c-Jun, a signal-transducing transcription factor of the AP-1 family, normally implicated in cell cycle progression, differentiation and cell transformation, recently has also been linked to apoptosis. To explore further the functional roles of c-Jun, a conditional allele was generated by fusion of c-Jun with the hormone-binding domain of the human estrogen receptor (ER). Here we demonstrate that increased c-Jun activity is sufficient to trigger apoptotic cell death in NIH 3T3 fibroblasts. c-Jun-induced apoptosis is evident at high serum levels, but is enhanced further in factor-deprived fibroblasts. Furthermore, apoptosis by c-Jun is not accompanied by an increase in DNA synthesis. Constitutive overexpression of the apoptosis inhibitor protein Bcl-2 delays the c-Jun-mediated cell death. The regions of c-Jun necessary for apoptosis induction include the amino-terminal transactivation and the carboxy-terminal leucine zipper domain, suggesting that c-Jun may activate cell death by acting as a transcriptional regulator. We further show that alpha-fodrin, a substrate of the interleukin 1beta-converting enzyme (ICE) and CED-3 family of cysteine proteases, becomes proteolytically cleaved in cells undergoing cell death by increased c-Jun activity. Moreover, cell-permeable irreversible peptide inhibitors of the ICE/CED-3 family of cysteine proteases prevented the cell death.

Cryo-electron tomography of frozen-hydrated biological samples offers a means of studying large and complex cellular structures in three-dimensions and with nanometer-scale resolution. The low contrast of unstained biological material embedded in amorphous ice and the need to minimise the exposure of these radiation-sensitive samples to the electron beam result in a poor signal-to-noise ratio. This poses problems not only in the visualisation and interpretation of such tomograms, it is also a problem in surveying the sample and in finding regions which contain the features of interest and which are suitable for recording tomograms. To address this problem, we have developed a correlative fluorescence light microscopy-electron microscopy approach, which guides the search for the structures of interest and allows electron microscopy to zoom in on them. With our approach, the total dose spent on locating regions of interest is negligible. A newly designed cryo-holder allows imaging of fluorescently labelled samples after vitrification. The absolute coordinates of structures identified and located by cryo-light microscopy are transferred to the electron microscope via a Matlab-based user interface. We have successfully tested the experimental setup and the whole procedure with two types of adherent fluorescently labelled cells, a neuronal cell line and keratinocytes, both grown directly on EM grids.

The structure of Sindbis virus was determined by electron cryomicroscopy. The virion contains two icosahedral shells of viral-encoded proteins separated by a membrane bilayer of cellular origin. The three-dimensional structure of the ice-embedded intact Sindbis virus, reconstructed from electron images, unambiguously shows that proteins in both shells are arranged with the same icosahedral lattice of triangulation number T = 4. These studies also provide structural evidence of contact between the glycoprotein and the nucleocapsid protein across the membrane bilayer. The structural organization of Sindbis virus has profound implications for the morphogenesis of the alphaviruses. The observed interactions confirm stoichiometric and specific protein associations that may be crucial for virion stability and predict a mechanism for assembly.

Recent studies suggest that HIV-1 budding occurs selectively from detergent-insoluble membrane domains, referred to as lipid rafts. Palmitoylation is thought to be one of the factors responsible for targeting membrane proteins to lipid rafts. The cytoplasmic domain of the HIV-1 envelope glycoprotein (gp160) contains two palmitoylated cysteine residues. In this work, we studied the solubility of gp160 after detergent extraction. We show that wild-type gp160 is mostly insoluble after ice-cold Triton X-100 extraction, but that it becomes almost completely soluble at 37 degrees C. In contrast, we find that a mutant gp160, in which the two palmitoylated cysteine residues are replaced by serine, is Triton X-100 soluble even under ice-cold extraction. These findings are consistent with the properties of proteins that localize to lipid rafts and strongly suggest that gp160 is associated with lipid rafts. Further, removal of both palmitoylation sites results in the formation of virus with low levels of gp160 incorporation as well as a decrease in viral infectivity by 60-fold. Our results strongly support the suggestion that HIV-1 buds from lipid rafts and point to a role for rafts as a viral assembly hub.

