Knowledge of how synapses alter their efficiency of communication is central to the understanding of learning and memory. The most extensively studied forms of synaptic plasticity are long-term potentiation (LTP) and its counterpart long-term depression (LTD) of AMPA receptor-mediated synaptic transmission. In the CA1 region of the hippocampus, it has been shown that LTP often involves a rapid increase in the unitary conductance of AMPA receptor channels. However, LTP can also occur in the absence of any alteration in AMPA receptor unitary conductance. In the present study we have used whole-cell dendritic recording, failures analysis and non-stationary fluctuation analysis to investigate the mechanism of depotentiation of LTP.We find that when LTP involves an increase in unitary conductance, subsequent depotentiation invariably involves the return of unitary conductance to pre-LTP values. In contrast, when LTP does not involve a change in unitary conductance then depotentiation also occurs in the absence of any change in unitary conductance, indicating a reduction in the number of activated receptors as the most likely mechanism.These data show that unitary conductance can be bi-directionally modified by synaptic activity. Furthermore, there are at least two distinct mechanisms to restore synaptic strength from a potentiated state, which depend upon the mechanism of the previous potentiation. These2+/calmodulin-kinase II (CaM-KII)-mediated phosphorylation of GluR1 which occurs during LTP [Recent studies have provided information on the possible mechanisms underlying postsynaptic alterations in synaptic strength. There is evidence that AMPA receptors are inserted into the postsynaptic membrane during LTP -15 and rring LTP , and whiring LTP . Therefode novo LTD .We have recently used peak-scaled non-stationary fluctuation analysis  of synaathways; ,23). In athways; . Indeed,N) [N never involves a change in γ (DPN). These data show, firstly, that there are two distinct molecular mechanisms for the reduction of synaptic strength that are dependent on the nature of the preceding LTP and, secondly, that γ can indeed be bi-directionally modified in response to synaptic activity.In the present study we have investigated a second form of LTD known as depotentiation (DP), which is a reversal of pre-established LTP -27. LTP N) . We wereUsing whole-cell recordings from the proximal apical dendrites of hippocampal CA1 pyramidal cells, minimal stimulation of nearby afferents evoked EPSCs that could be reliably resolved from failures  with a holding potential of 0 mV. This resulted in stable LTP (EPSC amplitude = 186 ± 16 % of baseline, n = 18).N). As noted previously [In agreement with a previous study , cells feviously , there wFigure rise: baseline = 1.6 ± 0.2 ms, LTPγ = 1.7 ± 0.3 ms, DPγ = 1.7 ± 0.2 ms, n = 11; τdecay: baseline = 8.3 ± 0.6 ms, LTPγ = 8.2 ± 0.6 ms, DPγ = 8.9 ± 0.7, n = 11; Figure For this group of cells, DP, induced by pairing stimulation (baseline frequency) with a holding potential of -40 mV, always resulted in a reversal of the γ increase, as indicated by the current-variance plot Figure . SimilarN), as indicated by the current-variance plot , a small depression of less than 50 %  was associated with no change in success rate  as well as a decrease in potency (potency ratio = 0.54 ± 0.04). This suggests that in these cells there was an additional decrease in Pr or n. Therefore, for DPγ, the change in γ did not fully account for the amplitude change in every cell . This il Figure  but did l Figure .N). As reported previously for different data sets from this age of rats [N). In the present study we saw a similar proportion of LTP expressed by changes in γ versus N. Strikingly, we observed a precise relationship between the mechanism of DP and the form of preceding LTP; LTPγ was always reversed by DPγ, and LTPN was always reversed by DPN.In this study we have shown that there are two mechanisms for NMDA receptor-dependent DP, a reduction in γ (DPγ) and a decrease in the number of activated AMPA receptors ? It is unlikely that this type of depression is due to a change in channel kinetics because there was no change in EPSC kinetics (see [open [What might be the mechanism underlying DP that is not associated with a decrease in γ (i.e., DPics see ,29). The. TheN)? ics see ,35. Post. PostN? ee [open or in theee [open .N are indistinguishable from those that we and others have reported recently for de novo LTD [de novo LTD were mutually occlusive, indicating a convergence of mechanisms. These data argue strongly for a postsynaptic mechanism of expression. Furthermore, since pep2m causes the removal of AMPA receptors from the membrane surface, as determined immunocytochemically [de novo LTD is due to the physical elimination of synaptic AMPA receptors. Other evidence for a postsynaptic mechanism for de novo LTD includes a reduction in the postsynaptic sensitivity to glutamate [N  a change from a single low conductance state to a single high conductance state, 2) changes in open times within a burst and, 3) changes in the proportion of time spent in different conductance states. Since AMPA receptors are known to have multiple conductance states ,33 we hade novo LTD was induced there was never a change in γ [de novo LTD and DP. Moreover, we have also investigated these possibilities using our standard compartmental model [decay which was never observed experimentally, and 2) pronounced changes in asynchrony necessary to cause an artefactual change in γ cause large deviations from the parabolic relationship in the current-variance plot (unpublished observations) and alterations in τrise [rise and τdecay [Other theoretical possibilities exist to explain DPγ. For example, the silencing of synapses close to the patch electrode leaving more distant synapses contributing a lower net γ due to electrotonic filtering, or a decrease in trial-to-trial asynchrony of transmitter release. We believe that these possibilities are unlikely because in every cell in which nge in γ . If suchal model . This shin τrise , also nein τrise . Such chin τrise , howeverd τdecay , which wIn summary, we have shown that there are two distinct molecular mechanisms for the reduction of synaptic strength. Although previous studies have provided evidence that LTD is associated with dephosphorylation of serine 845 on GluR1 ,17 it is4, 26 NaHCO3, 2 CaCl2, 1 MgSO4, 15 glucose, 2 ascorbic acid, 0.05 picrotoxin, saturated with 95% O2 / 5% CO2, at room temperature (23–25°C). Individual dendrites were visualised using infrared illumination and DIC optics and approached under visual control. Whole-cell dendritic recordings of synaptic currents were obtained at a holding potential of -70 mV using patch electrodes (6–10 MΩ) filled with a solution containing (in mM): 135 CsMeSO4, 8 NaCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 5 QX-314, pH 7.25, 285 mOsm. Schaffer collateral-commissural fibers were stimulated at 0.5 Hz using a platinum monopolar or concentric bipolar electrode, which was positioned 20–40 μm from the dendrite parallel to the input pathway. The stimulus intensity was set to evoke some failures to enable a failures analysis to be performed and to ensure that the majority of EPSCs were, for any given trial, evoked by release from a single site. However, to elicit sufficient EPSCs to obtain a baseline estimate of γ before "washout" of LTP it was usually necessary to set the success rate fairly high . As a result it is likely that multiple release sites contributed to the recordings. LTP was induced by pairing 40–60 stimuli (baseline frequency) with a holding potential of 0 mV. DP was induced following the induction of stable LTP by 100–200 stimuli (baseline frequency) at -40 mV holding potential. All recordings were made using an Axopatch 1B amplifier, signals were filtered at 5 kHz (8 pole Bessel filter), digitised at 10 kHz and stored on computer. EPSC amplitude and input resistance were analysed and displayed on-line using the 'LTP' program [Hippocampal slices (400 μm) were obtained from 12–15 day old rats and perfused with an extracellular solution containing (in mM): 124 NaCl, 3 KCl, 1.25 NaHPO program ). Seriesvs. the mean current amplitude and the single channel current was estimated by fitting the data to: σ2 = iI - I2/N + bl, where σ2 is the variance, I is the mean current, N is the number of channels activated at the peak, i is the single channel current and bl is the background variance. The single channel conductance (γ) is then γ = i/V, where V is the driving force . Response amplitude was measured in two ways. For failures, amplitude was estimated by measuring the difference between the average current over two time windows of equal length, one immediately before the stimulus artefact and the other centred on the peak of the mean EPSC. For estimation of the amplitude of successes the peak amplitude over a set time window was determined. Failures were identified visually, and potency was calculated as the mean EPSC amplitude excluding failures. For analysis of EPSC kinetics, rise time was estimated by the time-constant of a single exponential fit of the rising phase of the mean EPSC waveform. For decay, the time-constant of the single exponential fit to the decay phase was used. For display of individual EPSC traces in the figures, the stimulus artefact was digitally subtracted using an average of identified failures. All histograms are represented as smoothed line plots (SigmaPlot ver 5.0). Data are expressed as % of baseline (i.e., 100 % = no change). Statistical significance was assessed using the Student's t-test . All values are expressed as mean ± s.e.m.Non-SFA was performed as described previously . BrieflyAL performed most of the electrophysiology. MJP and MAW also contributed some recordings. PM provided some information that guided the work. TB performed the modelling that provided the theoretical frame-work. JI helped with the design and analysis of experiments. GLC coordinated the work and contributed to its design. All authors read and approved the manuscript.


