Caspase-3 feeds back on caspase-8, Bid and XIAP in type I Fas signaling in primary mouse hepatocytes.

The TNF-R1 like receptor Fas is highly expressed on the plasma membrane of hepatocytes and plays an essential role in liver homeostasis. We recently showed that in collagen-cultured primary mouse hepatocytes, Fas stimulation triggers apoptosis via the so-called type I extrinsic signaling pathway. Central to this pathway is the direct caspase-8-mediated cleavage and activation of caspase-3 as compared to the type II pathway which first requires caspase-8-mediated Bid cleavage to trigger mitochondrial cytochrome c release for caspase-3 activation. Mathematical modeling can be used to understand complex signaling systems such as crosstalks and feedback or feedforward loops. A previously published model predicted a positive feedback loop between active caspases-3 and -8 in both type I and type II FasL signaling in lymphocytes and Hela cells, respectively. Here we experimentally tested this hypothesis in our hepatocytic type I Fas signaling pathway by using wild-type and XIAP-deficient primary hepatocytes and two recently characterized, selective caspase-3/-7 inhibitors (AB06 and AB13). Caspase-3/-7 activity assays and quantitative western blotting confirmed that fully processed, active p17 caspase-3 feeds back on caspase-8 by cleaving its partially processed p43 form into the fully processed p18 species. Our data do not discriminate if p18 positively or negatively influences FasL-induced apoptosis or is responsible for non-apoptotic aspects of FasL signaling. However, we found that caspase-3 also feeds back on Bid and degrades its own inhibitor XIAP, both events that may enhance caspase-3 activity and apoptosis. Thus, potent, selective caspase-3 inhibitors are useful tools to understand complex signaling circuitries in apoptosis.

Niche differentiation of two sympatric species of Microdochium colonizing the roots of common reed.

BACKGROUND: Fungal endophyte communities are often comprised of many species colonizing the same host. However, little is known about the causes of this diversity. On the one hand, the apparent coexistence of closely related species may be explained by the traditional niche differentiation hypothesis, which suggests that abiotic and/or biotic factors mediate partitioning. For endophytes, such factors are difficult to identify, and are therefore in most cases unknown. On the other hand, there is the neutral hypothesis, which suggests that stochastic factors may explain high species diversity. There is a need to investigate to what extent each of these hypotheses may apply to endophytes. RESULTS: The niche partitioning of two closely related fungal endophytes, Microdochium bolleyi and M. phragmitis, colonizing Phragmites australis, was investigated. The occurrences of each species were assessed using specific nested-PCR assays for 251 field samples of common reed from Lake Constance, Germany. These analyses revealed niche preferences for both fungi. From three niche factors assessed, i.e. host habitat, host organ and season, host habitat significantly differentiated the two species. M. bolleyi preferred dry habitats, whereas M. phragmitis prevailed in flooded habitats. In contrast, both species exhibited a significant preference for the same host organ, i.e. roots. Likewise the third factor, season, did not significantly distinguish the two species. Differences in carbon utilization and growth temperature could not conclusively explain the niches. The inclusion of three unrelated species of Ascomycota, which also colonize P. australis at the same locations, indicated spatio-temporal niche partitioning between all fungi. None of the species exhibited the same preferences for all three factors, i.e. host habitat, host organ, and time of the season. CONCLUSIONS: The fungal species colonizing common reed investigated in this study seem to exploit niche differences leading to a separation in space and time, which may allow for their coexistence on the same host. A purely neutral model is unlikely to explain the coexistence of closely related endophytes on common reed.

A randomized, controlled trial of aerobic exercise in combination with paroxetine in the treatment of panic disorder.

