Type I antifreeze protein (AFP) from winter flounder is an alanine-rich, 37 amino acid, single alpha-helix that contains three 11 amino acid repeats (Thr-X(2)-Asx-X(7)), where X is generally Ala. The regularly spaced Thr, Asx and Leu residues lie on one face of the helix and have traditionally been thought to form hydrogen bonds and van der Waals interactions with the ice surface. Recently, substitution experiments have called into question the importance of Leu and Asn for ice-binding. Sequence alignments of five type I AFP isoforms show that Leu and Asn are not well conserved, whereas Ala residues adjacent to the Thr, at right angles to the Leu/Asn-rich face, are completely conserved. To investigate the role of these Ala residues, a series of Ala to Leu steric mutations was made at various points around the helix. All the substituted peptides were fully alpha-helical and remained as monomers in solution. Wild-type activity was retained in A19L and A20L. A17L, where the substitution lies adjacent to the Thr-rich face, had no detectable antifreeze activity. The nearby A21L substitution had 10% wild-type activity and demonstrated weak interactions with the ice surface. We propose a new ice-binding face for type I AFP that encompasses the conserved Ala-rich surface and adjacent Thr.

The Medieval Climate Anomaly (MCA) was the most recent pre-industrial era warm interval of European climate, yet its driving mechanisms remain uncertain. We present here a 947-year-long multidecadal North Atlantic Oscillation (NAO) reconstruction and find a persistent positive NAO during the MCA. Supplementary reconstructions based on climate model results and proxy data indicate a clear shift to weaker NAO conditions into the Little Ice Age (LIA). Globally distributed proxy data suggest that this NAO shift is one aspect of a global MCA-LIA climate transition that probably was coupled to prevailing La Niña-like conditions amplified by an intensified Atlantic meridional overturning circulation during the MCA.

Total mercury (THg), methylmercury (MeHg) and 22 other trace elements were measured in ice algae, three species of zooplankton, mixed zooplankton samples, Arctic cod (Boreogadus saida), ringed seals (Phoca hispida) and eight species of seabirds to examine the trophodynamics of these metals in an Arctic marine food web. All samples were collected in 1998 in the Northwater Polynya (NOW) located between Ellesmere Island and Greenland in Baffin Bay. THg and MeHg were found to biomagnify through the NOW food web, based on significant positive relationships between log THg and log MeHg concentrations vs. delta15N muscle and liver . The slope of these relationships for muscle THg and MeHg concentrations (slope=0.197 and 0.223, respectively) were similar to those reported for other aquatic food webs. The food web behavior of THg and delta15N appears constant, regardless of trophic state (eutrophic vs. oligotrophic), latitude (Arctic vs. tropical) or salinity (marine vs. freshwater) of the ecosystem. Rb in both liver and muscle tissue and Zn in muscle tissue were also found to biomagnify through this food web, although at a rate that is approximately 25% of that of THg. A number of elements (Cd, Pb and Ni in muscle tissue and Cd and Li in seabird liver tissue) were found to decrease trophically through the food web, as indicated by significantly negative relationships with tissue-specific delta15N. A diverse group of metals (Ag, Ba, La, Li, Sb, Sr, U and V) were found to have higher concentrations in zooplankton than seabirds or marine mammals due to bioconcentration from seawater. The remaining metals (As, Co, Cu, Ga, Mn, Mo and Se in muscle tissue) showed no relationship with trophic position, as indicated by delta15N values, although As in liver tissue showed significant biomagnification in the seabird portion of the food web.

