Tubulin is actively exported from the nucleus through the Exportin1/CRM1 pathway.

Microtubules of all eukaryotic cells are formed by α- and ß-tubulin heterodimers. In addition to the well known cytoplasmic tubulins, a subpopulation of tubulin can occur in the nucleus. So far, the potential function of nuclear tubulin has remained elusive. In this work, we show that α- and ß-tubulins of various organisms contain multiple conserved nuclear export sequences, which are potential targets of the Exportin 1/CRM1 pathway. We demonstrate exemplarily that these NES motifs are sufficient to mediate export of GFP as model cargo and that this export can be inhibited by leptomycin B, an inhibitor of the Exportin 1/CRM1 pathway. Likewise, leptomycin B causes accumulation of GFP-tagged tubulin in interphase nuclei, in both plant and animal model cells. Our analysis of nuclear tubulin content supports the hypothesis that an important function of nuclear tubulin export is the exclusion of tubulin from interphase nuclei, after being trapped by nuclear envelope reassembly during telophase.

Path selection rules for droplet trains in single-lane microfluidic networks.

We investigate the transport of periodic trains of droplets through microfluidic networks having one inlet, one outlet, and nodes consisting of T junctions. Variations of the dilution of the trains, i.e., the distance between drops, reveal the existence of various hydrodynamic regimes characterized by the number of preferential paths taken by the drops. As the dilution increases, this number continuously decreases until only one path remains explored. Building on a continuous approach used to treat droplet traffic through a single asymmetric loop, we determine selection rules for the paths taken by the drops and we predict the variations of the fraction of droplets taking these paths with the parameters at play including the dilution. Our results show that as dilution decreases, the paths are selected according to the ascending order of their hydrodynamic resistance in the absence of droplets. The dynamics of these systems controlled by time-delayed feedback is complex: We observe a succession of periodic regimes separated by a wealth of bifurcations as the dilution is varied. In contrast to droplet traffic in single asymmetric loops, the dynamical behavior in networks of loops is sensitive to initial conditions because of extra degrees of freedom.

[Programming nutritional and metabolic disorders: the diabetic environment during gestation]. / Déterminisme des troubles nutritionnels et métaboliques : impact de l'environnement diabétique durant la gestation.

During the last years, obesity and subsequent metabolic disorders and cardiovascular diseases have tremendously increased. Recent studies have shown that risk factors of cardiovascular diseases appear as soon as in infancy. In many situations, these disorders are programmed in early life during fetal development. These observations have lead to the concept of programming. The first studies on this subject underlined the link between poor fetal growth and the risk of nutritional and metabolic disorders during adulthood. But, it is now evident that excess of fetal growth as it is observed during pregnancy with maternal diabetes leads to the same consequences. The metabolic syndrome or syndrome X is the name for a clustering of risk factors for cardiovascular diseases and type II diabetes that are of metabolic origin. This syndrome, first described in the adults, is more and more studied during childhood and adolescence. Metabolic syndrome is now described in youth, particularly in subjects with risk factors as obesity. Alterations of intra-uterine environment lead to modified early development and represent short-term adaptations transmitted from one generation to another. This intergeneration effect contributes to the burden of adult metabolic disorders and cardiovascular diseases, as seen in the last decades. There is considerable evidence for the contribution of epigenetic mechanisms for the lifelong and the intergenerational alteration of gene transcription by variation in the early life environment. One of the major challenges in the following years is to promote public health programs which are aimed at prevention of long-term consequences of fetal programming.

Complications of endoscopic retrograde cholangiopancreatography during live endoscopy workshop demonstrations.