OBJECTIVES: Regular aerobic exercise (running) has been shown to be superior to a pill placebo in the treatment of panic disorder. Combined drug and exercise treatment has not been investigated in randomized controlled studies to date. METHODS: This is a randomized, 10-week, controlled, parallel group, pilot study. A total of 75 outpatients with panic disorder with or without agoraphobia (DSM-IV and ICD-10) received either (1) exercise plus paroxetine 40 mg/day (n=21), (2) relaxation plus paroxetine (n=17), (3) exercise plus pill placebo (n=20), or (4) relaxation plus pill placebo (n=17). Changes in the Panic and Agoraphobia Scale (P&A), and the Clinical Global Impression Scale (CGI) underwent repeated measure analysis. RESULTS: Effects sizes were large for all groups (d=1.53-3.87), however not significantly different. Paroxetine-treated patients were significantly more improved than placebo-treated patients. On the CGI, patients in the exercise groups (plus paroxetine or placebo) had a trend toward better improvement compared to relaxation (P=0.06). Response and remission rates were higher in the paroxetine compared to pill placebo groups. CONCLUSIONS: While paroxetine was superior to placebo, aerobic exercise did not differ from relaxation training in most efficacy measures.

Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes.

UNLABELLED: Fas/CD95-induced apoptosis of hepatocytes in vivo proceeds through the so-called type II pathway, requiring the proapoptotic BH3-only Bcl-2 family member Bid for mitochondrial death signaling. Consequently, Bid-deficient mice are protected from anti-Fas antibody injection induced fatal hepatitis. We report the unexpected finding that freshly isolated mouse hepatocytes, cultured on collagen or Matrigel, become independent of Bid for Fas-induced apoptosis, thereby switching death signaling from type II to type I. In such in vitro cultures, Fas ligand (FasL) activates caspase-3 without Bid cleavage, Bax/Bak activation or cytochrome c release, and neither Bid ablation nor Bcl-2 overexpression is protective. The type II to type I switch depends on extracellular matrix adhesion, as primary hepatocytes in suspension die in a Bid-dependent manner. Moreover, the switch is specific for FasL-induced apoptosis as collagen-plated Bid-deficient hepatocytes are protected from tumor necrosis factor alpha/actinomycin D (TNFalpha/ActD)-induced apoptosis. CONCLUSION: Our data suggest a selective crosstalk between extracellular matrix and Fas-mediated signaling that favors mitochondria-independent type I apoptosis induction.

Implementing a hospital-based smoking cessation programme: evidence for a learning effect.

OBJECTIVE: This study assessed a newly set-up, hospital-based smoking cessation clinic with regard to continuous abstinence rates and the effectiveness of concomittant nicotine replacement therapy. METHODS: Smoking status of 369 participants of this 8-week cognitive-behavioural smoking cessation group programme was obtained using exhaled carbon monoxide at the end of the course as well as self-report 6 months after the course. In addition to demographic data, FTND score, SDS score, and usage of nicotine replacement products were recorded. RESULTS: Overall, 29.8% of all participants reported to have been continuously abstinent for 6 months after the course. Success rates increased significantly during the first year after initiation of the programme (from 15 to 35%, p<0.001), indicating a learning process of the staff running the course. Nicotine replacement therapy was used by 51.3% of participants, but 58% of these discontinued its use within 5 weeks. Nicotine substitution for more than 5 weeks was associated with a 50% success rate after 6 months. CONCLUSIONS: Our data indicate a learning effect of smoking cessation course staff and a possible minimum duration required for nicotine replacement to be effective. PRACTICE IMPLICATIONS: The observed learning effect in smoking cessation programmes should be considered when evaluating newly established interventions of this kind. Patients tend to stop nicotine replacement therapy too early, thereby decreasing their chances of middle-term abstinence.

Sensitivity and specificity of qualitative muscle ultrasound in assessment of suspected neuromuscular disease in childhood.

Muscle ultrasound is considered a useful noninvasive technique for visualizing normal and pathological skeletal muscle. We determined the accuracy of qualitative muscle ultrasound in the discrimination of normal muscle from myopathic, neurogenic, and unspecifically abnormal tissue changes in the evaluation of suspected NMD in childhood. Sensitivity and specificity of muscle ultrasound were assessed by comparing sonographic classification of muscle tissue changes in 134 children with definitive diagnosis as provided by muscle histology or mutation analysis performed subsequently to the sonography. We found a sensitivity of 81% and a specificity of 96% for detection of any abnormal muscle tissue alteration by ultrasound. For detection of neurogenic changes, sensitivity was 77% with even higher specificity (98%). Accuracy was slightly lower for myopathic changes (79%) and clearly lower for unspecific abnormal tissue alterations (70%). Accuracy of ultrasound was lower in younger children. High reliability of muscle sonography justifies a more widespread use of this method in evaluation of suspected NMD in childhood.

