiotics upon many clinical isolates of Vibrio cholerae. In most cells, this approximately 100 Kb element is integrated into the host genome in a site-specific fashion; however, SXT can excise to form an extrachromosomal circle that is thought to be the substrate for conjugative transfer. Daughter cells lacking SXT can theoretically arise if cell division occurs prior to the element's reintegration. Even though approximately 2% of SXT-bearing cells contain the excised form of the ICE, cells that have lost the element have not been detected. Here, using a positive selection-based system, SXT loss was detected rarely at a frequency of approximately 1 x 10(-7). As expected, excision appears necessary for loss, and factors influencing the frequency of excision altered the frequency of SXT loss. We screened the entire 100 kb SXT genome and identified two genes within SXT, now designated mosA and mosT (for maintenance of SXT Antitoxin and Toxin), that promote SXT stability. These two genes, which lack similarity to any previously characterized genes, encode a novel toxin-antitoxin pair; expression of mosT greatly impaired cell growth and mosA expression ameliorated MosT toxicity. Factors that promote SXT excision upregulate mosAT expression. Thus, when the element is extrachromosomal and vulnerable to loss, SXT activates a TA module to minimize the formation of SXT-free cells.

The use of an alkane mixture that remains liquid at 77 K to freeze specimens has advantages over the use of a pure alkane that is solid at 77 K. It was found that a mixture of methane and ethane did not give a cooling rate adequate to produce vitreous ice, but a mixture of propane and ethane did result in vitreous ice. Furthermore, the latter mixture produced less damage to specimens mounted on a very thin, fragile holey carbon substrate.

Muscle injuries are one of the most common traumas occurring in sports. Despite their clinical importance, there are only a few clinical studies on the treatment of muscle injuries. Lack of clinical studies is most probably attributable to the fact that there is not only a high heterogeneity in the severity of injuries, but also the injuries take place in different muscles, making it very demanding to carry out clinical trials. Accordingly, the current treatment principles of muscle injuries have either been derived from experimental studies or been tested empirically only. Clinically, first aid for muscle injuries follows the RICE (Rest, Ice, Compression and Elevation) principle. The objective of RICE is to stop the injury-induced bleeding into the muscle tissue and thereby minimise the extent of the injury. Clinical examination should be carried out immediately after the injury and 5-7 days after the initial trauma, at which point the severity of the injury can be assessed more reliably. At that time, a more detailed characterisation of the injury can be made using imaging diagnostic modalities (ultrasound or MRI) if desired. The treatment of injured skeletal muscle should be carried out by immediate immobilisation of the injured muscle (clinically, relative immobility/avoidance of muscle contractions). However, the duration of immobilisation should be limited to a period sufficient to produce a scar of sufficient strength to bear the forces induced by remobilisation without re-rupture and the return to activity (mobilisation) should then be started gradually within the limits of pain. Early return to activity is needed to optimise the regeneration of healing muscle and recovery of the flexibility and strength of the injured skeletal muscle to pre-injury levels. The rehabilitation programme should be built around progressive agility and trunk stabilisation exercises, as these exercises seem to yield better outcome for injured skeletal muscle than programmes based exclusively on stretching and strengthening of the injured muscle.

Mice exposed to 100% O2 die after 3 or 4 d with diffuse alveolar damage and alveolar edema. Extensive cell death is evident by electron microscopy in the alveolar septa, affecting both endothelial and epithelial cells. The damaged cells show features of both apoptosis (condensation and margination of chromatin) and necrosis (disruption of the plasma membrane). The electrophoretic pattern of lung DNA indicates both internucleosomal fragmentation, characteristic of apoptosis, and overall degradation, characteristic of necrosis. Hyperoxia induces a marked increase in RNA or protein levels of p53, bax, bcl-x, and Fas, which are known to be expressed in certain types of apoptosis. However, we did not detect an increased activity of proteases belonging to the apoptosis "executioner" machinery, such as CPP32 (caspase 3), ICE (caspase 1), or cathepsin D. Furthermore, administration of an ICE-like protease inhibitor did not significantly enhance the resistance to oxygen. Additionally, neither p53-deficient mice nor lpr mice (Fas null) manifested an increased resistance to hyperoxia-induced lung damage. These results show that both necrosis and apoptosis contribute to cell death during hyperoxia. Multiple apoptotic pathways seem to be involved in this, and an antiapoptotic strategy does not attenuate alveolar damage.