We have shown previously that chronic exposure of HIT-T15 cells to supraphysiologic glucose concentrations causes decreased insulin gene transcription and decreased binding activities of two beta-cell specific transcription factors, STF-1 and the RIPE3b1 activator, and have suggested that these events may provide a mechanism for glucose toxicity on beta-cell function. However, this contention can be criticized because it is not clear whether these observations are unique to the HIT-T15 cell or generalizable to other beta-cell lines and the islet. Therefore, we cultured betaTC-6 cells for up to 41 wk in either 11.1 or 0.8 mM glucose. We observed a passage-dependent decrease in insulin content and insulin mRNA levels in betaTC-6 cells chronically cultured in 11.1 mM glucose. Cells chronically cultured in 0.8 mM glucose had higher insulin mRNA levels than cells chronically cultured in 11.1 mM glucose. The relative activity of a chloramphenicol acetyl transferase (CAT) reporter gene controlled by the 5' regulatory region of the human insulin gene was decreased in late passage betaTC-6 cells chronically cultured in 11.1 mM glucose, but was preserved in late passages of cells chronically cultured in 0.8 mM glucose. Electromobility shift assays demonstrated that binding of a specific nuclear protein that recognizes the RIPE3b1 binding site of the insulin gene was markedly diminished in late passage cells chronically exposed to 11.1 mM glucose, whereas binding activities of STF-1 and ICE activators were unchanged. RIPE3b1 binding activity was preserved in late passage cells chronically exposed to 0.8 mM glucose. Mutation of the RIPE3b1 binding site almost completely abolished insulin gene transcription as well as binding activity. We conclude that chronic exposure of betaTC-6 cells to high glucose concentrations paradoxically decreases insulin gene transcription, in part, by decreasing activity of the trans-activating factor which binds to the RIPE3b1 sequence. This study uniquely demonstrates that altered binding to the RIPE3b1 sequence mediates glucose toxicity in betaTC-6 cells, thus reinforcing the importance of this cis-acting element in the regulation of insulin gene transcription. We conclude that the phenomenon of glucose toxicity decreasing binding of transcription factors and thereby reducing insulin gene expression is not a feature solely of HIT-T15 cells and may be demonstrable generally in beta-cell lines.

Global climate change around 3-4 Myr ago is thought to have influenced the evolution of hominids, via the aridification of Africa, and may have been the precursor to Pleistocene glaciation about 2.75 Myr ago. Most explanations of these climatic events involve changes in circulation of the North Atlantic Ocean due to the closing of the Isthmus of Panama. Here we suggest, instead, that closure of the Indonesian seaway 3-4 Myr ago could be responsible for these climate changes, in particular the aridification of Africa. We use simple theory and results from an ocean circulation model to show that the northward displacement of New Guinea, about 5 Myr ago, may have switched the source of flow through Indonesia-from warm South Pacific to relatively cold North Pacific waters. This would have decreased sea surface temperatures in the Indian Ocean, leading to reduced rainfall over eastern Africa. We further suggest that the changes in the equatorial Pacific may have reduced atmospheric heat transport from the tropics to higher latitudes, stimulating global cooling and the eventual growth of ice sheets.

BACKGROUND

The high-quality sequence information and rich bioinformatics tools available for rice have contributed to remarkable advances in functional genomics. To facilitate the application of gene function information to the study of natural variation in rice, we comprehensively searched for articles related to rice functional genomics and extracted information on functionally characterized genes.

RESULTS

As of 31 March 2012, 702 functionally characterized genes were annotated. This number represents about 1.6% of the predicted loci in the Rice Annotation Project Database. The compiled gene information is organized to facilitate direct comparisons with quantitative trait locus (QTL) information in the Q-TARO database. Comparison of genomic locations between functionally characterized genes and the QTLs revealed that QTL clusters were often co-localized with high-density gene regions, and that the genes associated with the QTLs in these clusters were different genes, suggesting that these QTL clusters are likely to be explained by tightly linked but distinct genes. Information on the functionally characterized genes compiled during this study is now available in the O verview of Functionally Characterized G enes in R ice O nline database (OGRO) on the Q-TARO website ( http://qtaro.abr.affrc.go.jp/ogro ). The database has two interfaces: a table containing gene information, and a genome viewer that allows users to compare the locations of QTLs and functionally characterized genes.

CONCLUSIONS

OGRO on Q-TARO will facilitate a candidate-gene approach to identifying the genes responsible for QTLs. Because the QTL descriptions in Q-TARO contain information on agronomic traits, such comparisons will also facilitate the annotation of functionally characterized genes in terms of their effects on traits important for rice breeding. The increasing amount of information on rice gene function being generated from mutant panels and other types of studies will make the OGRO database even more valuable in the future.