BACKGROUND AND STUDY AIMS: Endoscopy workshops are thought to be associated with larger numbers of complications than routine clinical treatment. In this study, patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) during live demonstrations were compared with matched patients treated in an ERCP unit. PATIENTS AND METHODS: Patients who underwent ERCP during workshops over a 12-year period were reviewed. The control for each patient was the next patient admitted to the same ERCP unit with similar indications. Possible delays before treatment, ERCP indications, the use of general anesthesia, standard endoscopic and special treatments, success and complication rates for ERCP, prolonged hospitalization periods, and financial benefits for patients were assessed. RESULTS: A total of 168 workshop patients and 168 control patients were compared. ERCP was delayed in 18 patients to allow treatment during the workshops. General anesthesia was used in 87.5 % of the workshop patients, in comparison with 44 % of the control patients (P < 0.001). The duration of the endoscopies and radiation exposure did not differ, and the endoscopic treatments carried out also did not differ significantly, with the exception of cholangiopancreatoscopy (7 % in the workshop group versus 0 %; P < 0.01). The success and complication rates were similar in the workshop and control patients, as was the duration of hospitalization. Among the patients treated during workshops, 45 % benefited financially, as they were not charged for stents or other devices. CONCLUSIONS: These results suggest that, in this setting, ERCP performed during live demonstrations is safe and raises no major ethical problems.

Coat proteins of Rice tungro bacilliform virus and Mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin alpha.

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant pararetro- and geminiviruses. In both these virus types, the multifunctional coat protein (CP) is thought to be involved in this process. Here, a green fluorescent protein tagging approach was used to demonstrate nuclear import of the CPs of Rice tungro bacilliform virus (RTBV) and Mungbean yellow mosaic virus--Vigna (MYMV) in Nicotiana plumbaginifolia protoplasts. In both cases, at least two nuclear localization signals (NLSs) were identified and characterized. The NLSs of RTBV CP are located within both N- and C-terminal regions (residues 479KRPK/497KRK and 744KRK/758RRK), and those of MYMV CP within the N-terminal part (residues 3KR and 41KRRR). The MYMV and RTBV CP NLSs resemble classic mono- and bipartite NLSs, respectively. However, the N-terminal MYMV CP NLS and both RTBV CP NLSs show peculiarities in the number and position of basic residues. In vitro pull-down assays revealed interaction of RTBV and MYMV CPs with the nuclear import factor importin alpha, suggesting that both CPs are imported into the nucleus via an importin alpha-dependent pathway. The possibility that this pathway could serve for docking of virions to the nucleus is discussed.

A prospective, randomized multicenter trial comparing DoubleLayer and polyethylene stents for malignant distal common bile duct strictures.

BACKGROUND AND STUDY AIMS: Endoscopic biliary stenting is an established treatment for malignant obstructive jaundice. Stent clogging continues to be a major problem with plastic stents. The aim of this study was to carry out a prospective comparison of two stents with different materials and shapes: the Olympus DoubleLayer stent (DLS; perfluoro alkoxy, without sideholes) and the standard polyethylene (PE) stent (with sideholes). PATIENTS AND METHODS: A total of 120 patients (70 women; mean age 71, range 36 - 91) with jaundice due to malignant strictures of the middle to distal third of the common bile duct were randomly assigned to receive either DLS (n = 60) or PE (n = 60) biliary stents. Patients with cholangitis, hemobilia, previous biliary drainage, hilar stricture, or ampullary cancer were excluded. RESULTS: In all, 28 DLS patients (47 %) and 17 PE stent patients (29 %) died without clinical evidence of stent occlusion after a mean of 114 and 105 days, respectively ( P < 0.05). Twenty-six DLS patients (43 %) and 38 PE stent patients (63 %) had symptoms of stent clogging after a mean of 144 and 99 days, respectively ( P < 0.05). Stent dysfunction (stent orifice impacted on the bile duct or duodenal wall, stent migration) was recorded in six DLS patients (10 %) and five PE patients (8 %) (n. s.). Kaplan-Meier analysis of DLS and PE stent clogging-free survival showed a significantly longer patency period with the DLS stents (P = 0.0005). CONCLUSIONS: These results show that DoubleLayer stents have a longer patency period than PE stents. Patients who received PE stents had a higher risk of stent occlusion (relative risk 3.05; 95 % CI, 1.57 - 5.89) before death than DLS patients.

Clinical, social and rehabilitation status of long-term home parenteral nutrition patients: results of a European multicentre survey.