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was M. mucogenicum (formerly referred to as an M. chelonae-like organism).

Clovis, with its distinctive biface, blade and osseous technologies, is the oldest widespread archaeological complex defined in North America, dating from 11,100 to 10,700 (14)C years before present (bp) (13,000 to 12,600 calendar years bp). Nearly 50 years of archaeological research point to the Clovis complex as having developed south of the North American ice sheets from an ancestral technology. However, both the origins and the genetic legacy of the people who manufactured Clovis tools remain under debate. It is generally believed that these people ultimately derived from Asia and were directly related to contemporary Native Americans. An alternative, Solutrean, hypothesis posits that the Clovis predecessors emigrated from southwestern Europe during the Last Glacial Maximum. Here we report the genome sequence of a male infant (Anzick-1) recovered from the Anzick burial site in western Montana. The human bones date to 10,705 ± 35 (14)C years bp (approximately 12,707-12,556 calendar years bp) and were directly associated with Clovis tools. We sequenced the genome to an average depth of 14.4× and show that the gene flow from the Siberian Upper Palaeolithic Mal'ta population into Native American ancestors is also shared by the Anzick-1 individual and thus happened before 12,600 years bp. We also show that the Anzick-1 individual is more closely related to all indigenous American populations than to any other group. Our data are compatible with the hypothesis that Anzick-1 belonged to a population directly ancestral to many contemporary Native Americans. Finally, we find evidence of a deep divergence in Native American populations that predates the Anzick-1 individual.

Activation of proteolytic enzymes, including cysteine proteases of the ced-3/ICE family, is a characteristic feature of the apoptotic program. In contrast, the role of the proteasome as the major nonlysosomal machinery to degrade or process proteins by ATP/ubiquitin-dependent proteolysis in this process is less clear. In human leukemic HL60 cells, inhibition of proteasome-mediated proteolysis by specific proteasomal inhibitors leads to the rapid induction of apoptosis as judged by morphological changes as well as by nuclear condensation and DNA fragmentation. HL60 apoptosis is due to activation of CPP32, a member of the ced-3/ICE family of cysteine proteases, and appears to occur independently from ICE activity. HL60 apoptosis is accompanied by an increase in the concentration of the cyclin-dependent kinase inhibitor p27Kip1. Labeling of the cells by the TUNEL technique demonstrates that HL60 cells undergoing apoptosis are primarily in the G1 phase of the cell cycle. Proteasomal activity therefore appears to be required in proliferating, but not in quiescent, HL60 cells for cell survival as well as normal progression through the cell cycle.

BACKGROUND

It is widely accepted that the ancestors of Native Americans arrived in the New World via Beringia approximately 10 to 30 thousand years ago (kya). However, the arrival time(s), number of expansion events, and migration routes into the Western Hemisphere remain controversial because linguistic, archaeological, and genetic evidence have not yet provided coherent answers. Notably, most of the genetic evidence has been acquired from the analysis of the common pan-American mitochondrial DNA (mtDNA) haplogroups. In this study, we have instead identified and analyzed mtDNAs belonging to two rare Native American haplogroups named D4h3 and X2a.

RESULTS

Phylogeographic analyses at the highest level of molecular resolution (69 entire mitochondrial genomes) reveal that two almost concomitant paths of migration from Beringia led to the Paleo-Indian dispersal approximately 15-17 kya. Haplogroup D4h3 spread into the Americas along the Pacific coast, whereas X2a entered through the ice-free corridor between the Laurentide and Cordilleran ice sheets. The examination of an additional 276 entire mtDNA sequences provides similar entry times for all common Native American haplogroups, thus indicating at least a dual origin for Paleo- Indians.

CONCLUSIONS

A dual origin for the first Americans is a striking novelty from the genetic point of view, and it makes plausible a scenario positing that within a rather short period of time, there may have been several entries into the Americas from a dynamically changing Beringian source. Moreover, this implies that most probably more than one language family was carried along with the Paleo-Indians.

