BACKGROUND

We recently showed that enzymes of the TET family convert 5-mC to 5-hydroxymethylcytosine (5-hmC) in DNA. 5-hmC is present at high levels in embryonic stem cells and Purkinje neurons. The methylation status of cytosines is typically assessed by reaction with sodium bisulfite followed by PCR amplification. Reaction with sodium bisulfite promotes cytosine deamination, whereas 5-methylcytosine (5-mC) reacts poorly with bisulfite and is resistant to deamination. Since 5-hmC reacts with bisulfite to yield cytosine 5-methylenesulfonate (CMS), we asked how DNA containing 5-hmC behaves in bisulfite sequencing.

RESULTS

We used synthetic oligonucleotides with different distributions of cytosine as templates for generation of DNAs containing C, 5-mC and 5-hmC. The resulting DNAs were subjected in parallel to bisulfite treatment, followed by exposure to conditions promoting cytosine deamination. The extent of conversion of 5-hmC to CMS was estimated to be 99.7%. Sequencing of PCR products showed that neither 5-mC nor 5-hmC undergo C-to-T transitions after bisulfite treatment, confirming that these two modified cytosine species are indistinguishable by the bisulfite technique. DNA in which CMS constituted a large fraction of all bases (28/201) was much less efficiently amplified than DNA in which those bases were 5-mC or uracil (the latter produced by cytosine deamination). Using a series of primer extension experiments, we traced the inefficient amplification of CMS-containing DNA to stalling of Taq polymerase at sites of CMS modification, especially when two CMS bases were either adjacent to one another or separated by 1-2 nucleotides.

CONCLUSIONS

We have confirmed that the widely used bisulfite sequencing technique does not distinguish between 5-mC and 5-hmC. Moreover, we show that CMS, the product of bisulfite conversion of 5-hmC, tends to stall DNA polymerases during PCR, suggesting that densely hydroxymethylated regions of DNA may be underrepresented in quantitative methylation analyses.

Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the approximately 4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although approximately 80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5' or 3' untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.

We present a food pyramid that reflects Mediterranean dietary traditions, which historically have been associated with good health. This Mediterranean diet pyramid is based on food patterns typical of Crete, much of the rest of Greece, and southern Italy in the early 1960s, where adult life expectancy was among the highest in the world and rates of coronary heart disease, certain cancers, and other diet-related chronic diseases were among the lowest. Work in the field or kitchen resulted in a lifestyle that included regular physical activity and was associated with low rates of obesity. The diet is characterized by abundant plant foods (fruit, vegetables, breads, other forms of cereals, potatoes, beans, nuts, and seeds), fresh fruit as the typical daily dessert, olive oil as the principal source of fat, dairy products (principally cheese and yogurt), and fish and poultry consumed in low to moderate amounts, zero to four eggs consumed weekly, red meat consumed in low amounts, and wine consumed in low to moderate amounts, normally with meals. This diet is low in saturated fat (< or = 7-8% of energy), with total fat ranging from < 25% to>> 35% of energy throughout the region. The pyramid describes a dietary pattern that is attractive for its famous palatability as well as for its health benefits.

The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells. The first anatomical site from which cells with long-term HSC potential have been isolated is the aorta-gonad-mesonephros (AGM), more specifically the vicinity of the dorsal aortic floor. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen, others support the notion that HSCs first emerge within the underlying mesenchyme. Here we show by non-invasive, high-resolution imaging of live zebrafish embryos, that HSCs emerge directly from the aortic floor, through a stereotyped process that does not involve cell division but a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. The process is polarized not only in the dorso-ventral but also in the rostro-caudal versus medio-lateral direction, and depends on Runx1 expression: in Runx1-deficient embryos, the exit events are initially similar, but much rarer, and abort into violent death of the exiting cell. These results demonstrate that the aortic floor is haemogenic and that HSCs emerge from it into the sub-aortic space, not by asymmetric cell division but through a new type of cell behaviour, which we call an endothelial haematopoietic transition.

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1.

