Apoptosis occurs in the normal liver and in various forms of liver disease. The CD95 (APO-1/Fas) (CD95) receptor mediates apoptosis, and liver cells in animal models are acutely sensitive to apoptosis initiated by this receptor. We have used primary human hepatocytes as a model system to investigate CD95-mediated apoptotic liver damage. Treatment of fresh human hepatocytes with low concentrations of agonistic antibodies against CD95 resulted in apoptosis of>> 95% of the cultured liver cells within 4 and 7.5 h. Immunohistology of a panel of explanted liver tissues revealed that hepatocytes in normal livers (n = 5) and in alcoholic cirrhosis (n = 13) expressed low constitutive levels of CD95. CD95 receptor expression was highly elevated in hepatocytes in hepatitis B virus-related cirrhosis (n = 9) and in acute liver failure (n = 8). By in situ hybridization CD95 ligand messenger RNA expression was absent in normal liver but detected at high levels in livers with ongoing liver damage. In cases of hepatitis B virus-related cirrhosis and acute hepatic failure, ligand expression was found primarily in areas with lymphocytic infiltration. In contrast, in patients with alcoholic liver damage, high CD95 ligand messenger RNA expression was found in hepatocytes. These findings suggest that liver destruction in hepatitis B may primarily involve killing of hepatocytes by T lymphocytes using the CD95 receptor-ligand system. In alcoholic liver damage, death of hepatocytes might occur by fratricide and paracrine or autocrine mechanisms mediated by the hepatocytes themselves.

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in ail known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Kj = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathways.

We have recently identified alpha-galactosylceramide (alpha-GalCer) as a specific ligand for an invariant Valpha14/Vbeta8.2 T cell receptor exclusively expressed on the majority of Valpha14 NKT cells, a novel subset of lymphocytes. Here, we report that alpha-GalCer selectively activates Valpha14 NKT cells resulting in prevention of tumor metastasis. The effector mechanisms of the ligand-activated Valpha14 NKT cells seem to be mediated by natural killer (NK)-like nonspecific cytotoxicity. Indeed, the cytotoxic index obtained by alpha-GalCer-activated Valpha14 NKT cells was reduced by the addition of cold target tumor cells or by treatment with concanamycin A, which inhibits activation and secretion of perforin, but not by mAbs against molecules involved in the NKT cell recognition and conventional cytotoxicity, such as CD1d, Vbeta8, NK1. 1, Ly49C, Fas, or Fas ligand. These results suggest that the ligand-activated Valpha14 NKT cells kill tumor cells directly through a CD1d/Valpha14 T cell receptor-independent, NK-like mechanism.

Activation of the Fanconi anemia (FA) DNA damage-response pathway results in the monoubiquitination of FANCD2, which is regulated by the nuclear FA core ubiquitin ligase complex. A FANCD2 protein sequence-based homology search facilitated the discovery of FANCI, a second monoubiquitinated component of the FA pathway. Biallelic mutations in the gene coding for this protein were found in cells from four FA patients, including an FA-I reference cell line.

Consumption of foods high in saturated fatty acids (FAs) as well as elevated levels of circulating free FAs are known to be associated with T2D. Though previous studies showed inflammation is crucially involved in the development of insulin resistance, how inflammation contributes to β cell dysfunction has remained unclear. We report here the saturated FA palmitate induces β cell dysfunction in vivo by activating inflammatory processes within islets. Through a combination of in vivo and in vitro studies, we show β cells respond to palmitate via the TLR4/MyD88 pathway and produce chemokines that recruit CD11b(+)Ly-6C(+) M1-type proinflammatory monocytes/macrophages to the islets. Depletion of M1-type cells protected mice from palmitate-induced β cell dysfunction. Islet inflammation also plays an essential role in β cell dysfunction in T2D mouse models. Collectively, these results demonstrate a clear mechanistic link between β cell dysfunction and inflammation mediated at least in part via the FFA-TLR4/MyD88 pathway.