The notion of a neurally encoded “reward system” that reinforces pleasure-seeking behaviors first emerged fifty years ago. Psychologists James Olds and Peter Milner discovered this phenomenon when their “lack of aim” landed an electrode outside their target while studying the behavioral responses of rats given electrical stimulation to a particular brain region. It was known that stimulation of certain brain regions would induce an animal to avoid the behavior that produced the stimulus. But in the rat with the “misplaced” electrode, stimulation of this new region caused the rat to repeat the behavior that caused the stimulus. Stimulation of certain brain regions provides a very strong incentive to restimulate, creating a feedback loop that reinforces both the behavior and the neural response to it. When gentle shocks were delivered to the rat hypothalamus, for example, the animals would “self-stimulate” 2,000 times per hour by pushing a lever. The neurotransmitter dopamine, it was later discovered, plays an important role in the brain's reward system—and in laying the biochemical foundation of drug addiction.Essential for normal central nervous system function, dopamine signaling mediates physiological functions as diverse as movement and lactation. The dopamine transporter (DAT) is involved in terminating dopamine signaling by removing the dopamine chemical messenger molecules from nerve synapses and returning them into the releasing neurons . DAT can also bind amphetamine, cocaine, and other psychostimulants, which inhibit dopamine reuptake, and, in the case of amphetamine, also stimulate the release of dopamine through DAT. It's thought that abnormal concentrations of dopamine in synapses initiate a series of events that cause the behavioral effects of these drugs. The biochemical steps underlying amphetamine-induced dopamine release, however, are not well characterized. Now, a team led by Jonathan Javitch and Aurelio Galli has identified a chemical modification of DAT that is essential for DAT-mediated dopamine release in the presence of amphetamine. Since this modification does not inhibit the ability of DAT to accumulate dopamine, it may suggest a molecular target for treating drug addiction.Embedded in the membrane of nerve cells, the dopamine transporter has a “tail,” called the N-terminal domain, that protrudes into the cell interior and consists of a stretch of about 60 amino acids. Many of these amino acids are potential sites of phosphorylation, a chemical reaction in which a phosphate group is added through the action of enzymes called kinases. Amphetamine has been shown to increase kinase activity and Margaret Gnegy, a coauthor of the current research article, showed previously that inhibiting protein kinase C activity blocks amphetamine's ability to release dopamine. Therefore, Javitch, Galli, and Gnegy hypothesized that N-terminal phosphorylation of DAT might play a critical role in the dopamine overload caused by amphetamine.The researchers found that amphetamine-induced dopamine release was reduced by 80% in cells expressing a mutant dopamine transporter in which the first 22 amino acids of the N-terminal domain had been removed (del-22). Surprisingly, this truncated transporter displayed normal dopamine uptake. In a full-length DAT, mutation of the five N-terminal serine amino acids to alanine amino acids, which prevented phosphorylation, produced an effect similar to removing the 22 amino acids. In contrast, replacing these five serine residues with aspartate residues to mimic phosphorylation led to normal dopamine release as well as normal dopamine uptake.These findings suggest that phosphorylation of one or more of these serine residues is necessary for amphetamine to flood the synapses with dopamine. While phosphorylation is a normal mechanism for regulating protein activity in a cell—and DAT is “significantly phosphorylated” under normal conditions—amphetamine could increase the level of DAT phosphorylation. Elucidating the mechanisms through which phosphorylation of DAT's N-terminus facilitates dopamine overload could lead to the development of drugs that block the “rewarding” effects of amphetamines and other addictive psychostimulants without interfering with normal dopamine clearance.


Combination therapy of irinotecan, folinic acid (FA) and 5-fluorouracil (5-FU) has been proven to be highly effective for the treatment of metastatic colorectal cancer. However, in light of safety and efficacy concerns, the best combination regimen for first-line therapy still needs to be defined. The current study reports on the bimonthly FOLFIRI protocol consisting of irinotecan with continuous FA/5-FU in five German outpatient clinics, with emphasis on the safety and efficiency, quality of life, management of delayed diarrhea, and secondary resection of regressive liver metastases.2), L-FA (200 mg/m2) and 5-FU bolus (400 mg/m2) on day 1, followed by a 46-h continuous infusion 5-FU (2400 mg/m2). One cycle contained three fortnightly administrations. Staging was performed after 2 cycles. Dosage was reduced at any time if toxicity NCI CTC grade III/IV was observed. Chemotherapy was administered only to diarrhea-free patients.A total of 35 patients were treated for metastatic colorectal cancer. All patients received first-line treatment according to the FOLFIRI regimen, consisting of irinotecan . Dose reduction was necessary in ten patients. Grade III/IV toxicity was rare, with diarrhea (14%), nausea/vomiting (12%), leucopenia (3%), neutropenia (9%) and mucositis (3%). The overall response rate was 31% (4 CR and 7 PR), with disease control in 74%. After primary chemotherapy, resection of liver metastases was achieved in three patients. In one patient, the CR was confirmed pathologically. Median progression-free and overall survival were seven and 17 months, respectively.The FOLFIRI regimen proved to be safe and efficient. Outpatient treatment was well tolerated. Since downstaging was possible, combinations of irinotecan and continuous FA/5-FU should further be investigated in neoadjuvant protocols. ColoRectal Cancer (CRC) have been demonstrated to benefit from chemotherapeutic treatment in terms of both quality and duration of life [Patients with advanced  of life . Of thes of life ,3. 5-Flu of life  -, was a of life -9. TheseIrinotecan (CPT-11), a potent inhibitor of the enzyme topoisomerase I, has demonstrated anti-tumorogenic activity in metastatic colorectal cancer, when used alone or in combinations with FA/5-FU, as adjuvant or palliative treatment (for review see 10 or 11). In randomized phase III clinical trials, second-line therapy with irinotecan significantly improved survival compared to supportive care  or to inHowever, the best regimen of irinotecan and FA/5-FU has yet to be defined. Altogether, irinotecan combined with continuous FA/5-FU infusions seemed to be superiorly active and less toxic than combination with FA/5-FU bolus regimens ,7. RecenFurthermore, irinotecan schedules of weekly and of once every two or three weeks demonstrated similar efficacy and quality of life, as well as significantly lower incidences of severe diarrhea in patients with 5-FU-refractory, metastatic colorectal cancer -17. In cTo date, little data is available regarding irinotecan combined with the simplified bi-monthly LV5-FU2 regimen as a first-line treatment in patients with metastatic colorectal cancer . TherefoAfter approval by the local ethical committee, patients were consecutively recruited from five German outpatient clinics . Eligibility requirements included (1) histologically documented adenocarcinoma of the colon or rectum and progressive measurable metastatic disease, (2) minimum life expectancy of three months, (3) Karnofsky performance status ≥ 60, (4) adequate hematologic, hepatic, and renal function, and (4) no prior chemotherapy for metastatic disease. Participants needed to be between 18 and 75 years of age. This study required that previous adjuvant 5-FU-based therapy with or without radiation therapy be completed at least 6 months prior to entry. Patients with CNS metastases, bowel obstruction, or ileus were excluded from the study. The study was approved by the ethics commission board responsible for all participating institutions. Prior to treatment, all patients gave written informed consent.2 d1, 5-FU bolus 400 mg/m2 d1, followed by 5-FU 46 h continuous infusion 2400 mg/m2 (simplified LV5-FU2 schedule). To prevent expected toxicities, patients were carefully informed of the potential risk of delayed diarrhea and neutropenia and the need for early intervention with loperamide [As previously described -16, treaperamide  and metoperamide . Atropinperamide . AntiemePrimary measures of the study were the overall objective response rate , overall survival, and quality of life. Secondary measures included the disease control rate (ORR + stable disease), time to progression, and frequency and severity of toxicities. Quality of life was assessed after inclusion into the study and as often as possible during the course of treatment, using the EORTC QLQ-C30 (version 2) questionnaire .Safety assessments and complete blood counts were performed weekly. Toxicity was graded according to National Cancer Institute Common Toxicity Criteria (NCI CTC). Toxicities not defined by NCI CTC criteria were classified as grade 0 (none), grade I (minor), grade II (moderate), grade III (severe), and grade IV (life-threatening). In case of any toxicity grade II, with the exception of hand-foot syndrome or alopecia, the next scheduled doses of irinotecan, folinic acid and 5-FU were delayed for a maximum of 1 week (or resolution of diarrhea for at least five days). In case of toxicity grade III/IV or if improvement from grade II to I (or resolution of diarrhea) was not achieved by two weeks, the following chemotherapy doses were reduced by 20 percent. If grade III/IV toxicity did