Conditional risk of diverticulitis after non-operative management.

BACKGROUND: The objective of this study was to describe conditional recurrence-free survival (RFS) of patients after an index episode of diverticulitis managed without surgery, and to estimate the difference in conditional RFS for diverticulitis according to specific risk factors. METHODS: This was a multicentre retrospective cohort study including all patients managed without surgery for acute sigmoid diverticulitis at two university-affiliated hospitals in Montreal, Quebec, Canada. Conditional RFS for diverticulitis was estimated over 10 years of follow-up. A Cox proportional hazards model was performed at the index episode and again 2 years later. RESULTS: In total, 991 patients were included for analysis. The 1, 2- and 3-year actuarial diverticulitis RFS rates were 81·1, 71·5 and 67·5 per cent respectively. Compared with the 1-year actuarial RFS rate of 81·1 per cent, the 1-year conditional RFS increased with each additional year survived recurrence-free, reaching 96·0 per cent after surviving the first 4 years recurrence-free. A similar phenomenon was observed for 2-year diverticulitis conditional RFS. Lower age (hazard ratio (HR) 0·98, 95 per cent c.i. 0·98 to 0·99), Charlson Co-morbidity Index score of 2 or above (HR 1·78, 1·32 to 2·39) and immunosuppression (HR 1·85, 1·38 to 2·48) were independently associated with recurrence of diverticulitis from the index episode. At 2 years from the index episode, immunosuppression was no longer associated with diverticulitis recurrence (HR 1·02, 0·50 to 2·09). CONCLUSION: The conditional RFS of patients with diverticulitis improved with each year that was survived recurrence-free. Although several factors at index presentation may be associated with early recurrence, the conditional probability of recurrence according to many of these risk factors converged with time.

ANTECEDENTES: El objetivo de este estudio fue describir la supervivencia condicional libre de recidiva de diverticulitis (diverticulitis recurrence-free survival, Div-RFS) en pacientes tras un episodio de diverticulitis tratado de forma conservadora, y calcular la diferencia en la Div-RFS condicional de acuerdo con factores de riesgo específicos. MÉTODOS: Estudio de cohorte retrospectivo multicéntrico que incluyó a todos los pacientes tratados de forma no quirúrgica por diverticulitis sigmoidea aguda en dos hospitales afiliados a la universidad en Montreal, Quebec, Canadá. La supervivencia condicional libre de recidiva de la diverticulitis se calculó durante 10 años de seguimiento. Se realizó un análisis mediante un modelo de riesgos proporcionales de Cox en el episodio índice y nuevamente 2 años después. RESULTADOS: En total, se incluyeron 991 pacientes en el análisis. La Div-RFS actuarial a 1, 2 y 3 años fue del 81,1%, 71,5% y 67,5%, respectivamente. En comparación con la Div-RFS actuarial a 1 año del 81,1%, la Div-RFS condicional a 1 año aumentó con cada año adicional sobrevivido sin recidiva, alcanzando el 96,0% después de sobrevivir los primeros 4 años sin recidiva. Se observó un fenómeno similar para Div-RFS condicional a los 2 años. Una menor edad (cociente de riesgos instantáneos, hazard ratio, HR: 0,98; i.c. del 95%: 0,98 a 0,99), la puntuación de comorbilidad de Charlson ≥ 2 (HR: 1,78; i.c. del 95%: 1,32 a 2,39) y la inmunosupresión (HR: 1,85; i.c. del 95%: 1,38 a 2,48) se asociaron de forma independiente con la recidiva de la diverticulitis desde el episodio índice. En la regresión de Cox a los 2 años del episodio índice, la inmunosupresión ya no se asoció con recidiva de diverticulitis (HR: 1,02; i.c. del 95% 0,50-2,09). CONCLUSIÓN: La Div-RFS condicional mejoró con cada año de supervivencia sin recidiva. Si bien varios factores en la presentación del episodio índice pueden estar asociados con una recidiva precoz, la probabilidad condicional de recidiva en relación con muchos de estos factores de riesgo coincidió con el tiempo.