Fanconi anemia (FA) is a genetically heterogeneous cancer-prone disorder associated with chromosomal instability and cellular hypersensitivity to DNA crosslinking agents. The FA pathway is suspected to play a crucial role in the cellular response to DNA replication stress. At a molecular level, however, the function of most of the FA proteins is unknown. FANCM displays DNA-dependent ATPase activity and promotes the dissociation of DNA triplexes, but the physiological significance of this activity remains elusive. Here we show that purified FANCM binds to Holliday junctions and replication forks with high specificity and promotes migration of their junction point in an ATPase-dependent manner. Furthermore, we provide evidence that FANCM can dissociate large recombination intermediates, via branch migration of Holliday junctions through 2.6 kb of DNA. Our data suggest a direct role for FANCM in DNA processing, consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks.

The fatty acid synthase (FAS) gene is significantly up-regulated in various types of cancers, and blocking the FAS expression results in apoptosis of tumor cells. Therefore, FAS is considered to be an attractive target for anticancer therapy. However, the molecular mechanism by which the FAS gene is up-regulated in tumor cells is poorly understood. We found that FAS was significantly up-regulated by hypoxia, which was also accompanied by reactive oxygen species (ROS) generation in human breast cancer cell lines. The FAS expression was also activated by H(2)O(2), whereas N-acetyl-L-cystein, a ROS inhibitor, suppressed the expression. We also found that the hypoxia significantly up-regulated sterol regulatory-element binding protein (SREBP)-1, the major transcriptional regulator of the FAS gene, via phosphorylation of Akt followed by activation of hypoxia-inducible factor 1 (HIF1). Moreover, our results of reporter assay and chromatin immunoprecipitation analysis indicate that SREBP-1 strongly bound to the SREBP binding site/E-box sequence on the FAS promoter under hypoxia. In our xenograft mouse model, FAS was strongly expressed in the hypoxic regions of the tumor. In addition, our results of immunohistochemical analysis for human breast tumor specimens indicate that the expressions of both FAS and SREBP-1 were colocalized with hypoxic regions in the tumors. Furthermore, we found that hypoxia-induced chemoresistance to cyclophosphamide was partially blocked by a combination of FAS inhibitor and cyclophosphamide. Taken together, our results indicate that FAS gene is up-regulated by hypoxia via activation of the Akt and HIF1 followed by the induction of the SREBP-1 gene, and that hypoxia-induced chemoresistance is partly due to the up-regulation of FAS.

Functional lateralization is a feature of human brain function, most apparent in the typical left-hemisphere specialization for language. A number of anatomical and imaging studies have examined whether structural asymmetries underlie this functional lateralization. We combined functional MRI (fMRI) and diffusion-weighted imaging (DWI) with tractography to study 10 healthy right-handed subjects. Three language fMRI paradigms were used to define language-related regions in inferior frontal and superior temporal regions. A probabilistic tractography technique was then employed to delineate the connections of these functionally defined regions. We demonstrated consistent connections between Broca's and Wernicke's areas along the superior longitudinal fasciculus bilaterally but more extensive fronto-temporal connectivity on the left than the right. Both tract volumes and mean fractional anisotropy (FA) were significantly greater on the left than the right. We also demonstrated a correlation between measures of structure and function, with subjects with more lateralized fMRI activation having a more highly lateralized mean FA of their connections. These structural asymmetries are in keeping with the lateralization of language function and indicate the major structural connections underlying this function.

Direct measurements revealed low oxygen tensions (0.5-4.5% oxygen) in murine lymphoid organs in vivo. To test whether adaptation to changes in oxygen tension may have an effect on lymphocyte functions, T cell differentiation and functions at varying oxygen tensions were studied. These studies show: 1) differentiated CTL deliver Fas ligand- and perforin-dependent lethal hit equally well at all redox conditions; 2) CTL development is delayed at 2.5% oxygen as compared with 20% oxygen. Remarkably, development of CTL at 2.5% oxygen is more sustained and the CTL much more lytic; and 3) hypoxic exposure and TCR-mediated activation are additive in enhancing levels of hypoxia response element-containing gene products in lymphocyte supernatants. In contrast, hypoxia inhibited the accumulation of nonhypoxia response element-containing gene products (e.g., IL-2 and IFN-gamma) in the same cultures. This suggests that T cell activation in hypoxic conditions in vivo may lead to different patterns of lymphokine secretion and accumulation of cytokines (e.g., vascular endothelial growth factor) affecting endothelial cells and vascular permeabilization. Thus, although higher numbers of cells survive and are activated during 20% oxygen incubation in vitro, the CTL which develop at 2.5% oxygen are more lytic with higher levels of activation markers. It is concluded that the ambient 20% oxygen tension (plus 2-ME) is remarkably well suited for immunologic specificity and cytotoxicity studies, but oxygen dependence should be taken into account during the design and interpretation of results of in vitro T cell development assays and gene expression studies in vivo.