The increasingly detailed biochemical definition of the protein complexes that regulate gene transcription has led to the re-emergence of questions about the role of histones. Much recent evidence suggests that transcriptional activation requires that transcription factors successfully compete with histones for binding to promoters, and that there may be more than one mechanism by which this is achieved.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid C-terminally alpha-amidated peptide that was first isolated 20 years ago from an ovine hypothalamic extract on the basis of its ability to stimulate cAMP formation in anterior pituitary cells (Miyata et al., 1989. PACAP belongs to the vasoactive intestinal polypeptide (VIP)-secretin-growth hormone-releasing hormone-glucagon superfamily. The sequence of PACAP has been remarkably well conserved during evolution from protochordates to mammals, suggesting that PACAP is involved in the regulation of important biological functions. PACAP is widely distributed in the brain and peripheral organs, notably in the endocrine pancreas, gonads, respiratory and urogenital tracts. Characterization of the PACAP precursor has revealed the existence of a PACAP-related peptide, the activity of which remains unknown. Two types of PACAP binding sites have been characterized: type I binding sites exhibit a high affinity for PACAP and a much lower affinity for VIP, whereas type II binding sites have similar affinity for PACAP and VIP. Molecular cloning of PACAP receptors has shown the existence of three distinct receptor subtypes: the PACAP-specific PAC1-R, which is coupled to several transduction systems, and the PACAP/VIP-indifferent VPAC1-R and VPAC2-R, which are primarily coupled to adenylyl cyclase. PAC1-Rs are particularly abundant in the brain, the pituitary and the adrenal gland, whereas VPAC receptors are expressed mainly in lung, liver, and testis. The development of transgenic animal models and specific PACAP receptor ligands has strongly contributed to deciphering the various actions of PACAP. Consistent with the wide distribution of PACAP and its receptors, the peptide has now been shown to exert a large array of pharmacological effects and biological functions. The present report reviews the current knowledge concerning the pleiotropic actions of PACAP and discusses its possible use for future therapeutic applications.

Results from recent studies of retrograde amnesia following damage to the hippocampal complex of human and non-human subjects have shown that retrograde amnesia is extensive and can encompass much of a subject's lifetime; the degree of loss may depend upon the type of memory assessed. These and other findings suggest that the hippocampal formation and related structures are involved in certain forms of memory (e.g. autobiographical episodic and spatial memory) for as long as they exist and contribute to the transformation and stabilization of other forms of memory stored elsewhere in the brain.

BACKGROUND

The spectrum of disorders of the brain is large, covering hundreds of disorders that are listed in either the mental or neurological disorder chapters of the established international diagnostic classification systems. These disorders have a high prevalence as well as short- and long-term impairments and disabilities. Therefore they are an emotional, financial and social burden to the patients, their families and their social network. In a 2005 landmark study, we estimated for the first time the annual cost of 12 major groups of disorders of the brain in Europe and gave a conservative estimate of €386 billion for the year 2004. This estimate was limited in scope and conservative due to the lack of sufficiently comprehensive epidemiological and/or economic data on several important diagnostic groups. We are now in a position to substantially improve and revise the 2004 estimates. In the present report we cover 19 major groups of disorders, 7 more than previously, of an increased range of age groups and more cost items. We therefore present much improved cost estimates. Our revised estimates also now include the new EU member states, and hence a population of 514 million people.

OBJECTIVE

To estimate the number of persons with defined disorders of the brain in Europe in 2010, the total cost per person related to each disease in terms of direct and indirect costs, and an estimate of the total cost per disorder and country.

METHODS

The best available estimates of the prevalence and cost per person for 19 groups of disorders of the brain (covering well over 100 specific disorders) were identified via a systematic review of the published literature. Together with the twelve disorders included in 2004, the following range of mental and neurologic groups of disorders is covered: addictive disorders, affective disorders, anxiety disorders, brain tumor, childhood and adolescent disorders (developmental disorders), dementia, eating disorders, epilepsy, mental retardation, migraine, multiple sclerosis, neuromuscular disorders, Parkinson's disease, personality disorders, psychotic disorders, sleep disorders, somatoform disorders, stroke, and traumatic brain injury. Epidemiologic panels were charged to complete the literature review for each disorder in order to estimate the 12-month prevalence, and health economic panels were charged to estimate best cost-estimates. A cost model was developed to combine the epidemiologic and economic data and estimate the total cost of each disorder in each of 30 European countries (EU27+Iceland, Norway and Switzerland). The cost model was populated with national statistics from Eurostat to adjust all costs to 2010 values, converting all local currencies to Euro, imputing costs for countries where no data were available, and aggregating country estimates to purchasing power parity adjusted estimates for the total cost of disorders of the brain in Europe 2010.