not improve by 2 weeks, treatment was discontinued. Dose reductions were mandatory from the first cycle of chemotherapy in case of toxicity higher than grade II, and chemotherapy was resumed only after complete recovery from diarrhea.Tumor response was assessed according to World Health Organization (WHO) criteria. Tumor reassessment by the same imaging method used to establish baseline tumor measurement was generally performed after every two courses of therapy until progression. A complete response (CR) was defined as complete disappearance of evidence of cancer. A partial response (PR) was defined as a reduction in the sum of the products of the bi-perpendicular diameters of all measurable lesions by at least 50%. Progressive disease (PD) was defined as an increase in the sum of the products of the greatest bi-perpendicular diameters of all measurable lesions by at least 25% or the appearance of new lesions. Stable disease was defined as any reduction or increase in measurable lesions which did not meet the criteria for PR or PD. Confirmed objective responses were those for which a follow-up scan obtained at least four weeks later demonstrated the persistence of the response. The assessment of response and progression was based on investigator-reported measurements.Statistical analysis including survival analysis according to Kaplan-Meier was performed with the SPSS software package. The deadline for data evaluation was March 15, 2004. Survival was measured from the time of diagnosis to the date of death or last follow-up. Progression-free survival was calculated from treatment onset to the time of progression, study withdrawal or death of any cause. Patients who received at least one dose of the treatment regimen were evaluated for toxicity, and patients who completed at least two chemotherapy cycles were evaluated for response.Between 10/2001 and 5/2003, 35 consecutive patients  with metastatic colorectal cancer were enrolled into the study. The median age of these patients was 62 years, ranging from 38 to 73. Baseline characteristics of all patients are summarized in table The 35 patients received a total of 151 chemotherapy cycles (mean 4,3 per patient), consisting of 451 administrations. Overall, 51 (12,3 %) of all administrations had to be delayed for one week. During the complete study period, 19 patients had a delay of therapy for a median of nine days and in 10 patients (29%) a dose reduction was necessary at some point during the treatment period. The most common cause for discontinuation of study treatment was disease progression (18 patients, 51%). In case of discontinuation, 16 (46%) patients received a second line treatment with either oxaliplatin plus a FA/5-FU consisting regimen or an epidermal growth factor receptor antagonist.Hematologic toxicity was mild to moderate in the majority of patients table . Only onWith regard to response, four complete (CR) and seven partial responses were seen, and thus an overall response rate of 31% was observed table . In addiQuality of life data were obtained before and at least once during treatment from 13 patients . The 13 In the current phase IV study we evaluated toxicity and efficacy of the FOLFIRI combination of irinotecan with FA/5-FU as first-line chemotherapy for metastatic colorectal cancer. This combination was established within a phase I clinical trial with a recommended irinotecan dose of 180 mg/qm ,16. IrinIn the majority of our patients the FOLFIRI regimen was well tolerated. Gastrointestinal toxicity or thrombembolic events were never fatal. Most hospitalizations were for prevention rather than treatment of life-threatening conditions. Delayed diarrhea, a well known side effect of irinotecan , was genOverall, we observed relatively low toxicity in our study, with NCI CTC grade III leucopenia amounting to 3%, diarrhea to 14% and nausea/vomiting to 11%. The toxicity observed in our study was lower than that reported by Douillard et al. in the pivotal European first-line trial in the patient group receiving weekly irinotecan (80 mg/qm), 24-h HD-5-FU (2300 mg/qm) preceded by 2-h FA 500 mg/qm . In thisThe response rate achieved in our study (31%) was quite comperable tp previously published data ,7,14,26.Three reasons may account for these differences in survival between the studies. The most important reason may be that a higher percentage of patients  in our study had two or more metastatic sites, indicating a larger tumor burden and consequently a worse prognosis regarding survival . Second,It appears particularly noteworthy that after chemotherapy three of our patients achieved surgical resectability of their metastases. To our knowledge these results are the first ever reported to suggest a potential role for the FOLFIRI regimen in the neoadjuvant setting. Thus far, studies of regional chemotherapy for initially unresectable colorectal liver metastases could demonstrate some success with secondary curative surgery. In two recently published retrospective studies chronomodulated chemotherapy with oxaliplatin and FA/5-FU was used as neoadjuvant treatment for patients with unresectable colorectal liver metastases ,28. TherThe FOLFIRI regimen, consisting of irinotecan with continuous FA/5-FU over 48 h, given on an outpatient basis was safe and well tolerated in our study. The rate of severe side effects was comparably low with this bi-monthly regimen. As tumor control was achieved in about 75% and downstaging of metastatic disease was possible in some cases, combinations of irinotecan plus continuous FA/5-FU should be further investigated in neoadjuvant protocols for initially unresectable liver metastases.CRC Colorectal CancerFOLFIRI chemotherapeutic regimen consisting of irinotecan combined with continuous FA/5-FU infusionsFA Folinic Acid5-FU 5-FluorouracilHD-5-FU high dose 5-FUNCI CTC National Cancer Institute Common Toxicity CriteriaCR Complete remissionPR Partial responseSD Stable diseasePD Progressive diseaseTTP Time to progressionOS Overall survivalMM, SS and AT drafted the manuscript. MM and MH initiated the study. JS, CZ, HH, BA, MS, OK, T, PG and MH were involved in the patient's treatment as well as the documentation of response and side effects.The pre-publication history for this paper can be accessed here:


Keratins are members of the intermediate filaments (IFs) proteins, which constitute one of the three major cytoskeletal protein families. In hepatocytes, keratin 8 and 18 (K8/18) are believed to play a protective role against mechanical and toxic stress. Post-translational modifications such as phosphorylation and glycosylation are thought to modulate K8/18 functions. Treatment of mouse with a diet containing griseofulvin (GF) induces, in hepatocytes, modifications in organization, expression and phosphorylation of K8/18 IFs and leads, on the long term, to the formation of K8/18 containing aggregates morphologically and biochemically identical to Mallory bodies present in a number of human liver diseases. The aim of the present study was to investigate the relationship between the level and localization of the stress inducible heat shock protein 70 kDa (HSP70i) and the level and localization of K8/18 phosphorylation in the liver of GF-intoxicated mice. The role of these processes in Mallory body formation was studied, too. The experiment was carried out parallely on two different mouse strains, C3H and FVB/n.GF-treatment induced an increase in HSP70i expression and K8 phosphorylation on serines 79 (K8 S79), 436 (K8 S436), and K18 phosphorylation on serine 33 (K18 S33) as determined by Western blotting. Using immunofluorescence staining, we showed that after treatment, HSP70i was present in all hepatocytes. However, phosphorylated K8 S79 (K8 pS79) and K8 S436 (K8 pS436) were observed only in groups of hepatocytes or in isolated hepatocytes. K18 pS33 was increased in all hepatocytes. HSP70i colocalized with MBs containing phosphorylated K8/18. Phophorylation of K8 S79 was observed in C3H mice MBs but was not present in FVB/n MBs.in vivo, there is no direct relationship between GF-induced stress and K8/18 phosphorylation on the studied sites. The K8/18 phosphorylation pattern indicates that different cell signaling pathways are activated in subpopulations of hepatocytes. Moreover, our results demonstrate that, in distinct genetic backgrounds, the induction of K8/18 phosphorylation can be different.Our results indicate that GF intoxication represents a stress condition affecting all hepatocytes, whereas induction of K8/18 phosphorylation is not occurring in every hepatocyte. We conclude that,  Intermediate filaments (IFs) with microtubules and actin microfilaments are the major cytoskeletal components of most vertebrate cells -4. IF prIt is now generally accepted that, in multilayered epithelia, one of the function for keratins IFs is to protect the tissue from mechanical stress -9. The fAs for epidermal keratins, the production of transgenic mice targeting K8 or K18 has been necessary to unravel the role of IFs in simple epithelium such as in the liver. In hepatocytes, K8/18 is the only keratin pair and thus both keratins are necessary to form an IF network. Transgenic mice expressing K8 or K18 carrying mutations that affect filament formation, develop mild hepatitis and display greater liver sensitivity to mechanical and toxic stress than wild type animals ,13.Recent studies from Ku et al. -16 have Long-term treatment of mice with a diet containing griseofulvin (GF) induces the development of an hepatitis associated with the formation of MBs, which are biochemically and morphologically similar to those found in humans ,28. ThisIn the present study, we investigated the effect of chronic GF intoxication on hepatocytes from C3H and FVB/n mouse strains. We monitored the expression of the stress inducible form of the heat shock protein 70 kDa (HSP70i) and the induction of K8/18 phosphorylation at specific