1. Visual transduction in macaque cones was studied by measuring the membrane current of single outer segments projecting from small pieces of retina. 2. The response to a brief flash of light was diphasic and resembled the output of a bandpass filter with a peak frequency near 5 Hz. After the initial reduction in dark current there was a rebound increase which resulted from an increase in the number of open light-sensitive channels. The response to a step of light consisted of a prominent initial peak followed by a steady phase of smaller amplitude. 3. Responses to dim light were linear and time-invariant, suggesting that responses to single photons were linearly additive. From the flash sensitivity and the effective collecting area the peak amplitude of the single photon response was estimated as about 30 fA. 4. With flashes of increasing strength the photocurrent amplitude usually saturated along a curve that was gentler than an exponential but steeper than a Michaelis relation. The response reached the half-saturating amplitude at roughly 650 photoisomerizations. 5. The response-intensity relation was flatter in the steady state than shortly after a light step was turned on, indicating that bright light desensitized the transduction with a delay. This desensitization was not due to a reduction in pigment content. In the steady state, a background of intensity I lowered the sensitivity to a weak incremental test flash by a factor 1/(1 + I/IO), where IO was about 2.6 x 10(4) photoisomerizations s-1, or about 3.3 log trolands for the red- and green-sensitive cones. 6. Bleaching exposures produced permanent reductions in flash sensitivity but had little effect on the kinetics or saturating amplitude of subsequent flash responses. The sensitivity reductions were consistent with the expected reductions in visual pigment content and gave photosensitivities of about 8 x 10(-9) microns2 (free solution value) for the red- and green-sensitive pigments. During a steady bleaching exposure the final exponential decline of the photocurrent had a rate constant given by the product of the light intensity and the photosensitivity. 7. In some cells it was possible to measure a light-induced increase in current noise. The power spectrum of the noise resembled the spectrum of the dim flash response and the magnitude of the noise was consistent with a single photon response roughly 20 fA in size. 8. The membrane current recorded in darkness was noisy, with a variance near 0.12 pA2 in the band 0-20 Hz.(ABSTRACT TRUNCATED AT 400 WORDS)

Integrin-mediated cell-matrix interactions are essential for development, tissue homeostasis, and repair. Upon ligand binding, integrins are recruited into focal adhesions (FAs). Integrin-linked kinase (ILK) is an FA component that interacts with the cytoplasmic domains of integrins, recruits adaptor proteins that link integrins to the actin cytoskeleton, and phosphorylates the serine/threonine kinases PKB/Akt and GSK-3beta. Here we show that mice lacking ILK expression die at the peri-implantation stage because they fail to polarize their epiblast and to cavitate. The impaired epiblast polarization is associated with abnormal F-actin accumulation at sites of integrin attachments to the basement membrane (BM) zone. Likewise, ILK-deficient fibroblasts showed abnormal F-actin aggregates associated with impaired cell spreading and delayed formation of stress fibers and FAs. Finally, ILK-deficient fibroblasts have diminished proliferation rates. However, insulin or PDGF treatment did not impair phosphorylation of PKB/Akt and GSK-3beta, indicating that the proliferation defect is not due to absent or reduced ILK-mediated phosphorylation of these substrates in vivo. Furthermore, expression of a mutant ILK lacking kinase activity and/or paxillin binding in ILK-deficient fibroblasts can rescue cell spreading, F-actin organization, FA formation, and proliferation. Altogether these data show that mammalian ILK modulates actin rearrangements at integrin-adhesion sites.