Prenatal maternal stress has been shown to impair functioning in nonhuman primate offspring. Little is known about the effects of prenatal stress on intellectual and language development in humans because it is difficult to identify sufficiently large samples of pregnant women who have been exposed to an independent stressor. We took advantage of a natural disaster (January 1998 ice storm in Québec, Canada) to determine the effect of the objective severity of pregnant women's stress exposure on general intellectual and language development of their children. Bayley Mental Development Index (MDI) scores and parent-reported language abilities of 58 toddlers of mothers who were exposed to varying levels of prenatal stress were obtained at 2 y of age. The hierarchical multiple regression analyses indicated that the toddlers' birth weight and age at testing accounted for 12.0% and 14.8% of the variance in the Bayley MDI scores and in productive language abilities, respectively. More importantly, the level of prenatal stress exposure accounted for an additional 11.4% and 12.1% of the variance in the toddlers' Bayley MDI and productive language abilities and uniquely accounted for 17.3% of the variance of their receptive language abilities. The more severe the level of prenatal stress exposure, the poorer the toddlers' abilities. The level of prenatal stress exposure accounted for a significant proportion of the variance in the three dependent variables above and beyond that already accounted for by non-ice storm-related factors. We suspect that high levels of prenatal stress exposure, particularly early in the pregnancy, may negatively affect the brain development of the fetus, reflected in the lower general intellectual and language abilities in the toddlers.

Caspase 12 has been cloned from rodent cells, in which it mediated apoptosis in response to endoplasmic reticulum stress. Based on experiments with murine cells it was suggested that this caspase plays a central role in the pathogenesis of Alzheimer's disease. By alignment of the murine caspase 12 cDNA with the human genome sequence we localized the human caspase 12 gene at a single locus within the caspase 1/ICE gene cluster on chromosome 11q22.3. RT-PCR and molecular cloning revealed that nine alternatively spliced transcripts of this gene are expressed. A frame shift mutation and a premature stop codon which is present in all splice variants preclude the expression of a full length protein. An additional loss-of-function mutation within the SHG box, a critical site in caspases, prohibits any proteins, if they are produced, from acting catalytically. Based on our data we conclude that functional caspase 12 is lost in humans and that it can therefore not play a role in Alzheimer's disease.

A detailed kinetic analysis of three extranuclear end points of apoptosis, phosphatidylserine exposure, alpha-fodrin degradation, and plasma membrane blebbing, was performed and compared with nuclear fragmentation and the activation of the interleukin-1beta-converting enzyme (ICE)-like proteases in Jurkat T lymphocytes stimulated by anti-Fas monoclonal antibody (anti-Fas mAb) and in monocytic U937 cells stimulated by tumor necrosis factor (TNF) and cycloheximide. Phosphatidylserine exposure was quantitated by plasma clotting time, as well as annexin V-fluorescein isothiocyanate binding, and the ICE-like protease activity was examined by the cleavage of a specific fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin. VAD-chloromethylketone (VAD-cmk), an inhibitor of ICE-like proteases, effectively inhibited ICE-like activity in both cell types studied, whereas the calpain inhibitor calpeptin was ineffective. VAD-cmk also effectively inhibited all three extranuclear events, as well as nuclear fragmentation, in Jurkat cells stimulated by anti-Fas monoclonal antibody, indicating that ICE-like proteases play an important role in the regulation of this apoptotic system. Calpain inhibitors were ineffective in this system. TNF-induced extranuclear and nuclear changes in U937 cells were inhibited by calpeptin but were not as effectively inhibited by VAD-cmk as in Jurkat cells. This suggests that ICE-like enzymes predominate in anti-Fas monoclonal antibody-stimulated Jurkat cells, whereas proteases affected by calpain inhibitors as well as the ICE-like enzymes are involved in the signaling of apoptotic events in TNF-induced U937 cells. Importantly, the two apoptotic systems seem to be regulated by different proteases.