sites: K8 on serine 79 (K8 S79), K8 on serine 436 (K8 S436), K18 on serine 52 (K18 S52) and K18 on serine 33 (K18 S33) (reviewed in ,29). We The modifications in the amount of HSP70i, K8/18, and phosphorylated keratins (K8 pS79, K8 pS436 and K18 pS33) were analyzed by Western Blotting of total proteins from control and GF-treated C3H and FVB/n mouse livers (2 weeks, 6 weeks and 5 months). GF intoxication induced an increase in keratin levels in livers from both mouse strains Fig. . HSP70i,Total proteins from C3H and FVB/n livers were probed with antibodies against K8 pS79, K8 pS436 and K18 pS33 Fig. . SignifiWe analyzed at the cellular level, by double immunofluorescence staining, the distribution of HSP70i and IFs on liver sections of control and GF-treated C3H and FVB/n mouse livers.In control hepatocytes, IFs formed a complex cytoplasmic network that was denser at the cell membrane and particularly around the bile canaliculi Fig. . Our bioCryosections of control and GF-treated C3H and FVB/n mouse livers were fixed with 4% paraformaldehyde and processed for double immunofluorescence staining. As mentioned above, control mice showed hepatocytes with a cytoplasmic IF network, which was denser at the cell periphery Fig. . K8 pS79After 2 weeks of GF-treatment, hepatocytes were enlarged and an increase in cytoplasmic IF network was observed Fig. ,4,5C. ThIn the case of K18 S33, its phosphorylation was increased in most  hepatocytes. Most of the staining was observed at the periphery of the cells, delimitating clearly the bile canaliculi. A few hepatocytes showed high levels of cytoplasmic K18 pS33 Fig. .After 6 weeks of GF-treatment, the distribution of hepatocytes containing K8 pS79 and K8 pS436 was different from the one observed after 2 weeks of treatment. Clusters of labeled cells were smaller, whereas labeled isolated cells became more prominent Fig. ,4F. SingAfter 5 months of GF-treatment, MBs were present in some hepatocytes in both mouse strains Fig. ,4G,5G. MDouble immunostaining with anti-HSP70i and anti-phosphorylated keratins (K8 pS79 or K8 pS436) was performed for studying the localization of HSP70i in relation to keratin phosphorylation. The results showed that HSP70i and phosphorylated K8 species colocalized in some cells  or clusters of cells. Labeled singlet or doublet cells were more numerous after staining with the anti-K8 pS79 than after staining with the anti-K8 pS436. These cells could correspond to cells that are undergoing mitosis. This is supported by previous studies which showed that the phosphorylation of K8 on S79 and S436 occurs during mitosis ,48. ThisThe presence of K8 pS79 and K8 pS436 was also detected in islets of cells. Interestingly, both antigens were present in the same clusters of cells surrounding unstained cells that were most likely undergoing apoptosis. These unstained cells are evocative of detached cells during anoikis, an apoptotic process that can be induced by loss of cell-cell anchorage. Stress and apoptosis has been shown to modulate K8 S79 and K8 S436 phosphorylation ,48. The via K8. Our study show that colocalization of HSP70i and IFs occurs only in a few hepatocytes. Since the hepatocytes, in which colocalization was observed, contained K8 pS79 or K8 pS436, HSP70i binding to IFs in these cells may be related to the presence of keratin phosphorylation and participates to cellular pathways involving phosphorylated K8/18 on specific sites. Ku et al. [Liao et al.  have shou et al.  have shou et al. , HSP70i u et al. ,53. HoweChronic intoxication of mice with GF induces the formation of MBs. Numerous studies have demonstrated the presence of different phosphorylated K8/18 species within MBs, suggesting that K8/18 phosphorylation could participate in the MB formation processes ,54. In oThe treatment with GF that represents a toxic stress, most likely, involves the activation of stress activated protein kinases (SAPKs) in some hepatocytes. SAPKs p38 and JNK are physiologic kinases for K8 S79 and K8 S436 ,55. We pOur results show that increases in HSP70i, K8/18 expression and K8/18 phosphorylation constitute early events in the response of hepatocytes to the presence of GF. These observations support a role for keratins in preserving cellular integrity during stress conditions induced by the presence of a chemical agent ,35. HSP7ad libitum. GF-treated mice were fed a diet containing 2.5% (w/w) GF (Schering Corp., Kenilworth, NJ) for different periods of time: 2 weeks, 6 weeks and 5 months according to the method of Denk et al. [Experiments were performed with adult C3H mice (Charles River Canada, St-Constant, QC) and FVB/n mice  weighing 25 to 30 g. Two mouse strains were used to minimize the potential effect of different genetic background on the response of hepatocytes and to facilitate the interpretation of the data. All animals were housed with a 12-hour light-dark cycle and allowed the consumption of water and of a standard mouse semi-synthetic diet (Texlad Test Diet, Madison, WI), both k et al. . Control® Laboratories Canada, Burlington, ON) were used to perform immunolabelling with mAbs LJ4 and 5B3. The secondary antibodies used for Western blotting were as follows: biotinylated goat anti-rat IgG, biotinylated donkey anti-mouse IgG and peroxydase donkey anti-rabbit IgG (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON). Other reagents used were: Horseradish Streptavidin Peroxydase-conjugated (SPC) (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON), Bovine Serum Albumin (BSA) (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON), Leupeptin (Sigma-Aldrich Canada, Oakville, ON), Pepstatin (Sigma-Aldrich Canada, Oakville, ON), Aprotinin (Sigma-Aldrich Canada, Oakville, ON), Normal Horse Serum (NHS) (Vector® Laboratories Canada, Burlington, ON), Luminol (Amersham Pharmacia Biotech, Oakville, ON).The antibodies used were as following: Troma 1, a rat monoclonal antibody (rAb) that recognizes K8 ; LJ4, a ®) in PBS , incubated with the primary antibodies for 45 min, at room temperature, washed in PBS containing 0.2% (v/v) Tween 20 and incubated for 45 min with the appropriate secondary antibody: biotinylated goat anti-rat IgG (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON), biotinylated donkey anti-mouse IgG (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON) and horseradish peroxydase donkey anti-rabbit IgG (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON). When biotinylated secondary antibodies were used, membranes were washed with PBS-Tween 20 and incubated with streptavidin conjugated with horseradish peroxydase (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON) for 30 min and washed with PBS-Tween 20. The chemiluminescent horseradish peroxydase substrate Luminol (Amersham Pharmacia Biotech, Oakville, ON) was added to the membranes according to recommendations of the company, and membranes were exposed to Blue X-Omat X-ray film sheets (Mandel Scientific Company, Guelph, ON) to localize antibody binding.Livers were homogenized in 62.5 mM Tris-HCl, pH 6.8 containing 2.3 % (w/v) SDS, 50 mM sodium fluoride, 10 mM EDTA, 1 mM sodium pyrophosphate, 1 mM DTT, 1 mM PMSF, 1 μM leupeptin (Sigma-Aldrich Canada, Oakville, ON), 1 μM pepstatin (Sigma-Aldrich Canada, Oakville, ON), 2.5 μg/ml aprotinin (Sigma-Aldrich Canada, Oakville, ON). Proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels . Protein® Laboratories Canada, Burlington, ON) and an Avidin/Biotin blocking kit (Vector® Laboratories Canada, Burlington, ON) according to recommendations of the company. Normal horse serum (Vector® Laboratories Canada, Burlington, ON) was added to solution during incubation step with secondary antibody. For heat shock proteins staining, liver sections were incubated with anti-HSP 70i diluted in TBS containing 10% BSA for 45 min at 37°C, washed in TBS and incubated for 45 min at 37°C with a FITC conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON) diluted in TBS containing 10% BSA. For detection of HSP70i, K8 pS79 and K8 pS436, the sections were treated with 1% (v/v) Nonidet P-40 (Sigma-Aldrich Canada, Oakville, ON) following fixing step with 4% paraformaldehyde.Cryosections (4 μm) of fresh liver were fixed with 4% (w/v) paraformaldehyde in PBS pH 7.2 for 20 min, at room temperature, and rinsed in PBS or TBS  upon staining protocols. Since fixation can affect antibody-binding capacity, cryosections were also fixed with cold acetone (-20°C) for 10 min. For the detection of K8, sections were incubated with rAb Troma 1 at room temperature, washed in PBS and incubated with a FITC or a TRITC conjugated goat anti-rat IgG (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON) for 45 min, at room temperature. For immunostaining of K18 pS33, sections were incubated for 1 hour, at room temperature with anti-K18 pS33 (8250) diluted in PBS containing 10% (w/v) BSA, washed in PBS and incubated for 45 min with a FITC conjugated donkey anti-rabbit IgG in PBS containing 10% BSA (Jackson Immunoresearch, Bio/Can Scientific, Mississauga, ON). Immunostaining with anti-K8 pS79 (LJ4) and anti-K8 pS436 (5B3) mAbs, was done using the M.O.M. (mouse on mouse) detection kit (Vector® BX60 photomicroscope.The tissues were mounted in P-phenylene diamine diluted in 50% (v/v) glycerol. The slides were kept at -20°C and photomicrographs were collected using an OlympusHSP70i – inducible form of 70 kDa Heat shock protein. GF – griseofulvin. IFs – intermediate filaments. K8 – keratin 8. K8/18 – keratin 8 and keratin 18. K8 S79 – serine 79 on keratin 8. K8 pS79 – phosphorylated serine 79 on keratin 8. MBs – Mallory bodies.MF carried out all western blotting analyses, performed the immunofluorescence studies and participated in drafting the manuscript. LV participated in the design of the study. MC participated in the design of study, its coordination and drafting the manuscript. All authors read and approved the final manuscript.