Apoptosis induced by DNA damage and other stresses can proceed via expression of Fas ligand (FasL) and ligation of its receptor, Fas (CD95). We report that activation of the two transcription factors NF-kappa B and AP-1 is crucially involved in FasL expression induced by etoposide, teniposide, and UV irradiation. A nondegradable mutant of I kappa B blocked both FasL expression and apoptosis induced by DNA damage but not Fas ligation. These stimuli also induced the stress-activated kinase pathway (SAPK/JNK), which was required for the maximal induction of apoptosis. A 1.2 kb FasL promoter responded to DNA damage, as well as coexpression with p65 Rel or Fos/Jun. Mutations in the relevant NF-kappa B and AP-1 binding sites eliminated these responses. Thus, activation of NF-kappa B and AP-1 contributes to stress-induced apoptosis via the expression of FasL.

OBJECTIVE

Multiple biomarkers are used to quantify the severity of traumatic brain injury (TBI) and to predict outcome. Few are satisfactory. CT and conventional MR imaging underestimate injury and correlate poorly with outcome. New MR imaging techniques, including diffusion tensor imaging (DTI), can provide information about brain ultrastructure by quantifying isotropic and anisotropic water diffusion. Our objective was to determine if changes in anisotropic diffusion in TBI correlate with acute Glasgow coma scale (GCS) and/or Rankin scores at discharge.

METHODS

Twenty patients (15 male, five Female; mean age, 31 years) were evaluated. Apparent diffusion coefficients (ADCs) and fractional anisotropy (FA) values were measured at multiple locations and correlated with clinical scores. Results were compared with those of 15 healthy control subjects.

RESULTS

ADC values were significantly reduced within the splenium (Delta18%, P =.001). FA values were significantly reduced in the internal capsule (Delta14%; P <.001) and splenium (Delta16%; P =.002). FA values were significantly correlated with GCS (r = 0.65-0.74; P <.001) and Rankin (r = 0.68-0.71; P <.001) scores for the internal capsule and splenium. The correlation between FA and clinical markers was better than for the corresponding ADC values. No correlation was found between ADC of the internal capsule and GCS/Rankin scores.

CONCLUSIONS

DTI reveals changes in the white matter that are correlated with both acute GCS and Rankin scores at discharge. DTI may be a valuable biomarker for the severity of tissue injury and a predictor for outcome.

Apoptosis occurs in response to different cellular stresses, including viral infection, inflammatory cytokines, growth factor deprivation, and UV light, but it is unclear whether these inducers share a common mechanism of induction. The interferon-induced, double-stranded RNA-activated protein kinase (PKR) has been implicated in processes that rely on apoptosis as control mechanisms in vivo, including antiviral activities, cell growth regulation, and tumorigenesis. Here we report that mouse embryo fibroblasts from mutant mice containing homozygous deletions in the PKR gene (Pkr(0/0) mice) were resistant to apoptotic cell death in response to double-stranded RNA, tumor necrosis factor-alpha, or lipopolysaccharide. The mechanism underlying the suppression of apoptosis in the Pkr(0/0) cells could be attributed to defects in the activation of DNA-binding activity for the transcription factor interferon regulatory factor-1 and in Fas mRNA induction. Thus, these results provide genetic evidence implicating a requirement for PKR in mediating different forms of stress-related apoptosis.

OBJECTIVE

The aim of treatment for attention-deficit/hyperactivity disorder (ADHD) is to decrease symptoms, enhance functionality, and improve well-being for the child and his or her close contacts. However, the measurement of treatment response is often limited to measuring symptoms using behavior rating scales and checklists completed by teachers and parents. Because so much of the focus has been on symptom reduction, less is known about other possible health problems, which can be measured easily using health-related quality-of-life (HRQL) questionnaires, which are designed to gather information across a range of health domains. The aim of our study was to measure HRQL in a clinic-based sample of children who had a diagnosis of ADHD and consider the impact of 2 clinical factors, symptom severity and comorbidity, on HRQL. Our specific hypotheses were that parent-reported HRQL would be poorer in children with ADHD than in normative US and Australian pediatric samples, in children with increasing severity of ADHD symptoms, and in children who had diagnoses of comorbid psychiatric disorders.

