Some diminutive colorectal polyps can be removed and discarded without pathological examination.

BACKGROUND AND STUDY AIMS: Pathological examination of colorectal polyps is useful if clinical management is affected (i. e. when invasive carcinoma is detected or postpolypectomy surveillance interval is guided). Our aim was to assess whether the pathological examination of some diminutive (measuring ≤ 5 mm) polyps can be omitted. PATIENTS AND METHODS: Consecutive patients undergoing a colonoscopy at Pasteur Hospital (Colmar, France) between January and August 2008 were included in this prospective study. Six senior gastroenterologists predicted the future surveillance interval without referring to the result of pathological examination. RESULTS: In all, 350 polyps from 175 patients were removed and analyzed. The endoscopist was able to predict the correct surveillance interval without referring to the result of pathological examination in 118 patients (67.4 %; 95 % confidence interval [CI] 60.5 - 74.4). The pathological examination of 18.4 % (95 % CI 13.7 - 23.1) of diminutive polyps either associated with a cancer or a polyp measuring ≥ 10 mm or removed in very old or frail patients could be omitted without any consequence for the patient. If diminutive polyps one or two in number were discarded without pathological examination in patients with a personal history of colorectal neoplasm, three patients out of 43 would have a 5-year instead of a 3-year surveillance interval. As a whole, if 44.1 % (95 % CI 38.0 - 50.1) of diminutive polyps were discarded, the surveillance interval would remain identical in 98.3 % (95 % CI 96.4 - 100) of patients. CONCLUSIONS: The pathological examination of up to 44 % of diminutive polyps (i. e. 33 % of all polyps), can be safely omitted. The pathological examination would be required only for those with suspicious gross appearance, those three or more in number, and those isolated one or two in number that are removed from people without personal history of colorectal neoplasm.

Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies.

Fluorescence imaging of two independently labelled proteins is commonly used to determine their co-localization in cells. Antibody-mediated crosslinking can mediate the patching of such proteins at the cell surface, and their co-localization can serve to determine complex formation among them. However, manual analysis of such studies is both tedious and subjective. Here we present a digital co-localization analysis that is independent of the fluorescence intensity, is highly consistent and reproducible between observers, and dramatically reduces the analysis time. The approach presented is based on a segmentation procedure that creates binary objects, and then determines whether objects belonging to two different groups (e.g. green- and red-labelled) are co-localized. Two methods are used to determine co-localization. The 'overlap' analysis defines two objects as co-localized if the centre of mass of one falls within the area of the other. The 'nearest-neighbour distance' analysis considers two objects as co-localized if their centres are within a threshold distance determined by the imaging modality. To test the significance of the results, the analysis of the actual images is tested against randomized images generated by a method that creates images with uncorrelated distributions of objects from the two groups. The applicability of the algorithms presented to study protein interactions in live cells is demonstrated by co-patching studies on influenza haemagglutinin mutants that do or do not associate into mutual oligomers at the cell surface via binding to AP-2 adaptor complexes. The approach presented is potentially applicable to studies of co-localization by other methods (e.g. electron microscopy), and the nearest-neighbour distance method can also be adapted to study phenomena of correlated placement.

Corticosteroid-resistant pyoderma gangrenosum associated with Crohn's disease: rapid cure with infliximab.

A 41-year-old woman with Crohn's disease had a severe and rapidly extensive corticosteroid-resistant pyoderma gangrenosum (PG) of the leg. She had been treated 2 years previously with antibiotics and surgery for a similar lesion of the back of the hand which had been diagnosed as a fulminating infection. Infliximab 5 mg/kg was given at weeks 0, 5 and 9. A dramatic response was observed within 72 h with a favourable effect persisting for 4 weeks after each infliximab infusion. A complete healing was achieved at week 11. This case illustrates that (1). PG of the hand is frequently misdiagnosed as an infection and treated with inappropriate therapies; (2). infliximab may be an interesting alternative in corticosteroid-resistant PG associated with Crohn's disease.

The tail domain of lamin Dm0 binds histones H2A and H2B.