RESULTS

The total cost of disorders of the brain was estimated at €798 billion in 2010. Direct costs constitute the majority of costs (37% direct healthcare costs and 23% direct non-medical costs) whereas the remaining 40% were indirect costs associated with patients' production losses. On average, the estimated cost per person with a disorder of the brain in Europe ranged between €285 for headache and €30,000 for neuromuscular disorders. The European per capita cost of disorders of the brain was €1550 on average but varied by country. The cost (in billion €PPP 2010) of the disorders of the brain included in this study was as follows: addiction: €65.7; anxiety disorders: €74.4; brain tumor: €5.2; child/adolescent disorders: €21.3; dementia: €105.2; eating disorders: €0.8; epilepsy: €13.8; headache: €43.5; mental retardation: €43.3; mood disorders: €113.4; multiple sclerosis: €14.6; neuromuscular disorders: €7.7; Parkinson's disease: €13.9; personality disorders: €27.3; psychotic disorders: €93.9; sleep disorders: €35.4; somatoform disorder: €21.2; stroke: €64.1; traumatic brain injury: €33.0. It should be noted that the revised estimate of those disorders included in the previous 2004 report constituted €477 billion, by and large confirming our previous study results after considering the inflation and population increase since 2004. Further, our results were consistent with administrative data on the health care expenditure in Europe, and comparable to previous studies on the cost of specific disorders in Europe. Our estimates were lower than comparable estimates from the US.

CONCLUSIONS

This study was based on the best currently available data in Europe and our model enabled extrapolation to countries where no data could be found. Still, the scarcity of data is an important source of uncertainty in our estimates and may imply over- or underestimations in some disorders and countries. Even though this review included many disorders, diagnoses, age groups and cost items that were omitted in 2004, there are still remaining disorders that could not be included due to limitations in the available data. We therefore consider our estimate of the total cost of the disorders of the brain in Europe to be conservative. In terms of the health economic burden outlined in this report, disorders of the brain likely constitute the number one economic challenge for European health care, now and in the future. Data presented in this report should be considered by all stakeholder groups, including policy makers, industry and patient advocacy groups, to reconsider the current science, research and public health agenda and define a coordinated plan of action of various levels to address the associated challenges.

CONCLUSIONS

Political action is required in light of the present high cost of disorders of the brain. Funding of brain research must be increased; care for patients with brain disorders as well as teaching at medical schools and other health related educations must be quantitatively and qualitatively improved, including psychological treatments. The current move of the pharmaceutical industry away from brain related indications must be halted and reversed. Continued research into the cost of the many disorders not included in the present study is warranted. It is essential that not only the EU but also the national governments forcefully support these initiatives.

Nutritional conditions during key periods of development, when the architecture and modus operandi of the body become established, are of profound importance in determining the subsequent life-history trajectory of an organism. If developing individuals experience a period of nutritional deficit, they can subsequently show accelerated growth should conditions improve, apparently compensating for the initial setback. However, recent research suggests that, although compensatory growth can bring quick benefits, it is also associated with a surprising variety of costs that are often not evident until much later in adult life. Clearly, the nature of these costs, the timescale over which they are incurred and the mechanisms underlying them will play a crucial role in determining compensatory strategies. Nonetheless, such effects remain poorly understood and largely neglected by ecologists and evolutionary biologists.

This paper describes a computer method for handling gel reading data produced by the shotgun method of DNA sequencing. The method greatly reduces the time the sequencer needs to spend checking and editing his data and yet it produces a consensus sequence for which the accuracy of determination of every base can be clearly shown. The program can take a batch of new gel readings, screen them against vector sequences removing any that match, and then compare and align all the sequences to produce a final consensus. No information is lost in this process as alignments are achieved by making only insertions and because all the individual gel readings are added to a database from which they can be retrieved and displayed lined up one above the other. This allows the user to check on the alignments achieved by the program and if necessary change them. As each gel reading is added to the database the consensus is automatically updated accordingly and used for the next comparisons. This is a much faster process than comparing each new gel against every individual gel in the database.

A monoclonal antibody was produced to human peripheral blood T cells. This hybridoma antibody, termed OKT4, was reactive by indirect immunofluorescence with only 55-60% of the peripheral blood T cell population (OKT4+) and unreactive with normal B cells, null cells, and macrophages. The OKT4- T cell population contained the previously described TH2+ subset that has been shown to contain cytotoxic/suppressor cells. With cell-sorter separation of OKT4+ and OKT4- cells, it was shown that these T cell subsets were functionally discrete. Both gave proliferative responses with concanavalin A, alloantigens, and phytohemagglutinin although OKT4+ cells were much more responsive to the latter. OKT4+ cells alone responded to soluble antigens whereas OKT4- cells alone were cytotoxic after alloantigenic sensitization of unfractionated T cells. However, both OKT4+ and OKT4- cells were required during sensitization for optimal development of cytotoxicity. These data suggest that the OKT4+ subset represents a helper population and that the OKT4- subset contains the cytotoxic effector population. OKT4 could be a valuable reagent for determining alterations of these functional subsets in human diseases.