Variation in gene content among strains of a bacterial species contributes to biomedically relevant differences in phenotypes such as virulence and antimicrobial resistance. Group A Streptococcus (GAS) causes a diverse array of human infections and sequelae, and exhibits a complex pathogenic behavior. To enhance our understanding of genotype-phenotype relationships in this important pathogen, we determined the complete genome sequences of four GAS strains expressing M protein serotypes (M2, M4, and 2 M12) that commonly cause noninvasive and invasive infections. These sequences were compared with eight previously determined GAS genomes and regions of variably present gene content were assessed. Consistent with the previously determined genomes, each of the new genomes is approximately 1.9 Mb in size, with approximately 10% of the gene content of each encoded on variably present exogenous genetic elements. Like the other GAS genomes, these four genomes are polylysogenic and prophage encode the majority of the variably present gene content of each. In contrast to most of the previously determined genomes, multiple exogenous integrated conjugative elements (ICEs) with characteristics of conjugative transposons and plasmids are present in these new genomes. Cumulatively, 242 new GAS metagenome genes were identified that were not present in the previously sequenced genomes. Importantly, ICEs accounted for 41% of the new GAS metagenome gene content identified in these four genomes. Two large ICEs, designated 2096-RD.2 (63 kb) and 10750-RD.2 (49 kb), have multiple genes encoding resistance to antimicrobial agents, including tetracycline and erythromycin, respectively. Also resident on these ICEs are three genes encoding inferred extracellular proteins of unknown function, including a predicted cell surface protein that is only present in the genome of the serotype M12 strain cultured from a patient with acute poststreptococcal glomerulonephritis. The data provide new information about the GAS metagenome and will assist studies of pathogenesis, antimicrobial resistance, and population genomics.

Insect antifreeze proteins (AFP) are much more effective than fish AFPs at depressing solution freezing points by ice-growth inhibition. AFP from the beetle Tenebrio molitor is a small protein (8.4 kDa) composed of tandem 12-residue repeats (TCTxSxxCxxAx). Here we report its 1.4-A resolution crystal structure, showing that this repetitive sequence translates into an exceptionally regular beta-helix. Not only are the 12-amino-acid loops almost identical in the backbone, but also the conserved side chains are positioned in essentially identical orientations, making this AFP perhaps the most regular protein structure yet observed. The protein has almost no hydrophobic core but is stabilized by numerous disulphide and hydrogen bonds. On the conserved side of the protein, threonine-cysteine-threonine motifs are arrayed to form a flat beta-sheet, the putative ice-binding surface. The threonine side chains have exactly the same rotameric conformation and the spacing between OH groups is a near-perfect match to the ice lattice. Together with tightly bound co-planar external water, three ranks of oxygen atoms form a two-dimensional array, mimicking an ice section.

Total homocysteine is defined as the sum of all homocysteine species in plasma/serum, including free and protein-bound forms. In the present review, we compare and evaluate several techniques for the determination of total homocysteine. Because these assays include the conversion of all forms into a single species by reduction, the redistribution between free and protein-bound homocysteine through disulfide interchange does not affect the results, and total homocysteine can be measured in stored samples. Total homocysteine in whole blood increases at room temperature because of a continuous production and release of homocysteine from blood cells, but artificial increase is low if the blood sample is centrifuged within 1 h of collection or placed on ice. Different methods correlate well, and values between 5 and 15 mumol/L in fasting subjects are considered normal. Total homocysteine in serum/plasma is increased markedly in patients with cobalamin or folate deficiency, and decreases only when they are treated with the deficient vitamin. Total homocysteine is therefore of value for the diagnosis and follow-up of these deficiency states and may compensate for weaknesses of the traditional laboratory tests. In addition, total homocysteine is an independent risk factor for premature cardiovascular diseases. These disorders justify introduction of the total homocysteine assay in the routine clinical chemistry laboratory.