Outcome of 12 drowned children with attempted resuscitation on cardiopulmonary bypass: an analysis of variables based on the "Utstein Style for Drowning".

BACKGROUND: In 2003, the International Liaison Committee on Resuscitation (ILCOR) published the "Utstein Style for Drowning" (USFD) to advance knowledge on the epidemiology, treatment, and outcome prediction after drowning. Applying the USFD and evaluating its data template for outcome analysis, we report here on the largest study published thus far of drowned children (age 0-14) who underwent attempted resuscitation on cardiopulmonary bypass (CPB). METHODS: We conducted a retrospective review of all drowned children admitted to Göttingen University Hospital between 1/1987 and 12/2005 in sustained cardiopulmonary arrest and resuscitation with CPB. We correlated eight outcome-affecting USFD variables and four additional variables not included in the USFD with potential impact on outcome to four outcome groups: survival, non-survival, survival with full recovery, and failed resuscitation. RESULTS: Out of 12 children (aged 22 months to 7.5 years), 5 survived to hospital discharge and 7 died in hospital. Two survivors recovered fully and three remained in a vegetative state. In two patients, resuscitation on CPB failed. Both children who fully recovered, compared to the 10 others, had relatively low serum K+ concentrations (2.6 and 3.7 mmol/l versus 5.8+/-3.8 mmol/l [mean+/-S.D.; n=10]), a relatively slow rewarming speed (1.9 and 1.2 degrees C/h versus 3.4+/-1.8 degrees C/h), were female (all three girls survived), received early basic life support (BLS) and showed idioventricular bradycardia. Both children with failed resuscitation had severe hyperkalaemia (11.7 and 13.3 mmol/l versus 10 others, 4.0+/-1.5 mmol/l), were relatively rapidly rewarmed (6.9 and 4.0 degrees C/h versus 10 others, 2.61+/-1.32 degrees C/h), male, and in asystole. We identified no outcome trends for age, pH, or water and core temperatures. CONCLUSIONS: Most variables relevant for outcome in drowned children can be documented with the use of the USFD. Additional variables not included in the USFD that have emerged from this study and may predict outcome include serum K+ concentration, rewarming speed, and initial cardiac rhythm.

Growth hormone response to low-dose apomorphine in restless legs syndrome.

INTRODUCTION: Low-dose apomorphine challenge has been shown to cause a rise in growth hormone (GH) in patients with Parkinson's disease (PD). This was interpreted as an increased postsynaptic sensitivity of hypothalamic dopamine receptors in the course of a generalized degeneration of dopaminergic neurons. The dopaminergic system in the restless legs syndrome (RLS) has been assumed to play a role in its pathophysiology. It is therefore the aim of this study to determine whether the GH response to subcutaneously applied low-dose apomorphine is generally altered in patients with RLS as compared to healthy controls. PATIENTS AND METHODS: We examined 40 patients with idiopathic RLS as well as 20 age- and sex-matched healthy control subjects by means of the low-dose apomorphine test. GH was analyzed at baseline, as well as 45 and 60 min after subcutaneous low-dose apomorphine injection in the morning. RESULTS: Forty RLS patients (58.3+/-11.9 years, 32 females) with a mean RLS severity scale score of 23.9+/-6.6 (range 10-37) were examined. GH was not significantly increased 45 and 60 min after injection (p=0.397) (2.44+/-2.35 ng/ml at baseline versus 2.71+/-2.29 ng/ml after 45 min and 2.18+/-1.83 ng/ml after 60 min). The results were independent of pre-treatment with levodopa. Age, sex, duration, and severity of the disease did not show a covariate effect with GH levels. There was no difference compared with healthy controls. CONCLUSIONS: RLS patients did not show an increase in GH after stimulation with low-dose apomorphine. Lack of sensitivity alteration of extrastriatal hypothalamic dopamine receptors suggests that RLS is not a general dopaminergic degenerative disease or might only show circadian alterations.

Total tau protein, phosphorylated tau (181p) protein, beta-amyloid(1-42), and beta-amyloid(1-40) in cerebrospinal fluid of patients with dementia with Lewy bodies.