Excessive or persistent proinflammatory cytokine production plays a central role in autoimmune diseases. Acute activation of the sympathetic nervous system attenuates the innate immune response. However, both the autonomic nervous system and innate immune system are regarded as systems that cannot be voluntarily influenced. Herein, we evaluated the effects of a training program on the autonomic nervous system and innate immune response. Healthy volunteers were randomized to either the intervention (n = 12) or control group (n = 12). Subjects in the intervention group were trained for 10 d in meditation (third eye meditation), breathing techniques (i.a., cyclic hyperventilation followed by breath retention), and exposure to cold (i.a., immersions in ice cold water). The control group was not trained. Subsequently, all subjects underwent experimental endotoxemia (i.v. administration of 2 ng/kg Escherichia coli endotoxin). In the intervention group, practicing the learned techniques resulted in intermittent respiratory alkalosis and hypoxia resulting in profoundly increased plasma epinephrine levels. In the intervention group, plasma levels of the anti-inflammatory cytokine IL-10 increased more rapidly after endotoxin administration, correlated strongly with preceding epinephrine levels, and were higher. Levels of proinflammatory mediators TNF-α, IL-6, and IL-8 were lower in the intervention group and correlated negatively with IL-10 levels. Finally, flu-like symptoms were lower in the intervention group. In conclusion, we demonstrate that voluntary activation of the sympathetic nervous system results in epinephrine release and subsequent suppression of the innate immune response in humans in vivo. These results could have important implications for the treatment of conditions associated with excessive or persistent inflammation, such as autoimmune diseases.

Acute lateral ankle ligament sprains are common in young athletes (15 to 35 years of age). Diagnostic and treatment protocols vary. Therapies range from cast immobilisation or acute surgical repair to functional rehabilitation. The lateral ligament complex includes 3 capsular ligaments: the anterior tibiofibular (ATFL), calcaneofibular (CFL) and posterior talofibular (PTFL) ligaments. Injuries typically occur during plantar flexion and inversion; the ATFL is most commonly torn. The CFL and the PTFL can also be injured and, after severe inversion, subtalar joint ligaments are also affected. Commonly, an athlete with a lateral ankle ligament sprain reports having 'rolled over' the outside of their ankle. The entire ankle and foot must be examined to ensure there are no other injuries. Clinical stability tests for ligamentous disruption include the anterior drawer test of ATFL function and inversion tilt test of both ATFL and CFL function. Radiographs may rule out treatable fractures in severe injuries or when pain or tenderness are not associated with lateral ligaments. Stress radiographs do not affect treatment. Ankle sprains are classified from grades I to III (mild, moderate or severe). Grade I and II injuries recover quickly with nonoperative management. A non-operative 'functional treatment' programme includes immediate use of RICE (rest, ice, compression, elevation), a short period of immobilisation and protection with a tape or bandage, and early range of motion, weight-bearing and neuromuscular training exercises. Proprioceptive training on a tilt board after 3 to 4 weeks helps improve balance and neuromuscular control of the ankle. Treatment for grade III injuries is more controversial. A comprehensive literature evaluation and meta-analysis showed that early functional treatment provided the fastest recovery of ankle mobility and earliest return to work and physical activity without affecting late mechanical stability. Functional treatment was complication-free, whereas surgery had serious, though infrequent, complications. Functional treatment produced no more sequelae than casting with or without surgical repair. Secondary surgical repair, even years after an injury, has results comparable to those of primary repair, so even competitive athletes can receive initial conservative treatment. Sequelae of lateral ligament injuries are common. After conservative or surgical treatment, 10 to 30% of patients have chronic symptoms, including persistent synovitis or tendinitis, ankle stiffness, swelling, pain, muscle weakness and 'giving-way'. Well-designed physical therapy programmes usually reduce instability. For individuals with chronic instability refractory to conservative measures, surgery may be needed. Subtalar instability should be carefully evaluated when considering surgery.

From 3-D reconstructions of automatically recorded tilt series of ice-embedded macromolecules, several hundred 3-D images of single particles can be extracted. Here we describe correlation-based techniques to align the particles with respect to translation and orientation in 3-D and the calculation of an averaged reconstruction after application of the correct weighting function to the particle projections. Multivariate statistical analysis and classification are applied to the set of three-dimensionally reconstructed particles to investigate interimage variations on the 3-D level. Copyright 1997 Academic Press. Copyright 1997 Academic Press

Cassini has identified a geologically active province at the south pole of Saturn's moon Enceladus. In images acquired by the Imaging Science Subsystem (ISS), this region is circumscribed by a chain of folded ridges and troughs at approximately 55 degrees S latitude. The terrain southward of this boundary is distinguished by its albedo and color contrasts, elevated temperatures, extreme geologic youth, and narrow tectonic rifts that exhibit coarse-grained ice and coincide with the hottest temperatures measured in the region. Jets of fine icy particles that supply Saturn's E ring emanate from this province, carried aloft by water vapor probably venting from subsurface reservoirs of liquid water. The shape of Enceladus suggests a possible intense heating epoch in the past by capture into a 1:4 secondary spin/orbit resonance.