Formins are multidomain proteins defined by a conserved FH2 (formin homology 2) domain with actin nucleation activity preceded by a proline-rich FH1 (formin homology 1) domain. Formins act as profilin-modulated processive actin nucleators conserved throughout a wide range of eukaryotes.Dictyostelium discoideum. With the exception of ForI and ForC all other formins conform to the domain structure GBD/FH3-FH1-FH2-DAD, where DAD is the Diaphanous autoinhibition domain and GBD/FH3 is the Rho GTPase-binding domain/formin homology 3 domain that we propose to represent a single domain. ForC lacks a FH1 domain, ForI lacks recognizable GBD/FH3 and DAD domains and ForA, E and J have additional unique domains. To establish the relationship between formins of Dictyostelium and other organisms we constructed a phylogenetic tree based on the alignment of FH2 domains. Real-time PCR was used to study the expression pattern of formin genes. Expression of forC, D, I and J increased during transition to multi-cellular stages, while the rest of genes displayed less marked developmental variations. During sexual development, expression of forH and forI displayed a significant increase in fusion competent cells.We present a detailed sequence analysis of the 10 formins (ForA to J) identified in the genome of the social amoeba Dictyostelium formins: all isoforms might display actin nucleation activity and, with the exception of ForI, might also be susceptible to autoinhibition and to regulation by Rho GTPases. The architecture GBD/FH3-FH1-FH2-DAD appears common to almost all Dictyostelium, fungal and metazoan formins, for which we propose the denomination of conventional formins, and implies a common regulatory mechanism.Our analysis allows some preliminary insight into the functionality of  Mice with mutant alleles fail to form proper limbs and kidneys [Diaphanous) [Eukaryotic cells rely on de novo nucleation mechanisms to generate actin filaments in order to elicit spatial and temporal remodeling of their actin cytoskeleton. Besides the Arp2/3 complex, nucleation activity has been recently demonstrated also for formins (reviewed in ). Forminphanous) ,5. Due tphanous) ).The FH2 (formin homology 2) domain is the defining feature of all formins. It is very well conserved and is almost invariably preceded by a proline-rich region, the FH1 (formin homology 1) domain ,7. In viIn most fungal and metazoan formins the FH1-FH2 core is accompanied by a less well conserved N-terminal FH3 (formin homology 3) domain involved in targeting . In planDictyostelium discoideum is an attractive model organism to investigate the components of the actin cytoskeleton and the signaling pathways involved in its regulation [Dictyostelium amoebae are equipped with a complex actin cytoskeleton that endows the cells with motile behavior comparable to that of human leukocytes. In fact, a genome-wide survey revealed that the repertoire of cytoskeletal components of Dictyostelium is more similar to metazoa followed by fungi than to plants . In Dictyostelium, nine formins have been previously identified but only three of them have been characterized to some extent [In vivo experiments with GFP fusions showed that the N-terminal region of ForC targets the protein to places of active actin reorganization, like macropinosomes, phagocytic cups and cell-to-cell contacts [The social amoeba gulation ,19. Dicte extent . Mutantscontacts .Dictyostelium sequencing projects in order to achieve a complete inventory of formin genes. A detailed sequence analysis of the 10 formins identified revealed that, with the exception of ForI and ForC, all other formins conform to the domain structure GBD/FH3-FH1-FH2-DAD present in almost all fungal and metazoan formins, for which we propose the denomination of conventional formins. Our sequence analysis also indicates that the GBD and FH3 domains constitute a single domain also found in two Dictyostelium RasGEFs (guanine nucleotide exchange factors). The expression pattern of formin genes during asexual and sexual development was studied using real-time PCR. Our analysis allows some preliminary insight into the functionality of Dictyostelium formins: all isoforms might display actin nucleation activity and, with the exception of ForI, might also be susceptible to autoinhibition and regulation by Rho GTPases.We have made use of the information released by the Dictyostelium [Dictyostelium we sought to exploit the available databases in order to achieve a complete inventory of formin genes in its entire length. The sequences already reported by Kitayama et al. [Dictyostelium genomic DNA database. This allowed assembly of complete genomic sequence for forA through forI. In order to verify the predicted amino acid sequence for each formin, Blast searches were performed against the Dictyostelium EST database. In cases where no EST sequences were available, like forG and forI, introns were verified after RT-PCR.In a previous publication 9 genes that potentially encode proteins of the formin family were identified in ostelium . For soma et al.  were useforJ, whose genomic sequence was also retrieved and inspected. Recent completion of the assembly of the Dictyostelium genome allowed us to confirm our gene predictions and map each formin gene to its corresponding chromosome locus . Formin genes are dispersed all over the six chromosomes (each chromosome harbors at least one formin gene), and in no case two or more genes are placed adjacent to each other  along with these two intron positions underscore the view that all Dictyostelium formin genes might have arisen from a common ancestor gene. After duplications and divergence from this ancestral formin gene introns were acquired or lost and additional domains and extensions were appended to some genes.Only two intron positions are conserved among es Figs.  and 4. IDictyostelium formins was determined by means of bioinformatics tools and visual inspection. Although formins vary considerably in length (935 residues of ForI versus 2546 of ForJ), with few exceptions they have in common a core of about 1100 residues that harbors a GBD/FH3-FH1-FH2-DAD structure characteristic of most fungal and metazoan formins  have one or more stretches of intervening repetitive sequences of variable length rich in Arg, Gln or Ser. Such repetitive sequences are characteristic of many Dictyostelium genes. The crystal structure of the FH2 domain of two formins, Bni1p and mDia1, has been recently solved [Dictyostelium formins in the context of these two structures. The FH2 domain fold is almost entirely α-helical. It is a stable dimer that forms a closed parallelogram-shaped ring. The structure of this domain can be subdivided into subdomains. At the N-terminus a so-called lasso is connected to a globular knob  but the important methionine residue (Met7) [Dictyostelium formins as well as in members of the FHOD (formin homology domain containing protein) and plant class1 subfamilies.All residues of the sequence motif GNY/FMN participate in dimerization. This motif is also highly conserved in almost all e (Met7)  is preseDictyostelium formins) and Arg5 (substituted by Lys in ForB and ForE). These residues were found mutated in temperature-sensitive yeast mutants [Also very conserved are some residues probably involved in binding to actin, like Ile3 (absolutely conserved) in the N-terminal subdomain and Lys9 in the post region (substituted by Arg in ForB and ForH). Mutation of these residues in Bni1p to Ala and Asp, respectively, abolished actin nucleation and barbed end capping activity of the FH2 domain , and rep mutants ,24, and  mutants .Dictyostelium formins, indicating that all ten formins might be functional actin nucleators.In summary, all essential residues in the FH2 domain revealed by structural and functional studies in metazoan and fungal formins are conserved in Dictyostelium and other organisms and to investigate whether different species share subfamilies of formins, we constructed a phylogenetic tree based on the alignment of complete sets of sequences of FH2 domains from selected organisms, including representatives of fungi, plants, invertebrates and vertebrates. We retrieved sequences of already characterized formins and additionally we made a search of further available sequences through the SMART server with the FH2 domain as query. Appart from the ten sequences of Dictyostelium, we collected a total of 62 sequences, 21 from plants, 5 from yeasts, 6 from D. melanogaster, 6 from C. elegans and 14 from human. Taking into account that some genes might not have been predicted accurately and that predicted proteins not supported by EST sequences were not considered for our analysis, further metazoan formins, especially from human, most probably went unidentified in our search.In order to establish the relationship between formins of C. elegans members are more divergent. The phylogenetic analysis reveals clustering of most formins into well defined classes. Yeast formins form a separate class whereas plant formins significantly group into any of two classes. Metazoan formins do not constitute a single cluster, rather they distribute into a number of subfamilies. The FHOD, Diaphanous and FMNL (formin in leukocytes) subfamilies have representatives in human, D. melanogaster and C. elegans. The Cappuccino/Formin and DAAM (Dishevelled-asssociated activator of morphogenesis) subfamilies, as well as a novel subfamily, is present in human and D. melanogaster, but seems to be absent in C. elegans. Delphilin constitutes a subfamily with a unique member present only in human. Finally, C. elegans has some additional divergent formins apparently unique to this organism.The phylogenetic tree Fig.  supportsDictyostelium formins are 45.5% similar  to each other, with ForC being only slightly more divergent (40.0%/20.4% similarity/identity to the rest of Dictyostelium formins). A comparable degree of similarity (identity) was found to members of several subfamilies of metazoan formins, and ranged between 40% (20%) and 48% (24%). Similarity (identity) to plant and yeast formins was lower: 38% (19%) and 36% (17%) respectively. Dictyostelium ForC and ForG cluster together with the Cappuccino/Formin group (75% bootstraps), whereas ForI very weakly clusters with the FHOD subfamily (53% bootstraps). However, taking into account that the FH2 domain is highly conserved, the position of these three Dictyostelium formins in the tree does not necessarily mean functional relationship with the