METHODS

Cross-sectional survey was conducted in British Columbia, Canada. The sample included 165 respondents of 259 eligible children (63.7% response rate) who were referred to the ADHD Clinic in British Columbia between November 2001 and October 2002. Children who are seen in this clinic come from all parts of the province and are diverse in terms of socioeconomic status and case mix. ADHD was diagnosed in 131 children, 68.7% of whom had a comorbid psychiatric disorder. Some children had >1 comorbidity: 23 had 2, 5 had 3, and 1 had 4. Fifty-one children had a comorbid learning disorder (LD), 45 had oppositional defiant disorder or conduct disorder (ODD/CD), and 27 had some other comorbid diagnosis. The mean age of children was 10 years (standard deviation: 2.8). Boys composed 80.9% (N = 106) of the sample. We used the 50-item parent version of the Child Health Questionnaire to measure physical and psychosocial health. Physical domains include the following: physical functioning (PF), role/social limitations as a result of physical health (RP), bodily pain/discomfort (BP), and general health perception (GH). Psychosocial domains include the following: role/social limitations as a result of emotional-behavioral problems (REB), self-esteem (SE), mental health (MH), general behavior (BE), emotional impact on parent (PTE), and time impact on parents (PTT). A separate domain measures limitations in family activities (FA). There is also a single-item measure of family cohesion (FC). Individual scale scores and summary scores for physical (PhS) and psychosocial health (PsS) can be computed. Symptom severity data (parent and teacher) came from the Child/Adolescent Symptom Inventory 4. These checklists provide information on symptoms for the 3 ADHD subtypes (inattentive, hyperactive, and combined). Each child underwent a comprehensive psychiatric assessment by 1 of 4 child psychiatrists. Documentation included a full 5-axis Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition diagnosis on the basis of a comprehensive assessment. Clinical information for each child was extracted from hospital notes.

RESULTS

Compared with both population samples, children with ADHD had comparable physical health but clinically important deficits in HRQL in all psychosocial domains, FA, FC, and PsS, with effect sizes as follows: FC = -0.66, SE = -0.90, MH = -0.97, PTT = -1.07, REB = -1.60, BE = -1.73, PTE = -1.87, FA = -1.95, and PsS = -1.98. Poorer HRQL for all domains of psychosocial health, FA, and PsS correlated significantly with more parent-reported inattentive, hyperactive, and combined symptoms of ADHD. Children with>> or =2 comorbid disorders differed significantly from those with no comorbidity in most areas, including RP, GH, REB, BE, MH, SE, PTT, FA, and PsS, and from those with 1 comorbid disorder in 3 domains, including BE, MH, and FA and the PsS. The mean PsS score for children in the ODD/CD group (mean difference: -12.9; effect size = -1.11) and children in the other comorbidity group (-9.0; effect size = -.77) but not children in the LD group were significantly lower than children with no comorbid disorder. Predictors of physical health in a multiple regression model included child's gender (beta = .177) and number of comorbid conditions (beta = -.197). These 2 variables explained very little variation in the PhS. Predictors of psychosocial health included the number of comorbid conditions (beta = -.374) and parent-rated combined ADHD symptoms (beta = -.362). These 2 variables explained 31% of the variation in the PsS.