Assessing the readability, quality and accuracy of online health information for patients with low anterior resection syndrome following surgery for rectal cancer.

AIM: Management of low anterior resection syndrome (LARS) requires a high degree of patient engagement. This process may be facilitated by online health-related information and education. The aim of this study was to systematically review current online health information on LARS. METHOD: An online search of Google, Yahoo and Bing was performed using the search terms 'low anterior/anterior resection syndrome' and 'bowel function/movements after rectal cancer surgery'. Websites were assessed for readability (eight standardized tests), suitability (using the Suitability Assessment of Materials instrument), quality (the DISCERN instrument), accuracy and content (using a LARS-specific content checklist). Websites were categorized as academic, governmental, nonprofit or private. RESULTS: Of 117 unique websites, 25 met the inclusion criteria. The median readability level was 10.4 (9.2-11.7) and 11 (44.0%) websites were highly suitable. Using the DISCERN instrument, seven (28.0%) websites had clear aims, two (8.0%) divulged the sources used and four (16.0%) had high overall quality. Only eight (32.0%) websites defined LARS and ten (40.0%) listed all five major symptoms associated with the LARS score. There was variation in the number of websites that discussed dietary modifications (80.0%), self-help strategies (72.0%), medication (68.0%), pelvic floor rehabilitation (60.0%) and neuromodulation (8.0%). The median accuracy of websites was 93.8% (88.2-96.7%). Governmental websites scored highest for overall suitability (P = 0.0079) and quality (P < 0.001). CONCLUSIONS: Current online information on LARS is suboptimal. Websites are highly variable, important content is often lacking and material is too complex for patients.

Study protocol evaluating the use of bowel stimulation before loop ileostomy closure to reduce postoperative ileus: a multicenter randomized controlled trial.

AIM: Postoperative ileus is the most commonly observed morbidity following ileostomy closure. Studies have demonstrated that the defunctionalized bowel of a loop ileostomy undergoes a series of functional and structural changes, such as atrophy of the intestinal villi and muscular layers, which may contribute to ileus. A single-centre study in Spain demonstrated that preoperative bowel stimulation via the distal limb of the loop ileostomy decreased postoperative ileus, length of stay and time to gastrointestinal function. METHOD: A multicentre randomized controlled trial involving patients from Canadian institutions was designed to evaluate the effect of preoperative bowel stimulation before ileostomy closure on postoperative ileus. Stimulation will include canalizing the distal limb of the ileostomy loop with an 18Fr Foley catheter and infusing it with a solution of 500 ml of normal saline mixed with 30 g of a thickening agent (Nestle© Thicken-Up© ). This will be performed 10 times over the 3 weeks before ileostomy closure in an outpatient clinic setting by a trained Enterostomal Therapy nurse. Surgeons and the treating surgical team will be blinded to their patient's group allocation. Data regarding patient demographics, and operative and postoperative variables, will be collected prospectively. Primary outcome will be postoperative ileus, defined as an intolerance to oral food in the absence of clinical or radiological signs of obstruction, that either requires nasogastric tube insertion or is associated with two of the following on or after post-operative day 3: nausea/vomiting; abdominal distension; and the absence of flatus. Secondary outcomes will include length of stay, time to tolerating a regular diet, time to first passage of flatus or stool and overall morbidity. A cost analysis will be performed to compare the costs of conventional care with conventional care plus preoperative stimulation. DISCUSSION: This manuscript discusses the potential benefits of preoperative bowel stimulation in improving postoperative outcomes and outlines our protocol for the first multicenter study to evaluate preoperative bowel stimulation before ileostomy closure. The results of this study could have considerable implications for the care of patients undergoing ileostomy closure.

[Development and validation of an analytical method to quantify residues of cleaning products able to inactivate prion]. / Développement et validation d'une méthode de dosage des traces de détergents inactivants totaux du prion.