mammalian counterpart, because other domains are probably responsible for diversity of localization and function. Bootstrapping does not support a significant clustering of the rest of the Dictyostelium formins, and only few members cluster together with a reasonably high number of bootstraps (ForE, D, A and F, 51% bootstraps).On average, Dictyostelium, with the notable exception of ForC. The length of the FH1 domains is very variable among formins (10 to >500 amino acids). It constitutes a binding site for the actin monomer binding protein profilin, as well as for SH3 and WW domain containing signaling proteins [Dictyostelium formis have a variable number of these motifs, between 1 in ForD and 8 in ForA , RasGEFs, fungal formins and members of the Diaphanous, DAAM, FMNL and FHOD subfamilies .We constructed a phylogenetic tree based on a multiple alignment of the GBD/FH3 domain of ies Fig. . With feain Fig. . In manyDictyostelium formins revealed a DAD in all members with the exception of ForI  or downstream of the FH2 domain (ForD and ForJ) that might constitute potential protein interaction sites with regulatory or targeting functions.Like metazoan and fungal formins, most nds Fig. . For exaDictyostelium formins have additional recognizable domains at their N-terminus  or gradually increased (forF) or decreased (forA, B and E) after the onset of development.The expression patterns observed during asexual development can be classified into two major groups. Expression of forH and forI displayed a significant increase of about 3-fold in fusion competent cells compared to fusion incompetent cells. Cells cultured in light submerged conditions have a reduced sexual fusion competency [forH and forI were enriched in fusion competent compared to light submerged cells, indicating that this enrichment is related to the acquisition of the fusion competence rather than to the submerged condition that was included to induce the fusion competence.When expression was analyzed during sexual development only mpetency ,33. In pDictyostelium, which in this organism comprises 10 genes. A comparison of the domain composition of formins from diverse phyla allows their grouping into four major classes , which includes all fungal and almost all metazoan formins. This is in agreement with a genome wide analysis that places Dictyostelium closer to fungi and metazoa than to plants .We have performed a detailed sequence and expression analysis of the formin family of ses Fig. . In geneWith very few exceptions all formins have in common a FH2 domain immediately preceded by a FH1 domain. The FH1-FH2 combination constitutes the minimal core that is fully functional in terms of actin nucleation and elongation activity . We therefore propose the use of the name conventional formins for those subfamilies with the general structure GBD/FH3-FH1-FH2, although in particular members or in alternatively spliced variants a domain (but never the FH2) might be absent, indicating whether the protein is Rho-regulated where documented experimentally. The name Diaphanous-related formin should be restricted to the metazoan members of the Diaphanous subfamily, like human DRF1 to 3.The designation Diaphanous-related formin has been applied to those formins that interact with activated Rho GTPases . HoweverDictyostelium formins is scarce. Only three isoforms, formins A, B and C have been characterized to some extent. Mutants lacking ForA, ForB or both showed no detectable phenotype, whereas deletion of forC led to formation of aberrant fruiting bodies with short stalks and unlifted sori, suggesting this formin mediates actin remodeling during multicellular stages [Dictyostelium formins are expected to be functional according to their highly conserved FH1-FH2 structure; therefore a certain degree of functional redundancy is expected. However, diversity might arise through specific targeting and activation by Rho GTPases conferred by the GBD/FH3 domain, through interaction of specific SH3-domain containing proteins with the FH1 domain and by virtue of unique additional domains. These issues need to be addressed experimentally in the future.Functional data on r stages . DictyosForC and ForI might be exceptions in terms of regulation. ForC lacks a FH1 domain and consequently does not bind to profilins . AlthougDictyostelium formins have domains at their N-termini that are not found in other formins and might confer unique additional functions or ways of regulation or targeting. The C2 and C1 domains of ForA and ForE, respectively, might regulate activation or targeting of the molecule through interaction with specific lipids [Three c lipids ,30, whilc lipids . In genec lipids ,16 and tforC, D, I and J, displayed an increase in expression during transition to multi-cellular stages. During this phase cells acquire aggregation competence in parallel with maturation of signaling pathways involved in remodeling of the cytoskeleton. At least for forC gene expression data correlate with a developmental role, as mentioned above [rac1b and racF2 was found increased during the analysis of a gamete-enriched cDNA library [Functional diversity might also be related to different patterns of local and temporal gene expression. Our gene expression analyses also suggest specific roles during asexual and sexual development. Four genes in particular, ed above . ForH an library . It is tDictyostelium discoideum expresses 10 formins that with few exceptions conform to the domain structure GBD/FH3-FH1-FH2-DAD. This arhitecture and the high degree of conservation of the FH2 domain allow some preliminary conclusions about the functionality of Dictyostelium formins: all isoforms may display actin nucleation activity and, with the exception of ForI, may also be susceptible to autoinhibition and to regulation by Rho GTPases. Although functional redundancy may be expected to occur to some extent among Dictyostelium formins, specific roles may be conferred by the GBD/FH3 domain, which is less well conserved than the FH2 domain, and by specific patterns of gene expression during asexual and sexual development.The social amoeba Dictyostelium, fungal and most metazoan formins can be grouped within the class of what we designate conventional formins, characterized by the structure GBD/FH3-FH1-FH2-DAD. The GBD and FH3 domains, whose boundaries had not been defined previously, probably constitute a single domain. The architecture shared by conventional formins implies a common regulatory mechanism based on autoinhibition through intramoleculr interaction of the GBD/FH3 and the DAD domains and activation through release of this interaction upon binding of Rho GTPases. Formins of the other classes (plant formins and Delphilin) lack GBD/FH3 and DAD domains and must therefore have other mechanisms of activation.We propose four major classes of formins based on a comparison of the domain composition of proteins from diverse phyla. Note. While our manuscript was under review a phylogenetic analysis of the FH2 domain by H. N Higgs and K. J. Peterson has been published. These authors used a larger set of FH2 domains that includes only three formins from Dictyostelium. The topology of the phylogenetic tree described in that article and that of our tree are essentially coincident, and all seven metazoan groups identified by their authors can be found in our tree, with the novel subfamily INV comprising our HsKIAA1727, DmAE003560 and CeAF106580 sequences. Higgs and Peterson, however, do not recognize the GBD/FH3 region as a domain present in a larger number of formin subfamilies.Dictyostelium formins were used as query for BLAST searches [Dictyostelium genome project databases at The Welcome Trust Sanger Institute, Baylor College of Medicine, The University of Cologne and the Department of Genome Analysis of the Institute of Molecular Biotechnology in Jena. Nearly all of this data was generated at the aforementioned institutes with a small part of it produced at the Institute Pasteur. After assembly of the genome further analyses were performed through the Dictybase server [Dictyostelium formins can be found in Table The amino acid or DNA sequences of searches  of the De server . BLAST se server . AccessiS. cerevisiae Bni1p, P41832; Bnr1p, P40450; S. pombe Fus1, L37838; Cdc12, 786133; For3, AL035247. D. melanogaster Cappuccino, U34258; Diaphanous, U11288; FHOD, AE003554; FMNL, BT003654; DAAM, AAF45601; a novel formin, AE003560. C. elegans FHOD, U88314; Cyk-1, U40187; FMNL, AC024798; novel formins, Z78013, AF106580 and Z22174. H. sapiens Formin 1, AK127078; Formin 2, XM_351329; FHOD1, AF113615; FHOD3/FHOS2, KIAA1695; DRF1, AF05187; DRF2, Y15909; DRF3, BC034952; Delphilin, XM_353725; FMNL1, AF432213; FMNL2/FHOD2, KIAA1902; WBP3/FMNL3, NM_175736; DAAM1, NM_014992; DAAM2, AL833083; a novel formin KIAA1727. Sequences of plant formins were obtained from Cvrčková et al. [Dictyostelium RasGEF-L and RasGEF-V can be accessed at Dictybase [Accession numbers of the sequences retrieved for phylogenetic analyses are as follows. Protein sequences were aligned using the ClustalX  program D. discoideum AX2 strain was grown at 21°C in shaking suspension in axenic HL5 medium [K. aerogenes in BSS either in the darkness or in the light. Cells cultured for 15 hours in the dark become fusion-competent cells. However, cells cultured for 15 hours in the light condition exhibit reduced fusion competency, and are designated light submerged cells. Cells on SM agar plates [5 medium . AX2 cel5 medium . In brier plates  are fusiTotal RNA was purified from both asexually and sexually developing cells with the TRIZOL reagent (GIBCO BRL, USA). Asexually developing cells on phosphate agar plates  were colFor RT-PCR, first strand cDNA synthesis was performed with M-MLV reverse transcriptase (Promega Corporation, Madison, WI) on poly A+ mRNA purified with the Oligotex system (Qiagen GmbH, Hilden, Germany) from total RNA. PCR fragments were cloned into the pGEM-T Easy vector system (Promega Corporation, Madison, WI) and sequenced. DNA sequencing was done at the service laboratory of the Center for Molecular Medicine, Cologne, using an automated sequencer .FR conceived the study, performed the assembly of genomic sequences and the sequence alignments and drafted the manuscript. TM and HU carried out the gene expression studies. AKM performed RT-PCR and cloning. CK and TQPU participated in the design of the study and the assembly of genomic sequences. All authors read and approved the final manuscript.