A hallmark of the human motor system is its ability to adapt motor patterns for different environmental conditions, such as when a skilled ice-hockey player accurately shoots a puck with or without protective equipment. Each object (stick, shoulder pad, elbow pad) imparts a distinct load upon the limb, and a key problem in motor neuroscience is to understand how the brain controls movement for different mechanical contexts. We addressed this issue by training non-human primates to make reaching movements with and without viscous loads applied to the shoulder and/or elbow joints, and then examined neural representations in primary motor cortex (MI) for each load condition. Even though the shoulder and elbow loads are mechanically independent, we found that some neurons responded to both of these single-joint loads. Furthermore, changes in activity of individual neurons during multi-joint loads could be predicted from their response to subordinate single-joint loads. These findings suggest that neural representations of different mechanical contexts in MI are organized in a highly structured manner that may provide a neural basis for how complex motor behaviour is learned from simpler motor tasks.

A high-resolution oxygen-isotope record from a thorium-uranium-dated stalagmite from southern Oman reflects variations in the amount of monsoon precipitation for the periods from 10.3 to 2.7 and 1.4 to 0.4 thousand years before the present (ky B.P.). Between 10.3 and 8 ky B.P., decadal to centennial variations in monsoon precipitation are in phase with temperature fluctuations recorded in Greenland ice cores, indicating that early Holocene monsoon intensity is largely controlled by glacial boundary conditions. After approximately 8 ky B.P., monsoon precipitation decreases gradually in response to changing Northern Hemisphere summer solar insolation, with decadal to multidecadal variations in monsoon precipitation being linked to solar activity.

OBJECTIVE

To assess the effect of therapeutic inhibition of interleukin 1beta-converting enzyme (ICE) in an experimental model of severe acute pancreatitis (SAP).

BACKGROUND

Several lines of evidence suggest that cytokines activated by ICE play an instrumental role in the course of acute pancreatitis. Recent studies have shown that pharmacologic or genetic blockade of ICE significantly ameliorates the overall severity of and the death rate in SAP.

METHODS

Severe acute pancreatitis was induced by infusion of 5% sodium taurocholate in Wistar rats. A new, highly selective, irreversible inhibitor of ICE was intraperitoneally applied at a dosage of 0.25 mg every 12 hours. Control animals in group 1 received treatment with saline; in group 2 rats, ICE inhibition was started 6 hours after the onset of pancreatitis; and in group 3 rats, ICE inhibition was started 12 hours after the onset of pancreatitis. After a 7-day observation period, surviving rats were killed and blood, plasma, pancreas, lung, and liver were used for subsequent analysis.

RESULTS

Inhibition of ICE decreased the 7-day death rate from 87.5% to 38.9% irrespective whether treatment was started 6 hours or 12 hours after induction of SAP. Morphometric analysis revealed a significant reduction of acinar cell necrosis in both treated groups, whereas pancreatitis-associated pulmonary and hepatic damage was uniformly low and not influenced by ICE inhibition. Tissue myeloperoxidase concentrations were dramatically decreased in the pancreas and the lung after either regimen of ICE inhibitor treatment. In contrast to lung and liver, marked upregulation of interleukin 1beta, tumor necrosis factor alpha, and ICE mRNA was observed in the pancreas, with levels of interleukin 1beta and tumor necrosis factor alpha being reduced in ICE-inhibited animals. Compared with nontreated rats, plasma amylase levels were higher in both treatment groups, whereas lipase and hematocrit showed no difference. Changes of the differential white blood count including neutrophils, lymphocytes, and monocytes were attenuated in both groups after ICE inhibitor treatment.

CONCLUSIONS

Pharmacologic inhibition of ICE significantly improves the overall severity of and the death rate in SAP. A substantial reduction of neutrophil-mediated tissue injury in pancreas and lung seems to contribute to the beneficial effects of this approach. Moreover, ICE inhibition is still effective after a therapeutic window of 12 hours. Based on the current findings, future studies on the clinical application of ICE-inhibiting substances in acute pancreatitis seem to be promising.