The bacterial populations associated with sea ice sampled from Antarctic coastal areas were investigated by use of a phenotypic approach and a phylogenetic approach based on genes encoding 16S rRNA (16S rDNA). The diversity of bacteria associated with sea ice was also compared with the bacterial diversity of seawater underlying sea ice. Psychrophilic (optimal growth temperature, < or = 15 degrees C; no growth occurring at 20 degrees C) bacterial diversity was found to be significantly enriched in sea ice samples possessing platelet and bottom ice diatom assemblages, with 2 to 9 distinct (average, 5.6 +/- 1.8) psychrophilic taxa isolated per sample. Substantially fewer psychrophilic isolates were recovered from ice cores with a low or negligible population of ice diatoms or from under-ice seawater samples (less than one distinct taxon isolated per sample). In addition, psychrophilic taxa that were isolated from under-ice seawater samples were in general phylogenetically distinct from psychrophilic taxa isolated from sea ice cores. The taxonomic distributions of psychrotrophic bacterial isolates (optimal growth temperature,>> 20 degrees C; growth can occur at approximately 4 degrees C) isolated from sea ice cores and under-ice seawater were quite similar. Overall, bacterial isolates from Antarctic sea ice were found to belong to four phylogenetic groups, the alpha and gamma subdivisions of the Proteobacteria, the gram-positive branch, and the Flexibacter-Bacteroides-Cytophaga phylum. Most of the sea ice strains examined appeared to be novel taxa based on phylogenetic comparisons, with 45% of the strains being psychrophilic. 16S rDNA sequence analysis revealed that psychrophilic strains belonged to the genera Colwellia, Shewanella, Marinobacter, Planococcus, and novel phylogenetic lineages adjacent to Colwellia and Alteromonas and within the Flexibacter-Bacteroides-Cytophaga phylum. Psychrotrophic strains were found to be members of the genera Pseudoalteromonas, Psychrobacter, Halomonas, Pseudomonas, Hyphomonas, Sphingomonas, Arthrobacter, Planococcus, and Halobacillus. From this survey, it is proposed that ice diatom assemblages provide niches conducive to the proliferation of a diverse array of psychrophilic bacterial species.

Proteolytic cleavage of key substrates appears to be an important biochemical mechanism underlying the apoptotic process, and the centrality of interleukin 1 beta-converting enzyme (ICE)-like proteases as mediators of apoptosis has been suggested. The identification of the relevant substrates of the ICE protease family during apoptosis therefore constitutes a major challenge. Using human autoantibodies, we demonstrate here that a subset of autoantigens is specifically cleaved early during apoptosis. One of these cleaved molecules is identified as the catalytic subunit of the DNA-dependent protein kinase. The time courses of all proteolytic cleavages are identical and coincide with the onset of morphologic apoptosis. Furthermore, all cleavages share the same inhibition characteristics, which implicate an ICE-like activity(ies). We propose that cleavage of these autoantigens targets these molecules for an autoimmune response by revealing immunocryptic fragments in a proimmune apoptotic setting. Study of the immunogenicity of these fragments may yield insights into the autoimmune targeting of molecules. Moreover, the autoantibodies described will be valuable tools for the elucidation of mechanistically important proteolytic steps along the apoptotic pathway.

Many mountain ranges have been strongly glaciated during the Quaternary ice ages, and the locations of glacial refugia of mountain plants have been debated for a long time. A series of detailed molecular studies, investigating intraspecific genetic variation of mountain plants in the European Alps, now allows for a first synopsis. A comparison of the phylogeographic patterns with geological and palaeoenvironmental data demonstrates that glacial refugia were located along the southwestern, southern, eastern and northern border of the Alps. Additional glacial refugia were present in central Alpine areas, where high-elevation plants survived the last glaciation on ice-free mountain tops. The observed intraspecific phylogeographies suggest general patterns of glacial survival, which conform to well-known centres of Alpine species diversity and endemism. This implies that evolutionary or biogeographic processes induced by climatic fluctuations act on gene and species diversity in a similar way.