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo, and lamins bind specifically to chromatin in vitro. Deletion mutants of Drosophila lamin Dm0 were expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm0 tail domain. The apparent Kd of binding of the lamin Dm0 tail domain was found to be approximately 1 microM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm0. Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displaced the binding of lamin Dm0 tail to chromosomes. This displacement was specific, because polyamines or proteins such as histones H1, H3, or H4 did not displace the binding of the lamin Dm0 tail to chromosomes. In addition, DNA sequences, including M/SARs, did not interfere with the binding of lamin Dm0 tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm0 and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomes in vivo.

Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography.

We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feed-stock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography. The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration.

Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer.

It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [3H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [3H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [3H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [3H]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on cholesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process.

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids.

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.

Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA.

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA's COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.

The site of airway irritation during induction of anaesthesia.

The aim of this investigation was to study the role of the nasal airway in mediating upper airway reflexes during induction of anaesthesia when the commonly used irritant inhalational anaesthetic agent enflurane is used. In a prospective randomised study, 40 ASA 1 & 2 day-case patients undergoing body surface surgery were recruited. Following intravenous induction using propofol, 20 patients received enflurane administered via a laryngeal mask airway (LMA), the anaesthetic vapour therefore bypassing the nasal airway. In the other group, 20 patients received enflurane anaesthesia administered using a face mask, the nasal airway therefore being exposed to inhalation anaesthetic. We were unable to demonstrate any significant (p < 0.05) differences between the two groups in relation to upper airway complications (cough, breath holding, laryngeal spasm, bronchospasm and excitement). Previous work has identified the nose as a possible important reflexogenic site for upper airway reflexes in humans during anaesthesia. We have been unable to demonstrate any difference in upper airway complications when the nasal airway was included or excluded from exposure to irritant anaesthetic vapours, when administered in a clinical setting.

Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development.

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.

Mechanical stress of the carotid artery at the early phase of spontaneous hypertension in rats.

Common carotid artery (CCA) hypertrophy has long been recognized in the neonatal period of development in spontaneously hypertensive rats (SHR), but the mean circumferential and shear stresses acting on the arterial wall have never been investigated in vivo. We investigated intra-arterial blood pressure in conscious rats, CCA diameter (echotracking techniques), blood flow velocity (pulsed Doppler), wall thickness (histomorphometry), and ganglionic blockade (hexamethonium) in Wistar rats and SHR at 5 and 12 weeks of age. During this interval, weight gain was identical in the strains, whereas the increase in wall thickness and blood pressure was greater in SHR. CCA diameter was identical at week 5 and increased similarly at week 12 in both strains. During ganglionic blockade, a larger diameter was observed in SHR at week 5 for the same BP level, whereas equivalent values were observed at week 12. Blood flow velocity decreased with age but to a significantly greater extent in SHR. Mean circumferential stress and shear stress index were identical in both strains at week 12. However, from weeks 5 to 12, mean circumferential stress increased with age similarly in both strains, whereas the age-related decrease in mean shear stress index was much greater in SHR than Wistar rats. Thus, despite a higher blood pressure, SHR exhibit the same carotid diameter as Wistar rats during early development. Because the kinetics of shear stress are different in both strains, altered flow-dilatation mechanisms, and possibly resulting endothelial dysfunction, may be involved in the diameter changes.

Distinct regions specify the targeting of otefin to the nucleoplasmic side of the nuclear envelope.

Otefin is a 45-kDa nuclear envelope protein with no apparent homology to other known proteins. It includes a large hydrophilic domain, a single carboxyl-terminal hydrophobic sequence of 17 amino acids, and a high content of serine and threonine residues. Cytological labeling located otefin on the nucleoplasmic side of the nuclear envelope. Chemical extraction of nuclei from Drosophila embryos revealed that otefin is a peripheral protein whose association with the nuclear envelope is stronger than that of lamin. Deletion mutants of otefin were expressed in order to identify regions that direct otefin to the nuclear envelope. These experiments revealed that the hydrophobic sequence at the carboxyl terminus is essential for correct targeting to the nuclear envelope, whereas additional regions in the hydrophilic domain of otefin are required for its efficient targeting and stabilization in the nuclear envelope.

[Primary signet ring adenocarcinoma ofa diverted neurogenic bladder]. / Adénocarcinome primitif à cellules en bague à chaton sur vessie neurologique exclue. A propos d'un cas.