Nine different enzyme-linked immunosorbent assays (ELISAs) with a sonicate or recombinant proteins of Treponema pallidum as antigen have been evaluated comparatively by testing 52 highly selected sera from patients with primary syphilis, all negative in the microhemagglutination test for T. pallidum (MHA-TP). Eight tests exhibited greater sensitivity (48.5 to 76.9%) than the commonly used Venereal Disease Research Laboratory test (44.2%). Higher sensitivity could be related to (i) the volume and dilution of the serum, (ii) the design of the assay (capture and competitive tests showed higher sensitivity than sandwich-based assays), and (iii) the ability to detected specific immunoglobulin M antibodies. The specificity of the ICE Syphilis and the Enzygnost Syphilis tests was 99.5 and 99.8%, respectively, as determined by routine testing of 2, 053 unselected sera in comparison with the MHA-TP test. ELISAs tested offered high sensitivity in patients with primary syphilis; however, recommendations to use these tests as screening assays do need further data on specificity and reactivity in late stages of the disease.

In the recent past, there has been an explosion of probiotic health-based products. Many reports indicated that there is poor survival of probiotic bacteria in these products. Further, the survival of these bacteria in the human gastro-intestinal system is questionable. Providing probiotic living cells with a physical barrier against adverse environmental conditions is therefore an approach currently receiving considerable interest. The technology of micro-encapsulation of probiotic bacterial cells evolved from the immobilised cell culture technology used in the biotechnological industry. Several methods of micro-encapsulation of probiotic bacteria have been reported and include spray drying, extrusion, emulsion and phase separation. None of these reported methods however, has resulted in the large numbers of shelf-stable, viable probiotic bacterial cells necessary for use in industry for development of new probiotic products. The most commonly reported micro-encapsulation procedure is based on the calcium-alginate gel capsule formation. Kappa-carrageenan, gellan gum, gelatin and starch are also used as excipients for the micro-encapsulation of probiotic bacteria. The currently available equipment for micro-encapsulation is not able to generate large quantities of uniform sized micro or nano capsules. There is a need to design and develop equipment that will be able to generate precise and uniform micro or nano capsules in large quantities for industrial applications. The reported food vehicles for delivery of encapsulated probiotic bacteria are yoghurt, cheese, ice cream and mayonnaise. Studies need to be done on the application of micro-encapsulation of probiotic bacteria in other food systems. The number of probiotic supplements will increase in the future. More studies, however, need to be conducted on the efficacy of micro-encapsulation to deliver probiotic bacteria and their controlled or targeted release in the gastrointestinal tract.

The cysteine protease interleukin-1beta converting enzyme (ICE) is implicated as an effector of apoptosis in mammalian cells. Proteolytic activity of ICE can be blocked in vitro by the cytokine response modifier A (crmA), a serpin-like protease inhibitor encoded by cowpox virus. Here we show that CD2 enhancer-driven expression of crmA in T lymphocytes of transgenic mice (CD2-crmA mice) reduces CD95 (Fas/APO-1)-transduced apoptosis in vitro to the level seen in CD95-deficient mutant lpr mice, but does not protect against gamma-radiation or corticosteroid-induced cell death. Unlike lpr mice, CD2-crmA transgenic mice developed neither T cell hyperplasia nor serum autoantibodies. These results provide evidence that the phenotype of lpr mice is not simply due to failure of CD95 to trigger T cell apoptosis mediated by ICE.

The Bcl-2 family and the ICE family of cysteine proteases play important roles in regulating cell death. We show here that induction of cell death by a Ca2+ ionophore or hypoxia results in increased levels and activity of active ICE(-like) proteases and the subsequent activation of CPP32/Yama(-like) proteases, and that inhibition of these protease activities reduces the extent of cell death. Overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-xL inhibits the cell death and the activation of ICE(-like) and CPP32/Yama(-like) proteases, indicating that Bcl-2 and Bcl-xL act upstream of these proteases. We also show that specific inhibition of ICE(-like) proteases in vivo prevents activation of CPP32/Yama(-like) proteases, whereas inhibition of CPP32/Yama(-like) proteases does not prevent activation of ICE(-like) proteases, suggesting the existence of a protease cascade in vivo that requires ICE(-like) proteases for activation of CPP32/Yama(-like) proteases. Induction of necrotic cell death by KCN also induces activation of ICE(-like) proteases but not of CPP32/Yama(-like) proteases, and Bcl-2 and Bcl-xL inhibit the activation and the cell death, suggesting that the functional site of Bcl-2 and Bcl-xL is also upstream of ICE(-like) proteases in at least some forms of necrosis.