Anxiety and depression co-occur in children and adolescents with anxiety commonly preceding depression. Although there is some evidence to suggest that the association between early anxiety and later depression is explained by a shared genetic aetiology, the contribution of environmental factors is less well examined and it is unknown whether anxiety itself is a phenotypic risk factor for later depression. These explanations of the association between early anxiety and later depression were evaluated.Anxiety and depressive symptoms were assessed longitudinally in a U.K. population-based sample of 676 twins aged 5–17 at baseline. At baseline, anxiety and depression were assessed by parental questionnaire. Depression was assessed three years later by parental and adolescent questionnaire.Shared genetic effects between early anxiety and later depression were found. A model of a phenotypic risk effect from early anxiety on later depression provided a poor fit to the data. However, there were significant genetic effects specific to later depression, showing that early anxiety and later depression do not index entirely the same genetic risk.Anxiety and depression are associated over time because they share a partly common genetic aetiology rather than because the anxiety phenotype leads to later depression. Anxiety and depressive disorders are some of the most common psychiatric diagnoses in children and adolescents respectively. Both are known to be associated with major impairment in childhood and adverse consequences in later life -4. EstimDepression and anxiety co-occur more commonly than would be expected by chance in children and adolescents. This co-occurrence has been identified both in clinical studies of children and adolescents and general population samples that have examined sub-clinical levels of depression and anxiety symptoms ,17. MoreCross-sectional twin studies of children ,29 and aIn the present study, we set out to examine two hypotheses that may explain the observed associations between early anxiety and later depression.1. Early anxiety symptoms and later depression symptoms are associated because of shared risk factors.2. The association between anxiety symptoms and later depression symptoms is mediated by a risk effect of the phenotype of anxiety.Families from a systematically ascertained, population-based register of all twin births between 1980 and 1991 in South Wales, U.K. were invited to participate. This register forms a sub-sample of the Cardiff Study of All-Wales and North West of England Twins (CASTANET). Twins who had emigrated were excluded, as were cases in which one of the twins had died or had a serious illness. At the first wave of data collection in 1997, there were a total of 1109 pairs of twins aged 5–17 years although not all of these individuals were eligible to participate at both time points (see below). Data were collected by postal questionnaire. Families received three reminders and telephone reminders when numbers could be traced. The same methods were used three years later to collect longitudinal data except that families received four reminders. To be invited to participate in the follow-up study, we required that the twins were living together in the same home and were under the age of 18 years. Twins were required to live in the same home in order to minimise heterogeneity of environmental risk factors that can impact on genetic and environmental parameter estimates. The focus of the follow-up study was childhood psychopathology and for that reason young people aged 18 and over were not included. At time 1, there were 986 twin pairs who were within the age range of the study at both time points.In the first wave of the study , 670 families provided questionnaire responses giving a response rate of 61%. Comparison of responders and non-responders using Townsend Scores  which inOf these, 338 families replied, giving a total response rate of 75%. There were no significant socio-demographic differences between responders and non-responders at time 2 (t = 1.71, p = 0.09). Zygosity was assigned using a twin similarity questionnaire which has been shown to be over 90% accurate in distinguishing identical  from fraternal  twins [At time 1, parents were asked to complete the Children's Revised Manifest Anxiety Scale  which asFor descriptive statistics, (correlations and mean comparisons), the survey commands in the program STATA  were use2 critical value for the number of degrees of freedom gained in the reduced model.Analysing data from twins provides a means of estimating the relative contribution of genetic and environmental effects on individual variation in behaviour. In the basic (ACE) model, variation can arise from three sources: 1) additive genetic effects (A); 2) common environmental effects (C); 3) unique environmental effects (E). Common environmental effects are non-genetic factors that serve to make twins more similar to one another while unique environmental effects are non-genetic factors that uniquely influence one individual within a twin pair and tend to make the individuals in a twin pair different from each other. Model fitting was carried out using the programs Mx  and LISRBivariate analysis allows the covariance of two traits to be partitioned into covariance that is due to additive genetic factors, common environmental factors and unique environmental factors. The covariance parameters for the Cholesky model presented . Qualitative sex differences test whether the set of genes influencing the phenotype differs by gender i.e. different genes , this is done by estimating the genetic correlation for opposite sex DZ pairs and comparing the fit of this model to one that constrained the genetic correlation to 0.5. In addition, a bivariate sex limitation model was tested . This moThere were no significant mean differences on anxiety or depressive symptoms by gender . Age was not associated with anxiety or depressive symptoms . The mean age of children at time 1 was 10.58, range 5.58–17.83, and at time 2, was, 12.64, range 8.75–17.25. Symptoms of early anxiety and later depression were strongly correlated (parent-rated symptoms r = .479. The correlation was slightly lower for symptoms across rater (parent rated anxiety and adolescent rated depression), r = .335.2 = 1.505, Δ df = 3), nor were there qualitative genetic differences (Δ χ2 = 0, Δ df = 1). For depression, univariate models for parent-rated and self-rated scores indicated no significant gender effects for the magnitude of genetic effects  nor qualitative gender differences . Finally, for parent rated symptoms, results from the bivariate sex limitation Cholesky model showed no significant gender differences in the covariation between anxiety and depression in that the genetic and environmental covariation could be equated across the genders with no significant deterioration in fit (parent-rated, Δ χ2 = 0.445, Δ df = 3). However, for self-rated symptoms, one parameter, i.e., the non-shared environmental covariation parameter, could not be equated across the genders (Δ χ2 = 6.107, Δ df = 1). Estimates from this model for boys were; Aanx = 50, Canx = 17, Eanx = 32, Ac = 10, Cc = 47, Ec = -11, Adep = 9, Cdep = 5, Edep = 40; and for girls were; Aanx = 47, Canx = 22, Eanx = 30, Ac = 12, Cc = 13, Ec = 8, Adep = 30, Cdep = 12, Edep = 24; χ2 = 30.93, df = 32, AIC = -33.07). These results are presented in addition to those for the combined sample  = 11, p = .001).Table It has been shown that the aetiology of depression varies significantly according to age, with greater common environmental effects in children aged 8–10 than in adolescents aged 11–17 ,46,47. TFollowing this finding of significant common environmental covariation the nature of this latent factor was further examined. Information on a potential common environmental variable, general family functioning  was available. Family functioning was correlated with anxiety symptoms at time 1 (r = .210, p = .001). Bivariate analyses controlling for family functioning are shown in table 2 = 48.82, Δ df = 1, p < .001). Thus, it does not appear that anxiety leads to depression through direct phenotypic effects, but that, anxiety and depression symptoms are associated over time because they share aetiological factors in common.Finally, a model that included a direct causal path from anxiety to depression was fitted  a common genetic/environmental aetiology or 2) a phenotypic risk effect of early anxiety. There was significant genetic covariation between anxiety and later depression. (Moreover, the genetic covariation was not explained by the effects of early depression). This result is consistent with the association between early anxiety and later depression being due to a common genetic aetiology. However, the genetic overlap between early anxiety and later depression was far from complete in that there were significant, separate genetic effects on anxiety and genetic influences specific to later depression. Thus, in this sample, symptoms of anxiety and depression in children and adolescents share only a partly common genetic aetiology.In addition to genetic covariation, significant shared environmental covariation between anxiety and depression was also observed. It appeared that some of the shared environmental covariation between anxiety and depression observed in parent ratings of anxiety and depression was due to shared rater effects as the common environmental covariation (Cc) was no longer significant when analyses were performed with data across different informants. However, including family adversity as a measured environmental risk factor into a model with data from different informants also resulted in a small decrease in the Cc parameter estimate.The model that included a direct causal path from early anxiety symptoms to later depression symptoms provided a significantly poorer fit than the general bivariate model. These results suggest that anxiety is not an aetiologically distinct phenotype that is in itself a risk factor for future depression symptoms, but rather that the covariation over time arises from the common genetic and environmental architecture. It should be noted though, that some of the common environmental covariation is likely due to shared rater