BACKGROUND: Home parenteral nutrition (HPN) is a lifesaving treatment in patients with intestinal failure. Dependency of nutritional support becomes permanent for the majority of patients who had received HPN for at least 2 years. The alternative to long term HPN in selected patients is intestinal transplantation. AIMS: To study some of the clinical, social and rehabilitation aspects of long-term HPN treatment. METHODS: A survey was performed in nine European HPN centres. The questionnaire covered epidemiologic data, underlying diseases, intestinal anatomy, nutritional support and status, marital status, rehospitalization rate, HPN complications, rehabilitation score, drugs use, coexistent diseases and interest in intestinal transplantation. For some items, data were collected within 12 months prior to the evaluation. RESULTS: This survey included 228 patients with a median age of 49 years. The median duration of HPN was 7 years (range 2--24 years). Short bowel length less than 100 cm was reported in 65% of patients with a predominance of end-jejunostomy or jejuno-colonic anastomosis. Global subjective nutritional status was normal in 79% of the patients, who were supplied with a mean number of 5.6 bags of parenteral nutrition weekly. Rehospitalizations within the 12 months prior to evaluation accounted for a mean period of 23 days and were due to HPN complications in half of the cases. Catheter-related sepsis was the most frequent HPN-complication. Bone metabolism disorders, which seemed to be more common than liver diseases, were directly related to HPN duration. One-third of the HPN patients was regularly consuming analgesics or opiates. HPN impair complete rehabilitation status but may improve the status of patients who had a very low rehabilitation score before starting HPN. An interest of intestinal transplantation was noted in only 10% of medical teams and in 8% of HPN patients. CONCLUSIONS: This study is the largest European survey on long-term HPN patients with long standing or permanent intestinal failure. Data enlighten clinical, social and rehabilitation aspects of patients who could face the option of intestinal transplantation in the future.

Differential mRNA degradation of two beta-tubulin isoforms correlates with cytosolic Ca2+ changes in glucan-elicited soybean cells.

Transgenic soybean (Glycine max) culture cells expressing apoaequorin, a Ca2+ indicator, were exposed to glucan fragments derived from Phytophthora sojae or to chitin oligomers. The effects of these elicitors on cytosolic Ca2+ concentrations and on mRNA levels of two beta-tubulin isoforms, tubB1 and tubB2, were investigated. The glucan elicitors, to which the cells are known to react with a biphasic cytosolic Ca2+ increase, induced a down-regulation of the tubB1 mRNA levels while the tubB2 mRNA level remained constant. The decrease of tubB1 mRNA level was observed after 1 hour of glucan treatment. In contrast, chitin oligomers, known to provoke a monophasic Ca2+ increase of short duration, did not affect the tubB1 mRNA level. Pre-incubation with 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an extracellular Ca2+ chelator, blocked the cytosolic Ca2+ increase as well as the decrease of tubB1 mRNA levels induced by glucan elicitors. Likewise, pre-incubation with 1 mM neomycin, which reduced only the second glucan-induced Ca2+ peak, blocked the decrease of tubB1 mRNA level. Experiments with cordycepin, a transcription inhibitor, indicated that glucan fragments induced the degradation of tubB1 mRNA. In conclusion, the glucan-induced cytosolic Ca2+ changes are correlated with a strong increase in tubB1 mRNA degradation.

Higher plant cells: gamma-tubulin and microtubule nucleation in the absence of centrosomes.

The assembly of the higher plant cytoskeleton poses several fundamental questions. Since different microtubule arrays are successively assembled during the cell cycle in the absence of centrosomes, we can ask how these arrays are assembled and spatially organized. Two hypotheses are under debate. Either multiple nucleation sites are responsible for the assembly and organization of microtubule arrays or microtubule nucleation takes place at one site, the nuclear surface. In the latter case, microtubule nucleation and organization would be two distinct but coregulated processes. During recent years, novel approaches have provided entirely new insights to understand the assembly and dynamics of the plant cytoskeleton. In the present review, we summarize advances made in microscopy and in molecular biology which lead to novel hypotheses and open up new fields of investigation. From the results obtained, it is clear that the higher plant cell is a powerful model system to investigate cytoskeletal organization in acentrosomal eukaryotic cells.

Diversion of intestinal flow decreases the numbers of interleukin 4 secreting and interferon gamma secreting T lymphocytes in small bowel mucosa.