Members of the CED-3/interleukin-1beta-converting enzyme (ICE) protease (caspase) family are synthesized as proforms, which are proteolytically cleaved and activated during apoptosis. We report here that caspase-2 (ICH-1/NEDD-2), a member of the ICE family, is activated during apoptosis by another ICE member, a caspase-3 (CPP32)-like protease(s). When cells are induced to undergo apoptosis, endogenous caspase-2 is first cleaved into three fragments of 32-33 kDa and 14 kDa, which are then further processed into 18- and 12-kDa active subunits. Up to 50 microM N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), a caspase-3-preferred peptide inhibitor, inhibits caspase-2 activation and DNA fragmentation in vivo, but does not prevent loss of mitochondrial function, while higher concentrations of DEVD-CHO (>50 microM) inhibit both. In comparison, although the activity of caspase-3 is very sensitive to the inhibition of DEVD-CHO (<50 nM), inhibition of caspase-3 activation as marked by processing of the proform requires more than 100 microM DEVD-CHO. Our results suggest that the first cleavage of caspase-2 is accomplished by a caspase-3-like activity, and other ICE-like proteases less sensitive to DEVD-CHO may be responsible for activation of caspase-3 and loss of mitochondrial function.

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Substituting GTP for GTPgammaS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 microM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1.GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37 degreesC, whereas AP-1 recruited with GTPgammaS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1. GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1.GTP first primes the Golgi membrane at 37 degreesC, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1.GTP hydrolysis point. Thus, hydrolysis of ARF1.GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

Two cell permeable peptide fluoromethyl ketone inhibitors of Interleukin-1 beta converting enzyme (ICE) family proteases were tested as inhibitors of apoptotic cell death of T lymphocytes at various stages of differentiation. The CPP-32-like protease activity in apoptotic cell lysates was blocked by both the ICE inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethyl ketone (ZVAD-FMK) as well as its truncated analog Boc-Asp(OMe)-fluoromethyl ketone (BD-FMK), which failed to block ICE. In vitro apoptotic death in murine thymocytes triggered by the independent agents dexamethasone, etoposide, radiation, anti-Fas, and anti-CD3 was blocked equally well by BD-FMK and ZVAD-FMK, but not by the control reagent Cbz-Phe-Ala-fluoromethyl ketone. In activated T cell blasts, while anti-CD3/ Fas-induced death was almost completely inhibited by both ZVAD-FMK, and BD-FMK, death induced by dexamethasone, etoposide, or irradiation was more sensitive to inhibition by BD-FMK. In the murine T cell line CTLL-2, apoptotic death induced by IL-2 withdrawal, etoposide, or dexamethasone was inhibited by BD-FMK, while ZVAD-FMK was without effect. These data indicate that ICE-family proteases comprise a common functional step in distinct T cell apoptotic death pathways, but suggest that different family members are likely to be critical in various differentiated T cell types, even when triggered by the same stimulus.

BACKGROUND

Dairy foods and their constituents (lactose and calcium) have been hypothesized to promote ovarian carcinogenesis. Although case-control studies have reported conflicting results for dairy foods and lactose, several cohort studies have shown positive associations between skim milk, lactose, and ovarian cancer.

METHODS

A pooled analysis of the primary data from 12 prospective cohort studies was conducted. The study population consisted of 553,217 women among whom 2,132 epithelial ovarian cases were identified. Study-specific relative risks and 95% confidence intervals were calculated by Cox proportional hazards models and then pooled by a random-effects model.

RESULTS

No statistically significant associations were observed between intakes of milk, cheese, yogurt, ice cream, and dietary and total calcium intake and risk of ovarian cancer. Higher lactose intakes comparing>> or = 30 versus <10 g/d were associated with a statistically significant higher risk of ovarian cancer, although the trend was not statistically significant (pooled multivariate relative risk, 1.19; 95% confidence interval, 1.01-1.40; P(trend) = 0.19). Associations for endometrioid, mucinous, and serous ovarian cancer were similar to the overall findings.