The structure of the lysozyme from bacteriophage T4 has been refined at 1.7 A resolution to a crystallographic residual of 19.3%. The final model has bond lengths and bond angles that differ from "ideal" values by 0.019 A and 2.7 degrees, respectively. The crystals are grown from electron-dense phosphate solutions and the use of an appropriate solvent continuum substantially improved the agreement between the observed and calculated structure factors at low resolution. Apart from changes in the conformations of some side-chains, the refinement confirms the structure of the molecule as initially derived from a 2.4 A resolution electron density map. There are 118 well-ordered solvent molecules that are associated with the T4 lysozyme molecule in the crystal. Four of these are more-or-less buried. There is a clustering of water molecules within the active site cleft but, other than this, the solvent molecules are dispersed around the surface of the molecule and do not aggregate into ice-like structures or pentagonal or hexagonal clusters. The apparent motion of T4 lysozyme in the crystal can be interpreted in terms of significant interdomain motion corresponding to an opening and closing of the active site cleft. For the amino-terminal domain the motion can be described equally well (correlation coefficients approx. 0.87) as quasi-rigid-body motion either about a point or about an axis of rotation. The motion in the crystals of the carboxy-terminal domain is best described as rotation about an axis (correlation coefficient 0.80) although in this case the apparent motion seems to be influenced in part by crystal contacts and may be of questionable relevance to dynamics in solution.

The rise in Arctic near-surface air temperatures has been almost twice as large as the global average in recent decades-a feature known as 'Arctic amplification'. Increased concentrations of atmospheric greenhouse gases have driven Arctic and global average warming; however, the underlying causes of Arctic amplification remain uncertain. The roles of reductions in snow and sea ice cover and changes in atmospheric and oceanic circulation, cloud cover and water vapour are still matters of debate. A better understanding of the processes responsible for the recent amplified warming is essential for assessing the likelihood, and impacts, of future rapid Arctic warming and sea ice loss. Here we show that the Arctic warming is strongest at the surface during most of the year and is primarily consistent with reductions in sea ice cover. Changes in cloud cover, in contrast, have not contributed strongly to recent warming. Increases in atmospheric water vapour content, partly in response to reduced sea ice cover, may have enhanced warming in the lower part of the atmosphere during summer and early autumn. We conclude that diminishing sea ice has had a leading role in recent Arctic temperature amplification. The findings reinforce suggestions that strong positive ice-temperature feedbacks have emerged in the Arctic, increasing the chances of further rapid warming and sea ice loss, and will probably affect polar ecosystems, ice-sheet mass balance and human activities in the Arctic.

The pack ice of Earth's polar oceans appears to be frozen white desert, devoid of life. However, beneath the snow lies a unique habitat for a group of bacteria and microscopic plants and animals that are encased in an ice matrix at low temperatures and light levels, with the only liquid being pockets of concentrated brines. Survival in these conditions requires a complex suite of physiological and metabolic adaptations, but sea-ice organisms thrive in the ice, and their prolific growth ensures they play a fundamental role in polar ecosystems. Apart from their ecological importance, the bacterial and algae species found in sea ice have become the focus for novel biotechnology, as well as being considered proxies for possible life forms on ice-covered extraterrestrial bodies.

BACKGROUND

Diabetes mellitus causes multiple cardiovascular complications. High glucose can induce reactive oxygen species and apoptosis in endothelial cells. Little is known about the molecular mechanisms in high glucose-induced endothelial cell apoptosis.

RESULTS

We elucidated the signaling pathway of high glucose-induced apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with media containing 5.5, 19, or 33 mmol/L of glucose in the presence or absence of an antioxidant, ascorbic acid. The level of intracellular H(2)O(2) was measured by flow cytometry. For detection of apoptosis, the cell death detection ELISA assay and the morphological Hoechst staining were used. High glucose was capable of inducing the activity of c-Jun NH(2)-terminal kinase (JNK) but not extracellular signal-regulated kinase 1/2 or p38 mitogen-activated protein kinase during the treatment periods, as evidenced by immunocomplex kinase assay. Moreover, we found that the interleukin 1beta-converting enzyme (ICE)/CED-3 family protease (caspase-3) became activated in high glucose-induced apoptosis. Caspase-3/CPP32-specific inhibitor, Ac-DEVD-CHO, could inhibit high glucose-induced apoptosis. Furthermore, we found that JNK1 specific antisense oligonucleotide could suppress caspase-3 activity but not affect H(2)O(2) generation and could block apoptosis induced by high glucose. Also, H(2)O(2) generation, JNK activity, caspase-3 activity, and the subsequent apoptosis induced by high glucose could be suppressed by ascorbic acid.

CONCLUSIONS

The present study indicates that reactive oxygen species induced by high glucose may be involved in JNK activation, which in turn triggers the caspase-3 that facilitates the apoptosis in HUVECs.