CONCLUSIONS

Our study shows that ADHD has a significant impact on multiple domains of HRQL in children and adolescents. In support of our hypotheses, compared with normative data, children with ADHD had more parent-reported problems in terms of emotional-behavioral role function, behavior, mental health, and self-esteem. In addition, the problems of children with ADHD had a significant impact on the parents' emotional health and parents' time to meet their own needs, and they interfered with family activities and family cohesion. The differences that we found represent clinically important differences in HRQL. Our study adds new information about the HRQL of children with ADHD in relation to symptom severity and comorbidity. Children with more symptoms of ADHD had worse psychosocial HRQL. Children with multiple comorbid disorders had poorer psychosocial HRQL across a range of domains compared with children with none and 1 comorbid disorder. In addition, compared with children with no comorbidity, psychosocial HRQL was significantly lower in children with ODD/CD and children in the other comorbidity group but not in children with an LD. The demonstration of a differential impact of ADHD on health and well-being in relation to symptom severity and comorbidity has important implications for policies around eligibility for special educational and other supportive services. Because the impact of ADHD is not uniform, decisions about needed supports should incorporate a broader range of relevant indicators of outcome, including HRQL. Although many studies focus on measuring symptoms using rating scales and checklists, in our study, using a multidimensional questionnaire, we were able to show that many areas of health are affected in children with ADHD. We therefore argue that research studies of children with ADHD should include measurement of these broader domains of family impact and child health.

The two major phospholipid classes, namely, phosphatidylethanolamines (PE) and phosphatidylcholines (PC), were studied in four different regions of human brain, i.e., in frontal gray matter, frontal white matter, hippocampus and in pons. The fatty acid (FA) compositions of these phospholipids were found to be specific for the different regions. PC contains mostly saturated and 18:1 FA, while PE is rich in polyunsaturated FA. Aging has no influence on the FA compositions, while in Alzheimer's disease (AD) PE is modified in all four regions, particularly in frontal gray matter and in hippocampus. The abundance of the major monounsaturated FA of PE, 18:1, is not significantly altered in Alzheimer's disease, but there is a substantial increase in the relative amounts of the saturated components 14:0, 16:0 and 18:0. This is paralleled by a decrease in the polyunsaturated FA 20:4, 22:4 and 22:6. It is not clear whether the changes observed are specific for AD. Changes in saturated/polyunsaturated FA ratio are likely to influence cellular function, which in turn may cause certain neural deficiencies. The findings do not support the hypothesis that AD reflects an accelerated aging process.

Single cell oils (SCOs) are now produced by various microorganisms as commercial sources of arachidonic acid (ARA) and docosahexaenoic acid (DHA). These oils are now used extensively as dietary supplements in infant formulas. An understanding of the underlying biochemistry and genetics of oil accumulation in such microorganisms is therefore essential if lipid yields are to be improved. Also an understanding of the biosynthetic pathways involved in the production of these polyunsaturated fatty acids (PUFAs) is also highly desirable as a prerequisite to increasing their content in the oils. An account is provided of the biosynthetic machinery that is necessary to achieve oil accumulation in an oleaginous species where it can account for lipid build up in excess of 70% of the cell biomass. Whilst PUFA production in most microorganisms uses a conventional fatty acid synthase (FAS) system followed by a series of desaturases and elongases, in Schizochytrium sp., and probably related thraustochytrid marine protists, PUFA synthesis now appears to be via a polyketide synthase (PKS) route. This route is discussed. It clearly represents a major departure from conventional fatty acid biosynthesis, possibly as a means of decreasing the amount of NADPH that is needed in the overall process.

In vivo study of white matter microstructural integrity through magnetic resonance diffusion tensor imaging (DTI) permits examination of degradation of axonal circuitry that may underlie functional decline of frontally-based processes in normal adult aging. Determination of the pattern of age-related degradation of white matter microstructure requires quantitative comparison of the rostral-caudal and superior-inferior extents of the brain's white matter. To date, this has not been accomplished, probably because of significant artifacts from spatial distortion and poor signal resolution that precludes accurate analysis in prefrontal and inferior brain regions. Here, we report a profile analysis of the integrity of white matter microstructure across the supratentorium and in selected focal regions using DTI data collected at high-field strength (3 T), with isotropic voxel acquisition, and an analysis based on a concurrently-acquired field map to permit accurate quantification of artifact-prone, anterior and inferior brain regions. The groups comprised 10 younger and 10 older individuals; all were high functioning, highly educated, and in excellent health. The DTI profile analysis revealed a robust frontal distribution of low white matter anisotropy and high bulk mean diffusivity in healthy older compared with younger adults. In contrast to frontal fiber systems, posterior systems were largely preserved with age. A second analysis, based on focal samples of FA, confirmed that the age-related FA decline was restricted to frontal regions, leaving posterior and inferior brain regions relatively intact. The selective decline of anterior anisotropy with advancing age provides evidence for the potential of a microstructural white matter mechanism for the commonly observed decline in frontally-based functions.