The heat shock proteins (HSPs) induced by cell stress are expressed at high levels in a wide range of tumors and are closely associated with a poor prognosis and resistance to therapy. The increased transcription of HSPs in tumor cells is due to loss of p53 function and to higher expression of the proto-oncogenes HER2 and c-Myc, and is crucial to tumorigenesis. The HSP family members play overlapping, essential roles in tumor growth both by promoting autonomous cell proliferation and by inhibiting death pathways. The HSPs have thus become targets for rational anti-cancer drug design: HSP90 inhibitors are currently showing much promise in clinical trials, whereas the increased expression of HSPs in tumors is forming the basis of chaperone-based immunotherapy.

Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress.

An explosion of new information linking activation of cell surface signal initiators to changes in gene expression has recently emerged. The focus of much of this information has centered around the agonist-dependent activation of the mitogen-activated protein (MAP) kinases. Although this intracellular signal transduction pathway is extremely complex, conservation of many of its components has been observed in yeast, nematodes, Drosophila, and mammals. Thus, these signaling proteins may participate in the regulation of a variety of cellular processes.

Dopamine (DA) D1 receptor (D1R) stimulation in prefrontal cortex (PFC) produces an 'inverted-U' dose-response, whereby either too little or too much D1R stimulation impairs spatial working memory. This response has been observed across species, including genetic linkages with human cognitive abilities, PFC activation states and DA synthesis. The cellular basis for the inverted U has long been sought, with in vitro intracellular recordings supporting a variety of potential mechanisms. The current study demonstrates that the D1R agonist inverted-U response can be observed in PFC neurons of behaving monkeys: low levels of D1R stimulation enhance spatial tuning by suppressing responses to nonpreferred directions, whereas high levels reduce delay-related firing for all directions, eroding tuning. These sculpting actions of D1R stimulation are mediated in monkeys and rats by cyclic AMP intracellular signaling. The evidence for an inverted U at the cellular level in behaving animals promises to bridge in vitro molecular analyses with human cognitive experience.

Multiple sclerosis (MS) is traditionally seen as an inflammatory demyelinating disease, characterized by the formation of focal demyelinated plaques in the white matter of the central nervous system. In this review we describe recent evidence that the spectrum of MS pathology is much broader. This includes demyelination in the cortex and deep gray matter nuclei, as well as diffuse injury of the normal-appearing white matter. The mechanisms responsible for the formation of focal lesions in different patients and in different stages of the disease as well as those involved in the induction of diffuse brain damage are complex and heterogeneous. This heterogeneity is reflected by different clinical manifestations of the disease, such as relapsing or progressive MS, and also explains at least in part the relation of MS to other inflammatory demyelinating diseases.

We did a systematic review and meta-analysis of observational studies of the risk of HIV-1 transmission per heterosexual contact. 43 publications comprising 25 different study populations were identified. Pooled female-to-male (0.04% per act [95% CI 0.01-0.14]) and male-to-female (0.08% per act [95% CI 0.06-0.11]) transmission estimates in high-income countries indicated a low risk of infection in the absence of antiretrovirals. Low-income country female-to-male (0.38% per act [95% CI 0.13-1.10]) and male-to-female (0.30% per act [95% CI 0.14-0.63]) estimates in the absence of commercial sex exposure (CSE) were higher. In meta-regression analysis, the infectivity across estimates in the absence of CSE was significantly associated with sex, setting, the interaction between setting and sex, and antenatal HIV prevalence. The pooled receptive anal intercourse estimate was much higher (1.7% per act [95% CI 0.3-8.9]). Estimates for the early and late phases of HIV infection were 9.2 (95% CI 4.5-18.8) and 7.3 (95% CI 4.5-11.9) times larger, respectively, than for the asymptomatic phase. After adjusting for CSE, presence or history of genital ulcers in either couple member increased per-act infectivity 5.3 (95% CI 1.4-19.5) times versus no sexually transmitted infection. Study estimates among non-circumcised men were at least twice those among circumcised men. Low-income country estimates were more heterogeneous than high-income country estimates, which indicates poorer study quality, greater heterogeneity of risk factors, or under-reporting of high-risk behaviour. Efforts are needed to better understand these differences and to quantify infectivity in low-income countries.