OBJECTIVES: In this study, a novel analytical method to quantify prion inactivating detergent in rinsing waters coming from the washer-disinfector of a hospital sterilization unit has been developed. The final aim was to obtain an easy and functional method in a routine hospital process which does not need the cleaning product manufacturer services. METHODS: An ICP-MS method based on the potassium dosage of the washer-disinfector's rinsing waters was developed. Potassium hydroxide is present on the composition of the three prion inactivating detergent currently on the French market. The detergent used in this study was the Actanios LDI(®) (Anios laboratories). A Passing and Bablok regression compares concentrations measured with this developed method and with the HPLC-UV manufacturer method. RESULTS: According to results obtained, the developed method is easy to use in a routine hospital process. The Passing and Bablok regression showed that there is no statistical difference between the two analytical methods during the second rinsing step. Besides, both methods were linear on the third rinsing step, with a 1.5ppm difference between the concentrations measured for each method. CONCLUSIONS: This study shows that the ICP-MS method developed is nonspecific for the detergent, but specific for the potassium element which is present in all prion inactivating detergent currently on the French market. This method should be functional for all the prion inactivating detergent containing potassium, if the sensibility of the method is sufficient when the potassium concentration is very low in the prion inactivating detergent formulation.

Lasting alterations of the sodium current by short-term hyperlipidemia as a mechanism for initiation of cardiac remodeling.

Clinical and animal studies indicate that increased fatty acid delivery to lean tissues induces cardiac electrical remodeling and alterations of cellular calcium homeostasis. Since this may represent a mechanism initiating cardiac dysfunction during establishment of insulin resistance and diabetes or anaerobic cardiac metabolism (ischemia), we sought to determine if short-term exposure to high plasma concentration of fatty acid in vivo was sufficient to alter the cardiac sodium current (INa) in dog ventricular myocytes. Our results show that delivery of triglycerides and nonesterified fatty acids by infusion of Intralipid + heparin (IH) for 8 h increased the amplitude of INa by 43% and shifted its activation threshold by -5 mV, closer to the resting membrane potential. Steady-state inactivation (availability) of the channels was reduced by IH with no changes in recovery from inactivation. As a consequence, INa "window" current, a strong determinant of intracellular Na+ and Ca2+ concentrations, was significantly increased. The results indicate that increased circulating fatty acids alter INa gating in manners consistent with an increased cardiac excitability and augmentation of intracellular calcium. Moreover, these changes could still be measured after the dogs were left to recover for 12 h after IH perfusion, suggesting lasting changes in INa. Our results indicate that fatty acids rapidly induce cardiac remodeling and suggest that this process may be involved in the development of cardiac dysfunctions associated to insulin resistance and diabetes.

Genome sequence of the edible cyanobacterium Arthrospira sp. PCC 8005.

We determined the genome sequence of Arthrospira sp. PCC 8005, a cyanobacterial strain of great interest to the European Space Agency for its nutritive value and oxygenic properties in the Micro-Ecological Life Support System Alternative (MELiSSA) biological life support system for long-term manned missions into space.

A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization.

Coordination of the different cytoskeleton networks in the cell is of central importance for morphogenesis, organelle transport, and motility. The Rho family proteins are well characterized for their effects on the actin cytoskeleton, but increasing evidence indicates that they may also control microtubule (MT) dynamics. Here, we demonstrate that a novel Cdc42/Rac effector, X-p21-activated kinase (PAK)5, colocalizes and binds to both the actin and MT networks and that its subcellular localization is regulated during cell cycle progression. In transfected cells, X-PAK5 promotes the formation of stabilized MTs that are associated in bundles and interferes with MTs dynamics, slowing both the elongation and shrinkage rates and inducing long paused periods. X-PAK5 subcellular localization is regulated tightly, since coexpression with active Rac or Cdc42 induces its shuttling to actin-rich structures. Thus, X-PAK5 is a novel MT-associated protein that may communicate between the actin and MT networks during cellular responses to environmental conditions.

Analysis of bovine sexed sperm for IVF from sorting to the embryo.