Impaired central pain modulation is implicated in the pathophysiology of chronic pain. In this controlled experiment, we evaluated whether partial sleep loss altered endogenous pain inhibition and reports of spontaneous pain. Thirty-two healthy females were studied polysomnographically for 7 nights. On Nights 1-2 (Baseline), subjects slept undisturbed for 8 hours. After Night 2, subjects were randomized to Control (N = 12), Forced Awakening (FA, N = 10), or Restricted Sleep Opportunity (RSO, N = 10) conditions. Controls continued to sleep undisturbed. FA underwent 8 forced awakenings (one per hour) on Nights 3-5. RSO subjects were yoked to FA on total sleep time (TST), receiving partial sleep deprivation by delayed bedtime. On Night 6, both FA & RSO underwent 36 hours total sleep deprivation (TSD), followed by 11-hour recovery sleep (Night 7). Subjects completed twice-daily psychophysical assessments of mechanical pain thresholds and pain inhibition (Diffuse Noxious Inhibitory Controls), via use of a conditioning stimulus (i.e., cold pressor) paradigm. FA and RSO demonstrated 50% reductions in total sleep time and increases in nonpainful somatic symptoms during partial sleep deprivation. While sleep deprivation had no effect on pain thresholds, during partial sleep deprivation the FA group demonstrated a significant loss of pain inhibition and an increase in spontaneous pain; neither of the other 2 groups showed changes in pain inhibition or spontaneous pain during partial sleep deprivation. These data suggest that sleep continuity disturbance, but not simple sleep restriction, impairs endogenous pain-inhibitory function and increases spontaneous pain, supporting a possible pathophysiologic role of sleep disturbance in chronic pain.

Nonalcoholic steatohepatitis (NASH) may progress to liver fibrosis and cirrhosis. Mechanisms directly involved in the development of fibrosis have been poorly investigated. Because connective tissue growth factor (CTGF) is an intermediate key molecule involved in the pathogenesis of fibrosing chronic liver diseases and is potentially induced by hyperglycemia, the aims of this study were to (1) study the expression of CTGF in vivo both in human liver biopsy specimens of patients with NASH and in an experimental model of obesity and type II diabetes (Zucker rats); and (2) analyze the effects of hyperglycemia and insulin in vitro on hepatic stellate cells. In vivo, CTGF overexpression was observed in the liver tissue of all of the 16 patients with NASH. CTGF immunostaining was mild in 7 cases (44%) and moderate or strong in 9 cases (56%). Staining was mainly detected in the liver extracellular matrix in parallel with the amount of liver fibrosis. Liver from fa/fa rats also showed CTGF overexpression by comparison with Fa/fa rats both at the messenger RNA (mRNA) level (3-fold increase) and protein level. In vitro, both CTGF mRNA and protein were significantly increased when hepatic stellate cells were incubated with either glucose or insulin. A slight increase in type I procollagen mRNA level was also observed in hepatic stellate cells incubated with glucose. In conclusion, this study suggests that hyperglycemia and insulin are key-factors in the progression of fibrosis in patients with NASH through the up-regulation of CTGF.

Cytotoxic drugs used in chemotherapy of leukemias and solid tumors cause apoptosis in target cells. In lymphoid cells the CD95 (APO-1/Fas)/CD95 ligand (CD95-L) system is a key regulator of apoptosis. Here we describe that doxorbicin induces apoptosis via the CD95/CD95-L system in human leukemia T-cell lines. Doxorubicin-induced apoptosis was completely blocked by inhibition of gene expression and protein synthesis. Also, doxorbicin strongly stimulates CD95-L messenger RNA expression in vitro at concentrations relevant for therapy in vivo. CEM and jurkat cells resistant to CD95-mediated apoptosis were also resistant to doxorbicin-induced apoptosis . Furthermore, doxorbicin-induced apoptosis was inhibited by blocking F(ab')2 anti-APO-1 (anti-CD95) antibody fragments. Expression of CD95-L mRNA and protein in vitro was also stimulated by other cytotoxic drugs such as methotrexate. The finding that apoptosis caused by anticancer drugs may be mediated via the CD95 system provides a new molecular insight into resistance and sensitivity toward chemotherapy in malignancies.