effects, because we found that this path became non significant in cross-informant analyses.The present findings are consistent with those of several other twin studies that have reported strong genetic correlations between symptoms of anxiety and depression in children and adolescents ,29,31 anThe lack of significant univariate and bivariate gender differences in genetic and environmental parameters estimates for parent reports in the present sample is of interest. The results for self reports of depression are less clear, previous analysis of a larger sample, from which the present sample was drawn, did find sex differences for self rated depressive symptoms as measured by the long version of the Mood and Feelings Questionnaire (MFQ) , which wHow do the present findings fit with results from family studies? Several family studies of the offspring of depressed parents have found increased rates of anxiety rather than depressive disorders ,25 and RAs mentioned previously, several groups have shown that the aetiology of depressive symptoms differs between children (8–10) and adolescents (11–17) ,46,47. TAnxiety and depressive symptoms are strongly associated over time. This association does not appear to be due to a phenotypic risk effect of early anxiety. Rather, early anxiety and later depression are associated due to a common aetiology. This was primarily a common genetic aetiology although family functioning and a single informant rating on both sets of symptoms also contributed to this association. Some of the common genetic aetiology may act as indirect genetic effects via behaviour (gene-environment correlation and gene-environment interaction).The author(s) declare that they have no competing interests.AT and FR conceived the paper. FR carried out statistical analysis and wrote the paper. AT and MBM wrote and edited the paper. MBM provided statistical support. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Teaching -- and testing -- students creatively is a challenge, but new public databases and more accessible literature are now helping to develop the critical thinking skills of students The next generation of life scientists are currently undergraduates—and the success of this generation depends upon the quality of the education they receive. It is clear the expectations for undergraduate education are changing . When thEarly in my teaching career, I discussed graduate student preparation with a colleague at MIT. He said new graduate students knew about the different methods, they could even recite fine definitions—but if you asked them which method would be best to answer a particular question, they were uncertain. This reinforced my attitude towards teaching and testing. I realized that teaching science to students should be modeled on the way all scientists learn new information: in the context of an interesting question and on a need-to-know basis. This new style of teaching, “applied education,” would require me to reorganize reading materials for students, since most textbooks are written by someone who already knows all the information and has organized it accordingly. For example, describing membrane structure, protein structure, and signal transduction in Chapters 5, 12, and 15, respectively (spanning 227 pages) is not helpful for most students. It makes more sense to cover these three topics in close succession.www.bio.davidson.edu/courses/Molbio/Publicschedule.html#anchor99574051). So, for example, in my classes students first read the elegant paper by www.bio.davidson.edu/courses/Molbio/molecular.html#2003exams). Based on this success, I have designed my genomics course on the “applied education” principle .Gradually, I converted all my courses over to this “applied education” format in which students were learning new information the same way all other scientists do. I began by asking questions that could be answered by learning the information provided by textbooks or the literature. With time, I realized that published research papers are ideal teaching tools because they cover information in the context of an interesting question and new material is presented as needed. This led me to collect series of related papers to create my own course materials  is a rich repository of and portal to open access articles, BioMed Central  publishes a growing number of open-access journals, and there are a few new open-access education journals such as Cell Biology Education (http://www.cellbioed.org) and the Journal of Undergraduate Neuroscience Education . As the newest player in the open-access arena, PLoS Biology has further enriched the growing espritdes-corps of publishing and has already improved undergraduate education. My students now have equal access to a growing portion of the literature that Nobel laureates and investigators at wealthy institutions enjoy.When I was a graduate student (in the late 1980s and early 1990s), PubMed was restricted to those institutions that could afford the subscription fee; now PubMed is freely available to all who have Internet access. This change in access to PubMed has significantly improved undergraduate training by providing students with the opportunities to do literature searches for their lab reports, papers, seminars, and of course original research. Free access to information in the life sciences has continued to evolve with the newest phenomenon in publishing—open-access journals. PubMed Central  is forced to make difficult choices about which journals we can afford. The number of journal subscriptions goes down in proportion to the rise of subscription costs, but fortunately this loss is being offset by the creation of new open-access journals.http://www.bio.davidson.edu/molecular). Now I use PDF files for students to print and for me to display in class with no loss of information due to reformatting or resolution problems  in which I capitalize on a confluence of two trends in the field: public domain databases and open-access journals (www.bio.davidson.edu/courses/genomics/2003/cain/home.html). But genomics courses are not the only beneficiaries, since other classes at many institutions (e.g., introductory biology, biochemistry, cell development, genetics, microbiology, molecular biology [see http://www.bio.davidson.edu/courses/Molbio/standardsHP.html#anchor78181983], and neuroscience) require students to mine public domain databases (www.bio.davidson.edu/people/macampbell/Hope/DQ/DQ9.html and www.bio.davidson.edu/people/macampbell/Hope/DQ/DQ10.html). First-year students use Genome Browser and BLAST to determine the molecular causes of cystic fibrosis and Huntington disease, respectively. The benefit of public databases and open-access literature to educators is obvious and immediate. Images can be used in lectures, and papers can be distributed easily and on short notice for class use. There is no need to worry about limited access due to subscription costs nor an obligation to obtain copyright permission from publishers, which is a bothersome and sometimes expensive process for busy faculty members. By reducing nonproductive busy work for faculty, open-access journals have already created an environment that is improving undergraduate education today with long-term benefits in creating research-ready graduate students.During the past three years, I have taught an undergraduate course in genomics . Open-access journals make these two educational goals much more feasible because students can utilize current findings immediately without having to wait for interlibrary loans, which can take up to two weeks, can cost up to $20 per article, and can result in poor-quality black-and-white photocopies.Two common educational goals are to encourage students to become skeptical of unsubstantiated claims and to enable students to evaluate data critically. One way to accomplish these goals is to capitalize on the natural curiosity of students and ask them to compare topics in the popular press to that in the scientific literature . Students were asked to combine what they learned from the paper and the course and choose new proteins . As a result of these professional interactions, students became participants in a community of scholars, interacting with investigators at the University of California, San Francisco, while taking their exams.At the end of their exam, students were given an opportunity for extra credit points (a maximum of three points out of 100 available on the exam) if they provided constructive criticism directly to the database curators. About 70% of the students sent comments, including this one: “In recently using your database, I found it difficult to search the The use of open-access journals for teaching and testing has already improved my courses. I can provide exam questions that are more interesting, more educational, and more current. Furthermore, I accomplish two tasks simultaneously: I keep abreast of new developments in my field and I write exam questions. But what do students think? While I have not formally assessed student attitudes, I have collected information from end-of-semester course evaluations, including the following comments: “One of the best parts of the entire course for me were the exams. The exams really gave me an opportunity to show how I could work through real problems. This class definitely increased my critical thinking skills. Each test presented me with new ideas and problems to work through. I enjoyed the idea that each exam would be a learning experience.”PLoS Biology, my students benefit from free access to published research results. Free access to research literature enhances student learning and helps produce the next generation of graduate students, who are then better trained. Open-access publishing provides the right mix of benefits for educators and students alike.Teaching is a lot like raising children. Like parents, teachers provide learning opportunities in part by modeling the behavior we want our students to learn. By choosing the most current literature as testing material, my students realize that I read the literature to stay current in my field and that there are always new opportunities to learn, analyze, and design experiments, etc. By my choosing open-access papers such as those published in