The objective of this study was to characterize bovine semen parameters and determine the best IVF conditions to produce a maximal percentage of blastocysts. Four types of semen were analyzed with CASA and flow cytometry: fresh and frozen non-sexed semen; fresh and frozen sexed semen. Semen was obtained from four Holstein bulls and two ejaculates from each bull were analyzed. Oocytes from slaughterhouse ovaries were matured and fertilized in vitro with all types of semen (for sexed semen, 2, 5 or 10microg/mL heparin was added to the IVF media while for non-sexed semen, 10microg/mL was added in the IVF medium). Presumptive zygotes were co-cultured with Buffalo rat liver cells in Menezo's B2 medium, and cleavage rates at Day 2, and blastocyst rates at Day 7 of culture, were recorded. Sexed semen resulted in fewer blastocysts than non-sexed semen (P<0.05), and certain bulls performed better in IVF. Freezing, and not sexing, had a more significant negative effect on semen quality. Compromised semen quality due to sexing and/or freezing can explain the reduced in vitro blastocyst rates when using frozen-thawed sexed semen. Sexed semen that appeared more capacitated seemed to require less heparin in IVF than sexed semen that appeared less capacitated to produce a maximal percentage of blastocyst. Flow cytometry sorting eliminates spermatozoa that possess compromised DNA, and therefore the reduced fertility seen in vitro is not due to an increased percentage of spermatozoa with compromised DNA. This study describes tools that can monitor semen parameters to optimize IVF conditions and thus obtain maximal blastocyst rates.

Contribution of estrogen receptors alpha and beta to the effects of estradiol in the brain.

Clinical and experimental studies show a modulatory role of estrogens in the brain and suggest their beneficial action in mental and neurodegenerative diseases. The estrogen receptors ERalpha and ERbeta are present in the brain and their targeting could bring selectivity and reduced risk of cancer. Implication of ERs in the effect of estradiol on dopamine, opiate and glutamate neurotransmission is reviewed. The ERalpha agonist, PPT, is shown as estradiol to modulate hippocampal NMDA receptors and AMPA receptors in cortex and striatum of ovariectomized rats whereas the ERbeta agonist DPN is inactive. Striatal DPN activity suggests implication of ERbeta in estradiol modulation of D2 receptors and transporters in ovariectomized rats and is supported by the lack of effect of estradiol in ERbeta knockout (ERKObeta) mice. Both ERalpha and ERbeta agonists modulate striatal preproenkephalin (PPE) gene expression in ovariectomized rats. In male mice PPT protects against MPTP toxicity to striatal dopamine; this implicates Akt/GSK3beta signaling and the apoptotic regulators Bcl2 and Bad. This suggests a role for ERalpha in striatal dopamine neuroprotection. ERKOalpha mice are more susceptible to MPTP toxicity and not protected by estradiol; differences in ERKObeta mice are subtler. These results suggest therapeutic potential for the brain of ER specific agonists.

Neutrophils invade lumbar dorsal root ganglia after chronic constriction injury of the sciatic nerve.

To test whether neutrophils (PMN) target lumbar dorsal root ganglia (DRG) following axonal injury leading to neuropathic pain, we visualized PMN infiltration in DRG tissue sections and estimated PMN count by flow cytometry following sciatic chronic constriction injury (CCI). Seven days after CCI, results show PMN within DRG where their count increased by three fold ipsilateral to injury compared to contralateral or sham, concomitant with peak neuropathic pain behavior. Superoxide burst in PMN isolated from rats d7 after CCI was elevated by 170% +/-18 compared to naïve and MCP-1 mRNA expression in DRG increased by 8.9+/-2.9 fold, but that of MIP-2, CINC-1, and RANTES did not change. We conclude that CCI causes PMN invasion of the DRG whereby the functional implication of their close proximity to neuronal axon and soma remains unknown.

Nuclear localization of the adenovirus DNA-binding protein: requirement for two signals and complementation during viral infection.