BACKGROUND

Damage to white matter tracts, resulting in "cerebral disconnection," may underlie age-related cognitive decline.

METHODS

Using diffusion tensor MRI (DTI) to investigate white matter damage, and magnetic resonance spectroscopy (MRS) to look at its underlying pathologic basis, the authors investigated the relationship between white matter structure and cognition in 106 healthy middle-aged and elderly adults. Fractional anisotropy (FA) and mean diffusivity (MD) values, whole brain white matter histograms, and regions of interest placed in the white matter of the centrum semiovale were analyzed. Correlations with executive function, working memory, and information-processing speed were performed.

RESULTS

There was a progressive reduction in FA and increase in diffusivity with age in both region of interest (r = 0.551, p < 0.001), and whole brain histograms (r = 0.625, p < 0.001). DTI values correlated with performance in all three cognitive domains. After controlling for age, DTI parameters correlated with working memory but not with the other two cognitive domains. MRS studies found a correlation of N-acetyl aspartate, a neuronal marker, with DTI parameters (r = 0.253, p < 0.05).

CONCLUSIONS

The results are consistent with white matter damage due to axonal loss, causing age- related cognitive decline. Working memory may be particularly dependent on complex networks dependent on white matter connections.

Fas-associated death domain protein (FADD) and caspase-8 (casp8) are vital intermediaries in apoptotic signaling induced by tumor necrosis factor family ligands. Paradoxically, lymphocytes lacking FADD or casp8 fail to undergo normal clonal expansion following antigen receptor cross-linking and succumb to caspase-independent cell death upon activation. Here we show that T cells lacking FADD or casp8 activity are subject to hyperactive autophagic signaling and subvert a cellular survival mechanism into a potent death process. T cell autophagy, enhanced by mitogenic signaling, recruits casp8 through interaction with FADD:Atg5-Atg12 complexes. Inhibition of autophagic signaling with 3-methyladenine, dominant-negative Vps34, or Atg7 shRNA rescued T cells expressing a dominant-negative FADD protein. The necroptosis inhibitor Nec-1, which blocks receptor interacting protein kinase 1 (RIP kinase 1), also completely rescued T cells lacking FADD or casp8 activity. Thus, while autophagy is necessary for rapid T cell proliferation, our findings suggest that FADD and casp8 form a feedback loop to limit autophagy and prevent this salvage pathway from inducing RIPK1-dependent necroptotic cell death. Thus, linkage of FADD and casp8 to autophagic signaling intermediates is essential for rapid T cell clonal expansion and may normally serve to promote caspase-dependent apoptosis under hyperautophagic conditions, thereby averting necrosis and inflammation in vivo.

Activation of CD40 is essential for thymus-dependent humoral immune responses and rescuing B cells from apoptosis. Many of the effects of CD40 are believed to be achieved through altered gene expression. In addition to Bcl-x, a known CD40-regulated antiapoptotic molecule, we identified a related antiapoptotic molecule, A1/Bfl-1, as a CD40-inducible gene. Inhibition of the NF-kappaB pathway by overexpression of a dominant-active inhibitor of NF-kappaB abolished CD40-induced up-regulation of both the Bfl-1 and Bcl-x genes and also eliminated the ability of CD40 to rescue Fas-induced cell death. Within the upstream promoter region of Bcl-x, a potential NF-kappaB-binding sequence was found to support NF-kappaB-dependent transcriptional activation. Furthermore, expression of physiological levels of Bcl-x protected B cells from Fas-mediated apoptosis in the absence of NF-kappaB signaling. Thus, our results suggest that CD40-mediated cell survival proceeds through NF-kappaB-dependent up-regulation of Bcl-2 family members.