Chromosomal fragile sites are specific loci that preferentially exhibit gaps and breaks on metaphase chromosomes following partial inhibition of DNA synthesis. Their discovery has led to novel findings spanning a number of areas of genetics. Rare fragile sites are seen in a small proportion of individuals and are inherited in a Mendelian manner. Some, such as FRAXA in the FMR1 gene, are associated with human genetic disorders, and their study led to the identification of nucleotide-repeat expansion as a frequent mutational mechanism in humans. In contrast, common fragile sites are present in all individuals and represent the largest class of fragile sites. Long considered an intriguing component of chromosome structure, common fragile sites have taken on novel significance as regions of the genome that are particularly sensitive to replication stress and that are frequently rearranged in tumor cells. In recent years, much progress has been made toward understanding the genomic features of common fragile sites and the cellular processes that monitor and influence their stability. Their study has merged with that of cell cycle checkpoints and DNA repair, and common fragile sites have provided insight into understanding the consequences of replication stress on DNA damage and genome instability in cancer cells.

Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line.

Conditioned fear responses to a tone paired with footshock extinguish when the tone is presented repeatedly in the absence of shock. Rather than erase the tone-shock association, extinction is thought to involve new learning accompanied by inhibition of conditioned responding. Despite much interest in extinction from a clinical perspective, little is known about the neural circuits that are involved. Although the prefrontal cortex has a well established role in the inhibition of inappropriate behaviors, previous reports have disagreed as to the role of the ventromedial prefrontal cortex (vmPFC) in extinction. We have reexamined the effects of electrolytic vmPFC lesions made before training on the acquisition, extinction, and recovery of conditioned fear responses in a 2 d experiment. On Day 1 vmPFC lesions had no effect on acquisition or extinction of conditioned freezing and suppression of bar pressing. On Day 2 sham rats recovered only 27% of their acquired freezing, whereas vmPFC-lesioned rats recovered 86%, which was indistinguishable from a control group that never received extinction. The high recovery in lesioned rats could not be attributed to decreased motivation or altered sensitivity to footshock. vmPFC lesions that spared the caudal infralimbic (IL) nucleus had no effect. Thus, the vmPFC (particularly the IL nucleus) is not necessary for expression of extinction, but it is necessary for the recall of extinction learning after a long delay. These data suggest a role of the vmPFC in consolidation of extinction learning or the recall of contexts in which extinction took place.

Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.

Set2 methylates Lys36 of histone H3. We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in slight sensitivity to 6-azauracil and much less beta-galactosidase produced by a reporter plasmid, resulting from a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.

Antioxidant enzyme expression was determined in rat pancreatic islets and RINm5F insulin-producing cells on the level of mRNA, protein, and enzyme activity in comparison with 11 other rat tissues. Although superoxide dismutase expression was in the range of 30% of the liver values, the expression of the hydrogen peroxide-inactivating enzymes catalase and glutathione peroxidase was extremely low, in the range of 5% of the liver. Pancreatic islets but not RINm5F cells expressed an additional phospholipid hydroperoxide glutathione peroxidase that exerted protective effects against lipid peroxidation of the plasma membrane. Regression analysis for mRNA and protein expression and enzyme activities from 12 rat tissues revealed that the mRNA levels determine the enzyme activities of the tissues. The induction of cellular stress by high glucose, high oxygen, and heat shock treatment did not affect antioxidant enzyme expression in rat pancreatic islets or in RINm5F cells. Thus insulin-producing cells cannot adapt the low antioxidant enzyme activity levels to typical situations of cellular stress by an upregulation of gene expression. Through stable transfection, however, we were able to increase catalase and glutathione peroxidase gene expression in RINm5F cells, resulting in enzyme activities more than 100-fold higher than in nontransfected controls. Catalase-transfected RINm5F cells showed a 10-fold greater resistance toward hydrogen peroxide toxicity, whereas glutathione peroxidase overexpression was much less effective. Thus inactivation of hydrogen peroxide through catalase seems to be a step of critical importance for the removal of reactive oxygen species in insulin-producing cells. Overexpression of catalase may therefore be an effective means of preventing the toxic action of reactive oxygen species.