CONCLUSIONS

Overall, no associations were observed for intakes of specific dairy foods or calcium and ovarian cancer risk. A modest elevation in the risk of ovarian cancer was seen for lactose intake at the level that was equivalent to three or more servings of milk per day. Because a new dietary guideline recommends two to three servings of dairy products per day, the relation between dairy product consumption and ovarian cancer risk at these consumption levels deserves further examination.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species.

OBJECTIVE

Gene expression microarray technologies have the potential to define molecular profiles that may identify specific phenotypes(diagnosis), establish a patient's expected clinical outcome (prognosis), and indicate the likelihood of a beneficial effect of a specific therapy (prediction). We wished to develop optimal tissue acquisition, processing, and analysis procedures for exploring the gene expression profiles of breast core needle biopsies representing cancer and noncancer tissues.

METHODS

Human breast cancer xenografts were used to evaluate several processing methods for prospectively collecting adequate amounts of high-quality RNA for gene expression microarray studies. Samples were assessed for the preservation of tissue architecture and the quality and quantity of RNA recovered. An optimized protocol was applied to a small study of core needle breast biopsies from patients, in which we compared the molecular profiles from cancer with those from noncancer biopsies. Gene expression data were obtained using Research Genetics, Inc. Named Genes cDNA microarrays. Data were visualized using simple hierarchical clustering and a novel principal component analysis-based multidimensional scaling. Data dimensionality was reduced by simple statistical approaches. Predictive neural networks were built using a multilayer perceptron and evaluated in an independent data set from snap-frozen mastectomy specimens.

RESULTS

Processing tissue through RNALater preserves tissue architecture when biopsies are washed for 5 min on ice with ice-cold PBS before histopathological analysis. Cell margins are clear, tissue folding and fragmentation are not observed, and integrity of the cores is maintained, allowing optimal pathological interpretation and preservation of important diagnostic information. Adequate concentrations of high-quality RNA are recovered; 51 of 55 biopsies produced a median of 1.34 microg of total RNA (range, 100 ng to 12.60 microg). Snap-freezing or the use of RNALater does not affect RNA recovery or the molecular profiles obtained from biopsies. The neural network predictors accurately discriminate between predominantly cancer and noncancer breast biopsies.

CONCLUSIONS

The approaches generated in these studies provide a simple, safe, and effective method for prospectively acquiring and processing breast core needle biopsies for gene expression studies. Gene expression data from these studies can be used to build accurate predictive models that separate different molecular profiles. The data establish the use and effectiveness of these approaches for future prospective studies.

The polar bear (Ursus maritimus) depends on sea ice for feeding, breeding, and movement. Significant reductions in Arctic sea ice are forecast to continue because of climate warming. We evaluated the impacts of climate change on polar bears in the southern Beaufort Sea by means of a demographic analysis, combining deterministic, stochastic, environment-dependent matrix population models with forecasts of future sea ice conditions from IPCC general circulation models (GCMs). The matrix population models classified individuals by age and breeding status; mothers and dependent cubs were treated as units. Parameter estimates were obtained from a capture-recapture study conducted from 2001 to 2006. Candidate statistical models allowed vital rates to vary with time and as functions of a sea ice covariate. Model averaging was used to produce the vital rate estimates, and a parametric bootstrap procedure was used to quantify model selection and parameter estimation uncertainty. Deterministic models projected population growth in years with more extensive ice coverage (2001-2003) and population decline in years with less ice coverage (2004-2005). LTRE (life table response experiment) analysis showed that the reduction in lambda in years with low sea ice was due primarily to reduced adult female survival, and secondarily to reduced breeding. A stochastic model with two environmental states, good and poor sea ice conditions, projected a declining stochastic growth rate, log lambdas, as the frequency of poor ice years increased. The observed frequency of poor ice years since 1979 would imply log lambdas approximately - 0.01, which agrees with available (albeit crude) observations of population size. The stochastic model was linked to a set of 10 GCMs compiled by the IPCC; the models were chosen for their ability to reproduce historical observations of sea ice and were forced with "business as usual" (A1B) greenhouse gas emissions. The resulting stochastic population projections showed drastic declines in the polar bear population by the end of the 21st century. These projections were instrumental in the decision to list the polar bear as a threatened species under the U.S. Endangered Species Act.