Diffusion of a G-protein coupled receptor, mu-opioid receptor (muOR), in the plasma membrane was tracked by single-fluorescent molecule video imaging and high-speed single-particle tracking. At variance with a previous publication, where gold-tagged muOR was found to be totally confined within a domain, which in turn underwent very slow diffusion itself, we found that muOR undergoes rapid hop diffusion over membrane compartments (210-nm and 730-nm nested double compartments in the case of normal rat kidney cell line), which are likely delimited by the actin-based membrane-skeleton "fence or corrals" and its associated transmembrane protein "pickets", at a rate comparable to that for transferrin receptor (every 45 and 760 ms on average, respectively), suggesting that the fence and picket models may also be applicable to G-protein coupled receptors. Further, we found that strong confinement of gold-labeled muOR could be induced by the prolonged on-ice preincubation of the gold probe with the cells, showing that this procedure should be avoided in future single-particle tracking experiments. Based on the dense, long trajectories of muOR obtained by high-speed single-particle tracking, the membrane compartments apposed and adjoined to each other could be defined that are delimited by rather straight boundaries, consistent with the involvement of actin filaments in membrane compartmentalization.

Marine teleosts at high latitudes can encounter ice-laden seawater that is approximately 1 degrees C colder than the colligative freezing point of their body fluids. They avoid freezing by producing small antifreeze proteins (AFPs) that adsorb to ice and halt its growth, thereby producing an additional non-colligative lowering of the freezing point. AFPs are typically secreted by the liver into the blood. Recently, however, it has become clear that AFP isoforms are produced in the epidermis (skin, scales, fin, and gills) and may serve as a first line of defense against ice propagation into the fish. The basis for the adsorption of AFPs to ice is something of a mystery and is complicated by the extreme structural diversity of the five antifreeze types. Despite the recent acquisition of several AFP three-dimensional structures and the definition of their ice-binding sites by mutagenesis, no common ice-binding motif or even theme is apparent except that surface-surface complementarity is important for binding. The remarkable diversity of antifreeze types and their seemingly haphazard phylogenetic distribution suggest that these proteins might have evolved recently in response to sea level glaciation occurring just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Not surprisingly, the expression of AFP genes from different origins can also be quite dissimilar. The most intensively studied system is that of the winter flounder, which has a built-in annual cycle of antifreeze expression controlled by growth hormone (GH) release from the pituitary in tune with seasonal cues. The signal transduction pathway, transcription factors, and promoter elements involved in this process are just beginning to be characterized.

NADH:ubiquinone oxidoreductase (complex I) is the first and largest complex in the electron transport chain of mitochondria. The bovine complex purified from cardiac muscle consists of at least 42 different subunits with a combined molecular mass of about 890 kDa. The three-dimensional structure of the complex was determined at 22 A from single particles embedded in vitrified ice using electron cryo-microscopy. The structure was calculated using a new program to align particles, to correct for the contrast transfer function of the microscope, and to carry out the three-dimensional reconstruction of the complex. The bovine complex has the overall L-shaped appearance found in earlier studies of the closely related complex I from Neurospora crassa, but it differs by having a thin stalk region linking the membrane-bound globular arm with the intrinsic membrane domain. Thus, the stalk which measures about 30 A in diameter is likely to contain part of the electron transfer pathway linking the NADH binding site in the globular arm with the ubiquinone binding site in the membrane domain. The globular domain of bovine complex I is significantly bigger than that of the N. crassa enzyme, suggesting that the apparent additional subunit complexity of the bovine enzyme is associated with the globular part.