The adenovirus DNA-binding protein (DBP) is an abundant multifunctional protein located primarily in the nuclei of infected cells. To define sequences involved in nuclear transport of DBP, a series of point and small deletion mutants were constructed via oligonucleotide-directed mutagenesis. Two short stretches of basic amino acids located in the amino-terminal domain (amino acids 42 to 46 and 84 to 89) were identified. Their importance, however, depended on the context in which DBP was expressed. Disruption of either site prevented nuclear localization after transient expression in transfected 293 cells, implying that two nuclear localization signals are necessary for transport of this nuclear protein. In contrast, the mutant DBPs synthesized during viral infection were located either primarily in the nucleus or in the nucleus and cytoplasm, depending on the mutation and the stage of the viral infection. Thus, the nuclear localization defect could be complemented by viral infection, perhaps through the interaction of the mutant polypeptide with a virus-encoded or -induced factor(s).

cis-acting elements and a trans-acting factor affecting alternative splicing of adenovirus L1 transcripts.

Expression of the L1 region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52- to 55-kilodalton (52-55K) and IIIa polypeptides. The distal acceptor site (IIIa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the L1 region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IIIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the IIIa exon to the 52-55K intron greatly enhanced splicing of the IIIa junction, suggesting that the IIIa exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the IIIa acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the IIIa splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of E1A and E1B genes, did not facilitate use of the IIIa acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IIIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.

Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase.

1,3-beta-D-Glucan is a major structural polymer of yeast and fungal cell walls and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-beta-D-glucan synthase. Previous work has shown that the FKS1 gene encodes a 215-kDa integral membrane protein (Fks1p) which mediates sensitivity to the echinocandin class of antifungal glucan synthase inhibitors and is a subunit of this enzyme. We have cloned and sequenced FKS2, a homolog of FKS1 encoding a 217-kDa integral membrane protein (Fks2p) which is 88% identical to Fks1p. The residual glucan synthase activity present in strains with deletions of fks1 is (i) immunodepleted by antibodies prepared against FKS2 peptides, demonstrating that Fks2p is also a component of the enzyme, and (ii) more sensitive to the echinocandin L-733,560, explaining the increased sensitivity of fks1 null mutants to this drug. Simultaneous disruption of FKS1 and FKS2 is lethal, suggesting that Fks1p and Fks2p are alternative subunits with essential overlapping function. Analysis of FKS1 and FKS2 expression reveals that transcription of FKS1 is regulated in the cell cycle and predominates during growth on glucose, while FKS2 is expressed in the absence of glucose. FKS2 is essential for sporulation, a process which occurs during nutritional starvation. FKS2 is induced by the addition of Ca2+ to the growth medium, and this induction is completely dependent on the Ca2+/calmodulin-dependent phosphoprotein phosphatase calcineurin. We have previously shown that growth of fks1 null mutants is highly sensitive to the calcineurin inhibitors FK506 and cyclosporin A. Expression of FKS2 from the heterologous ADH1 promoter results in FK506-resistant growth. Thus, the sensitivity of fks1 mutants to these drugs can be explained by the calcineurin-dependent transcription of FKS2. Moreover, FKS2 is also highly induced in response to pheromone in a calcineurin-dependent manner, suggesting that FKS2 may also play a role in the remodeling of the cell wall during the mating process.

Increased attachment of nucleic acids to eukaryotic and prokaryotic cells induced by chemical and physical carcinogens and mutagens.

Significantly enhanced attachment to Ehrlich ascites and Escherichia coli cells was observed for radioactive DNA and RNA in the presence of chemical mutagens and ultimate carcinogens. In some instances, formation of nucleic acid-protein adducts by these compounds further (or similarly) enhanced the binding. DNA irradiated with ultraviolet light in the presence of a protein bound more efficiently than either an unirradiated mixture of these two macromolecules or DNA irradiated alone. The spectrum of compounds tested and found active in this system includes alkylating agents, aromatic amines, and carcinogenic metals. Precarcinogens and nonultimate carcinogenic chemicals, as well as tumor-promoting agents, did not increase the binding. However, addition of extracts from mouse or rat livers activated precarcinogenic and proximate carcinogenic chemicals and resulted in enhanced cellular attachment of indicator nucleic acids in their presence. Possible usefulness of this test system for fast and efficient screening for environmental carcinogens and mutagens, as well as possible relevance of the observed phenomena to in vivo effects of chemical and physical carcinogens, is considered.