Primary signet-ring cell carcinoma of urinary bladder is an uncommon primitive bladder tumor. We report the first case occurring on a diverted neurogenic bladder. Except of adenocarcinoma of urachal origin, about 60 cases have been reported to date. The histogenesis of these tumors remains controversial.

Transcatheter oily chemoembolization for hepatocellular carcinoma. A 4-year study of 127 French patients.

BACKGROUND: In Western countries, only a small proportion of patients with hepatocellular carcinoma (HCC) can be treated with surgical resection. For other patients, locoregional management by transcatheter oily chemoembolization seems to be useful and warrants evaluation. METHODS: One hundred and twenty-seven French patients with an inoperable HCC were treated by transcatheter oily chemoembolization. The efficiency of the treatment was assessed by a comparison of this group with a group of 127 untreated patients. Each patient of the treated group was matched closely with an untreated patient for all the main clinical, anatomic, and biologic features that characterize the spontaneous evolution of HCC. RESULTS: The overall probabilities of survival in the treated group were 64%, 38%, 27%, and 27% at 1, 2, 3, and 4 years, respectively; those for the untreated group were 18%, 6%, and 5% at 1, 2 and 3 years, respectively (P < 0.0001). The survival was significantly increased in patients with Okuda Stage I and II disease (P < 0.0001), but not in those with Stage III. Karnofsky and Child-Pugh scores remained stable during the follow-up period and dropped only shortly before patients died. CONCLUSION: Transcatheter oily chemoembolization is an efficient treatment for unresectable HCC for the palliation of symptoms as well as for the prolongation of survival with a good quality of life.

Fluoxetine: activating and sedating effects at multiple fixed doses.

Serotonin uptake inhibitors are generally considered activating antidepressants. To assess rates and temporal patterns of activation and sedation as well as dose-effect relationships, adverse event data were evaluated from a fixed-dose study comparing placebo and fluoxetine 5, 20, and 40 mg/day in the treatment of major depressive disorder (N = 363) and two fixed-dose studies pooled together comparing placebo and fluoxetine 20, 40, and 60 mg/day in the treatment of major depressive disorder (N = 746). The adverse events nervousness, anxiety, agitation, and insomnia were considered indicative of activation; somnolence and asthenia were considered indicative of sedation. Activation and sedation were both statistically significant (p less than or equal to 0.05) treatment-emergent phenomena, but dose-effect relationships differed. Activation rates were relatively stable between 5 and 40 mg/day, and then increased at 60 mg/day. Sedation rates increased linearly to 40 mg/day and then were comparable at 40 and 60 mg/day. Discontinuations for either phenomenon were uncommon. The temporal patterns of first occurrences and persistence of activation and sedation differed. First occurrences of activation peaked early and declined over time with all doses. First occurrences of sedation also peaked early with all doses, but there may have been greater variability in first occurrences of sedation over time with lower doses. Persistent occurrences of sedation may decline less over time than persistent occurrences of activation.

Immunoaffinity purification: basic principles and operational considerations.

Immunoaffinity purification has become an important technique in biotechnology. In this review the basic principles of immunoaffinity separations are described with respect to the stages of operation and potential application. The most commonly used support materials, activation procedures, and coupling chemistries are compared to one another for suitability in various applications. Individual operational steps for fixed bed immunoadsorbents including loading, washing, elution and regeneration are described in terms of both theory and practice. Factors influencing adsorbent stability are identified, and alternative operation and configuration strategies are discussed in light of their application to immunoaffinity systems.

Fluoxetine in tricyclic refractory major depressive disorder.

Data regarding open-label treatment with fluoxetine following failure to respond to tricyclic antidepressants (TCAs) or intolerance of TCA side effects, suggest a response rate between 51.4% and 62.1%, depending on the definition of TCA refractoriness employed. Double-blind study of this issue would extend these findings. Fluoxetine is well tolerated in patients unable to tolerate TCAs. Within this population, more than 80% of patients unable to tolerate TCAs found fluoxetine acceptable. Fluoxetine, as an alternative to polypharmaceutical augmentation, may represent a logical choice as the next step in therapy for a patient who has initially been treated with a TCA and has proven refractory or intolerant.