BACKGROUND/AIMS: The intestinal immune system faces large amounts of antigens, and its regulation is tightly balanced by cytokines. In this study, the effect of intestinal flow diversion on spontaneous secretion of interleukin (IL)-4 and interferon (IFN)- gamma was analysed. METHODS: Eight patients (two with Crohn's disease, four with ulcerative colitis, and two with previous colon cancer) carrying a double lumen small bowel stoma after a total colectomy procedure were included in the study. For each patient, eight biopsy samples were taken endoscopically from both the diverted and non-diverted part of the small bowel. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were isolated separately and assayed for numbers of cells spontaneously secreting IL-4 and/or IFN-gamma by an ELISPOT technique. RESULTS: Compared with the non-diverted mucosa, a significant decrease in the number of spontaneously IFN-gamma secreting CD3 lymphocytes was observed in the diverted small bowel mucosa among both IELs (p = 0.008) and LPLs (p = 0.007). The same results, although less significant, were obtained for IL-4, especially in LPLs (p = 0.01). CONCLUSION: The intestinal content influences the spontaneous secretion of IFN-gamma and IL-4 by intestinal lymphocytes. These results could help to elucidate the anti-inflammatory role of split ileostomy in patients suffering from inflammatory bowel diseases.

"Missed" upper gastrointestinal tract lesions may explain "occult" bleeding.

BACKGROUND AND STUDY AIMS: Enteroscopy has been shown to be an effective diagnostic method in patients with obscure gastrointestinal bleeding. Arteriovenous malformations (AVMs) of the small bowel are the most common lesions discovered at enteroscopy. However, bleeding lesions may still be detected in the upper gastrointestinal tract even in patients who have previously undergone esophagogastroduodenoscopy. The aim of this study was to focus on these "missed" upper gastrointestinal lesions. PATIENTS AND METHODS: A retrospective review was conducted of all enteroscopic examinations carried out in patients with suspected gastrointestinal bleeding or overt gastrointestinal bleeding treated at our institution between 1993 and 1997. All patients had previously undergone an esophagogastroduodenoscopy. The push enteroscope (Olympus XSIF-100) was used in all of them. RESULTS: Push enteroscopy was performed in 233 patients (124 men and 109 women; mean age 63). A suspected bleeding lesion was observed in 53 % of the cases. AVMs represented 63% of the detected lesions. "Missed" upper gastrointestinal lesions were described in 25 patients (10.2 %). In half of these cases, the lesion was located in the upper part of the fundus. Wirsungorrhagia was the cause of bleeding in two cases. CONCLUSIONS: The study confirmed that push enteroscopy is an effective method of detecting lesions responsible for occult gastrointestinal bleeding. In this study, the overall diagnostic yield was 53%. In 10% of the patients, the lesion was located in the upper gastrointestinal tract, despite an initial esophagogastroduodenoscopy. The lesions were mainly located in the fundus. Although it is a rare condition, Wirsungorrhagia must be considered in patients with occult gastrointestinal bleeding.

Acute lower gastrointestinal bleeding in Crohn's disease: characteristics of a unique series of 34 patients. Belgian IBD Research Group.

OBJECTIVE: Acute lower gastrointestinal bleeding is a rare complication of Crohn's disease, which represents a diagnostic and therapeutic challenge. The aim of this study was to define epidemiological characteristics and therapeutic options of hemorrhagic forms of Crohn's disease. METHODS: Thirty-four cases of hemorrhagic forms of Crohn's disease were studied retrospectively. Acute lower gastrointestinal hemorrhage was defined as acute rectal bleeding originating in diseased bowel and requiring a transfusion of at least 2 units of red blood cells within 24 h. Upper gastrointestinal tract hemorrhage or anal lesions and postoperative bleeding were excluded. RESULTS: Mean age at time of hemorrhage was 34.2 +/- 14 yr. Mean duration of disease before the hemorrhage was 5.6 +/- 6 yr. The hemorrhage occurred during a flare up of the disease in 35% of cases. The hemorrhage revealed Crohn's disease in 23.5% of cases. The hemorrhage was more frequent in colonic disease (85%) than in isolated small bowel disease (15%) (p < 0.0001). The origin of bleeding was identified in 65% of cases, by colonoscopy (60%), by angiography (3 patients), or at surgery (1 patient). The bleeding lesion was an ulcer in 95% of cases, most often in the left colon. The treatment was surgical in 20.5% (colectomy in 36%), endoscopical (7 patients, including 5 successes), or medical. Hemorrhage recurred in 12 patients (35%) within a mean time of 3 yr (4 days-8 yr), requiring surgery in 3 cases. No death was observed. CONCLUSIONS: This study performed in a series characterized by a nonsurgical recruitment, the largest to date, shows that hemorrhagic forms of Crohn's disease may reveal disease in 23.5%, occurs in quiescent Crohn's disease in two-thirds of cases. Given the potential efficacy of endoscopical or medical treatment, as well as the absence of mortality, a conservative approach may be suggested as first-line therapy in the majority of patients.