Cysteine proteases of the interleukin 1 beta Converting Enzyme (ICE)/CED-3 family have been implicated in the effector process of apoptosis in several systems, including Fas-mediated apoptosis. We have recently isolated and partially characterized a protease present in extracts from anti-Fas antibody treated Jurkat T cells that promotes apoptotic changes in isolated nuclei (Schlegel, J., Peters, I., and Orrenius, S. (1995) FEBS Lett. 364, 139-142). We now show that this protease cleaves poly-(ADP-ribose) polymerase (PARP) with high efficiency and specificity. Both PARP proteolysis and the proapoptotic effects of the protease are inhibited by nanomolar concentrations of a selective inhibitor of apopain (CPP32), while an inhibitor of IL-1 beta converting enzyme is much less effective, requiring micromolar concentrations for the inhibition of the isolated protease. Kinetic analysis of the isolated protease reveals kinetic constants similar to those reported for apopain. The isolated protease is recognized by antibodies specific for CPP32/apopain but not by an anti-ICE antibody. Furthermore, a selective inhibitor of apopain prevents Fas-induced apoptosis in intact Jurkat T cells. We therefore conclude that CPP32/apopain is activated in Fas-induced apoptosis.

Changes in past atmospheric carbon dioxide concentrations can be determined by measuring the composition of air trapped in ice cores from Antarctica. So far, the Antarctic Vostok and EPICA Dome C ice cores have provided a composite record of atmospheric carbon dioxide levels over the past 650,000 years. Here we present results of the lowest 200 m of the Dome C ice core, extending the record of atmospheric carbon dioxide concentration by two complete glacial cycles to 800,000 yr before present. From previously published data and the present work, we find that atmospheric carbon dioxide is strongly correlated with Antarctic temperature throughout eight glacial cycles but with significantly lower concentrations between 650,000 and 750,000 yr before present. Carbon dioxide levels are below 180 parts per million by volume (p.p.m.v.) for a period of 3,000 yr during Marine Isotope Stage 16, possibly reflecting more pronounced oceanic carbon storage. We report the lowest carbon dioxide concentration measured in an ice core, which extends the pre-industrial range of carbon dioxide concentrations during the late Quaternary by about 10 p.p.m.v. to 172-300 p.p.m.v.

BACKGROUND

The role of pulmonary vein (PV) isolation in ablative treatment of atrial fibrillation (AF) has been debated in conflicting reports. We sought to compare PV conduction in patients who had no AF recurrence (group I), patients who could maintain sinus rhythm on antiarrhythmic medication (group II), and patients who had recurrent AF despite antiarrhythmic medication (group III) after PV antrum isolation (PVAI).

RESULTS

PV conduction was examined in consecutive patients undergoing second PVAI for AF recurrence. We also recruited some patients cured of AF to undergo a repeat, limited electrophysiological study at >3 months after PVAI. All patients underwent PVAI with an intracardiac echocardiography (ICE)-guided approach with complete isolation of all 4 PV antra (PVA). The number of PVs with recurrent conduction and the shortest atrial to PV (A-PV) conduction delay was measured with the use of consistent Lasso positions defined by ICE. Late AF recurrence was defined as AF >2 months after PVAI with the patient off medications. Patients in groups I (n=26), II (n=37), and III (n=44) did not differ at baseline (38% permanent AF; ejection fraction 53+/-6%). Recurrence of PV-left atrial (LA) conduction was seen in 1.7+/-0.8 and 2.2+/-0.8 PVAs for groups II and III but only in 0.2+/-0.4 for group I (P=0.02). In patients with recurrent PV-LA conduction, the A-PV delay increased from the first to second procedure by 69+/-47% for group III, 267+/-110% for group II, and 473+/-71% for group I (P<0.001). When pacing was at a faster rate, A-PV block developed in all 5 of the group I patients with recurrent PV-LA conduction.

CONCLUSIONS

The majority of patients with drug-free cure show no PV-LA conduction recurrence. Substantial A-PV delay is seen in patients able to maintain sinus rhythm on antiarrhythmic medication or cured of AF compared with patients who fail PVAI.

All currently available climate models predict a near-surface warming trend under the influence of rising levels of greenhouse gases in the atmosphere. In addition to the direct effects on climate--for example, on the frequency of heatwaves--this increase in surface temperatures has important consequences for the hydrological cycle, particularly in regions where water supply is currently dominated by melting snow or ice. In a warmer world, less winter precipitation falls as snow and the melting of winter snow occurs earlier in spring. Even without any changes in precipitation intensity, both of these effects lead to a shift in peak river runoff to winter and early spring, away from summer and autumn when demand is highest. Where storage capacities are not sufficient, much of the winter runoff will immediately be lost to the oceans. With more than one-sixth of the Earth's population relying on glaciers and seasonal snow packs for their water supply, the consequences of these hydrological changes for future water availability--predicted with high confidence and already diagnosed in some regions--are likely to be severe.