The intra vitam diagnosis of different dementias is still based on clinical grounds. So far, no technical investigations have been available to support these diagnoses. For tau protein and beta-amyloid(1-42) in cerebrospinal fluid (CSF), promising results for the diagnosis of Alzheimer's disease (AD) have been reported; however, their differential diagnostic spectrum is limited, as was recently shown for dementia with Lewy bodies (DLB) and for AD. Therefore, further marker proteins have to be established to ameliorate, support, and differentiate these clinical diagnoses. We evaluated beta-amyloid(1-40) and phosphorylated tau protein (181p), in addition to total tau protein and beta-amyloid(1-42), in 20 patients with DLB, 34 AD patients, and 20 non-demented neurological controls (NDCs). All markers could differentiate between the dementia groups (AD, DLB) and the controls. AD and DLB could be differentiated only by levels of total tau protein and by the ratio total tau protein/phosphorylated tau protein. However, values still overlapped markedly. In some cases, tau protein levels in CSF may contribute to the clinical distinction between DLB and AD, but the value of the markers is still limited, especially because of mixed pathology. We conclude that more specific markers have to be established to differentiate between these diseases.

Only a few fungal species dominate highly diverse mycofloras associated with the common reed.

Plants are naturally colonized by many fungal species that produce effects ranging from beneficial to pathogenic. However, how many of these fungi are linked with a single host plant has not been determined. Furthermore, the composition of plant-associated fungal communities has not been rigorously determined. We investigated these essential issues by employing the perennial wetland reed Phragmites australis as a model. DNA extracted from roots, rhizomes, stems, and leaves was used for amplification and cloning of internal transcribed spacer rRNA gene fragments originating from reed-associated fungi. A total of 1,991 clones from 15 clone libraries were differentiated by restriction fragment length polymorphism analyses into 345 operational taxonomical units (OTUs). Nonparametric estimators for total richness (Chao1 and ACE) and also a parametric log normal model predicted a total of about 750 OTUs if the libraries were infinite. Sixty-two percent of the OTUs sequenced were novel at a threshold of 3%. Several of these OTUs represented undocumented fungal species, which also included higher taxonomic levels. In spite of the high diversity of the OTUs, the mycofloras of vegetative organs were dominated by just a few typical fungi, which suggested that competition and niche differentiation influence the composition of plant-associated fungal communities. This suggestion was independently supported by the results of nested PCR assays specifically monitoring two OTUs over 3 years, which revealed significant preferences for host habitat and host organ.

Levodopa treatment does not affect low-dose apomorphine test in patients with Parkinson's disease.

Challenge with low-dose apomorphine causes a significant rise in growth hormone (GH) in patients with Parkinson's disease (PD) compared to controls and patients with multiple system atrophy (MSA) who have not previously received dopaminergic treatment. To date, it has not been demonstrated whether an apomorphine-induced rise in GH can still be detected in PD patients who are currently treated with levodopa. We investigated whether an ongoing treatment with levodopa influences the GH response to subcutaneously applied low-dose apomorphine in PD patients. We studied 44 patients with idiopathic PD using the low-dose apomorphine test. Twenty-three patients were under treatment with levodopa and 21 patients were without any dopaminergic therapy. GH and cortisol levels were analyzed at time of injection and 45 minutes and 60 minutes after subcutaneous apomorphine injection. Forty-five minutes after apomorphine injection, there was no significant difference between the mean rise in plasma GH in untreated PD patients compared with levodopa-treated patients (P = 0.235). There was no increase of cortisol levels in each treatment group. Age, sex, duration, and severity of the disease did not show a covariate effect with GH levels. A small group of PD patients (n = 8) treated with dopamine agonists and a small group of patients with MSA (n = 5) as well as patients with vascular parkinsonism (n = 5) did not show any increase of GH. Our data suggest that the apomorphine-induced rise in GH does not depend on previous levodopa treatment in PD patients but, as expected, is blocked by dopamine agonists and is not present in patients with other than idiopathic parkinsonian syndrome. Thus, the low-dose apomorphine test may also be a useful biological marker in the early differential diagnosis of PD patients who have already received levodopa treatment.

Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane.

It is assumed that the survival factors Bcl-2 and Bcl-x(L) are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-x(L) is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-x(L) requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-x(L) specifically functions on the MOM.