The growing cell plate of higher plants is a site of both actin assembly and vinculin-like antigen recruitment.

Compelling evidence supports the idea that actin filaments play an active role in the cytokinetic process of higher plant cells. However, the mechanisms that control the growth of the cell plate and its stabilization remain so far unknown. We show that a novel population of short actin filaments continuously assembles in the phragmoplast at the growing cell plate. Microinjection of rhodamine-phalloidin during these final stages of telophase revealed the dynamic assembly and organization of these actin filaments during vesicle fusion. Comparable data were obtained in endosperm syncytia during the development of the cell plate between non sister nuclei, i.e. independently of the formation of the mitotic phragmoplast. Concomitantly, plant polypeptides sharing epitopes with human vinculin are revealed within the forming cell plate, suggesting their recruitment during cytokinesis-associated actin assembly. These vinculin-like antigens may participate in membrane/F-actin anchorage protein complexes. Our data, in addition to the identification of plant integrin homologues reported by several authors, suggest the existence of a cell wall/extracellular matrix/plasma membrane/actin cytoskeleton continuum. Such an architecture may control cell-cell interactions during cell plate formation and may contribute to the establishment of polarity in higher plants.

Oral budesonide for lymphocytic colitis.

Lymphocytic colitis is a rare inflammatory colonic disease of unknown etiology accompanied by watery diarrhea. Diagnosis is based upon pathological examination of colonic biopsies. Treatment essentially involves antiinflammatory agents such as sulfasalazine/5-ASA or corticosteroids if necessary. We report the case of a female patient suffering from severe lymphocytic colitis who remained unresponsive after 5-ASA therapy but who improved dramatically after oral budesonide administration.

Short- and long-term efficacy of cyclosporin administration in patients with acute severe ulcerative colitis. Belgian IBD Group.

Cyclosporin (CsA) has been proposed in the management of patients with acute ulcerative colitis (UC) in whom standard therapy failed and who were candidates for colectomy. Seven academic hospitals contributed to this retrospective study that included 29 patients (median age: 33 y. (15-74 y.); 12 females and 17 males). The median duration of the disease was 4 y. (0.3 to 33 y.). Before initiating CsA, patients were unresponsive to treatment including i.v. corticosteroids (n = 29), 5-ASA or salazopyrine (n = 19), azathioprine (n = 3), antibiotics (n = 14). The i.v. mean dose was 4 mg/kg/day and was adapted to blood level. Concomitant treatment included corticosteroids (n = 27). The median duration of i.v. CsA administration was 10 days (4 to 41 days). At the end of CsA administration, a global improvement was described in 20 patients while a surgery had to be performed immediately in 8 patients because of exacerbation of symptoms (n = 7) or perforation (n = 1). One other patient (74 y.) died because of Pneumocystis carinii infection. For the responders, maintenance therapy included: tapering dose of steroids (n = 12), azathioprine (n = 12), 5-ASA or salazopyrine (n = 10), methotrexate (n = 1) or oral CsA (n = II). The median duration of follow-up was 12 months (4 to 48 months). Among the 20 responders, 7 were subsequently referred for colectomy either electively (n = 3) or because of recurrence of the disease (n = 4). Among the 12 patients treated by azathioprine as a maintenance therapy, only 3 had to be referred for surgery (25%). Among the 8 patients who did not receive azathioprine, 4 were subsequently referred for a colectomy (50%) (NS). In patients with acute refractory UC who received CsA, the short-term efficacy (avoidance of immediate colectomy) was obtained in 20 out of 29 patients (69%). However, after a median follow-up of 12 months, only 13 patients were colectomy free (45%).