The cold-pressor test (CPT) in which subjects immerse their hand in ice water is among the most commonly used laboratory stressors. While the CPT elicits strong sympathetic nervous system activation, cortisol elevations indicative for the reactivity of the hypothalamus-pituitary-adrenal (HPA) axis are moderate to low in response to the CPT. In the present study, we assessed whether cortisol responses to the CPT can be increased by adding social-evaluative elements. Therefore, 70 healthy young men immersed their hand in ice or warm water and were watched by a woman and videotaped during hand immersion or not. While the standard CPT and the socially evaluated cold-pressor test (SECPT) led to comparable increases in blood pressure and subjective stress ratings, saliva cortisol elevations and the proportion of subjects showing a saliva cortisol response (defined as increase >2nmol/l) were significantly higher after the SECPT. Social evaluation during hand immersion in warm water did not affect saliva cortisol levels suggesting that both social evaluation and a challenge are required for HPA axis activation. These findings indicate that the incorporation of social-evaluative elements increases HPA axis responses to the CPT. The SECPT can serve as a tool for future stress research.

To investigate the relation of dietary intakes of sucrose, meat, and fat, and anthropometric, lifestyle, hormonal, and reproductive factors to colon cancer incidence, data were analyzed from a prospective cohort study of 35,215 Iowa (United States) women, aged 55-69 years and without a history of cancer, who completed mailed dietary and other questionnaires in 1986. Through 1990, 212 incident cases of colon cancer were documented. Proportional hazards regression was used to adjust for age and other risk factors. Risk factors found to be associated significantly with colon cancer included: (i) sucrose-containing foods and beverages other than ice cream/milk; relative risks (RR) across the quintiles = 1.00, 1.73, 1.56, 1.54, and 2.00 (95% confidence intervals [CI] for quintiles two and five exclude 1.0); (ii) sucrose; RR across the quintiles = 1.00, 1.70, 1.81, 1.82, and 1.45 (CI for quintiles two through four exclude 1.0); (iii) height; RR = 1.23 for highest to lowest quintile (P for trend = 0.02); (iv) body mass index; RR = 1.41 for highest to lowest quintile (P for trend = 0.03); and (v) number of livebirths, RR = 1.59 for having had one to two livebirths and 1.80 for having had three or more livebirths compared with having had none (P for trend = 0.04). These data support hypotheses that sucrose intake or being tall or obese increases colon cancer risk; run contrary to the hypothesis that increased parity decreases risk; support previous findings of no association with demographic factors other than age, cigarette smoking, or use of oral contraceptives or estrogen replacement therapy; and raise questions regarding previous associations with meat, fat, protein, and physical activity.

Bacteria have important roles in freshwater food webs and in the cycling of elements in the ecosystem. Yet specific ecological features of individual phylogenetic groups and interactions among these are largely unknown. We used 454 pyrosequencing of 16S rRNA genes to study associations of different bacterioplankton groups to environmental characteristics and their co-occurrence patterns over an annual cycle in a dimictic lake. Clear seasonal succession of the bacterioplankton community was observed. After binning of sequences into previously described and highly resolved phylogenetic groups (tribes), their temporal dynamics revealed extensive synchrony and associations with seasonal events such as ice coverage, ice-off, mixing and phytoplankton blooms. Coupling between closely and distantly related tribes was resolved by time-dependent rank correlations, suggesting ecological coherence that was often dependent on taxonomic relatedness. Association networks with the abundant freshwater Actinobacteria and Proteobacteria in focus revealed complex interdependencies within bacterioplankton communities and contrasting linkages to environmental conditions. Accordingly, unique ecological features can be inferred for each tribe and reveal the natural history of abundant cultured and uncultured freshwater bacteria.