Inactivation of protein kinase A is not required for c-mos translation during meiotic maturation of Xenopus oocytes.

It has been shown previously that protein kinase A (PKA) maintains Xenopus oocytes arrested at G2, at least in part by preventing c-mos translation, but how PKA controls c-mos translation is not known. Using microinjection of recombinant c-mos, which still activates MAP kinase in the presence of active PKA, we have found that PKA does not exert any effect on translation of endogenous c-mos if MAP kinase is first activated. Even though they accumulate c-mos and contain MAP kinase activity as high as control oocytes, oocytes do not exit G2 in the presence of active PKA. These results are discussed in connection with recent findings on regulation of c-raf activity.

Is Cdk7/cyclin H/MAT1 the genuine cdk activating kinase in cycling Xenopus egg extracts?

Formation of active cdk (cyclin dependent kinase)/ cyclin kinases involves phosphorylation of a conserved threonine residue in the T loop of the cdk catalytic-subunit by CAK (Cdk Activating Kinase). CAK was first purified biochemically from higher eukaryotes and identified as a trimeric complex containing a cdk7 catalytic subunit, cyclin H and MAT1 (Ménage à trois), a member of the RING finger family. The same trimeric complex is also part of basal transcription factor TFIIH. In budding yeast, the closest homologs of cdk7 and cyclin H, KIN28 and CCL1, respectively, also associate with TFIIH. However, the KIN28/CCL1 complex does not display CAK activity and a distinct protein kinase able to phosphorylate monomeric CDC28 and GST-cdk2 was recently identified, challenging the identification of cdk7 as the physiological CAK in higher eukaryotes. Here we demonstrate that immunodepletion of cdk7 suppresses CAK activity from cycling Xenopus egg extracts, and arrest them before M-phase. We also show that specific translation of mRNAs encoding Xenopus cdk7 and its associated subunits restores CAK activity in cdk7-immunodepleted Xenopus egg extracts. Hence, the cdk7 complex is necessary and sufficient for activation of cdk-cyclin complexes in cycling Xenopus egg extracts.

Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins.

We have selectively mutagenized specific residues at the junction between the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 (HIV-1) to study the effects of PR-RT fusion proteins in the context of a full-length, infectious proviral construct. Mutant viruses derived from COS-7 cells transfected with this construct were analyzed in regard to each of viral replication, maturation, and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein remained functionally active, whereby HIV-1 precursor proteins were processed efficiently. Furthermore, the RT component of the fusion protein also retained its enzymatic activity as shown in RT assays. Electron microscopy revealed that the mutant viruses containing the PR-RT fusion protein possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of the HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20 times less infectious than wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N terminus of PR, a cleavage site mutation between PR and RT did not affect the enzymatic activities of either PR or RT and viruses containing PR-RT fusion proteins were viable.

Oestrogens prevent loss of dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) in substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice.

Previous results from our laboratory have shown that 17beta-oestradiol prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) striatal dopamine depletion. 17beta-oestradiol, oestriol and oestrone are the naturally occurring oestogens in humans. Using various dopamine markers, the present study investigated whether oestrone and oestriol such as 17beta-oestradiol have neuroprotective activity in MPTP-treated mice. Male mice were treated with 17beta-oestradiol, oestriol or oestrone for 5 days before and after MPTP administration, and were compared with nonlesioned mice receiving the same treatment. Striatal concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were assayed by high-performance liquid chromatography. Dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) specific binding were measured by autoradiography. DAT, VMAT2 and tyrosine hydroxylase mRNA levels were measured by in situ hybridisation. MPTP induced a loss of DAT and VMAT2 specific binding in the striatum and substantia nigra, as well as a decrease of VMAT2 mRNA in the substantia nigra. 17beta-oestradiol treatment prevented the loss of these dopaminergic markers, as well as striatal concentrations of dopamine, DOPAC and HVA. Mice receiving oestriol and oestrone showed catecholamine concentrations comparable to MPTP mice. Oestriol treatment had no effect on dopaminergic markers in MPTP mice whereas oestrone prevented striatal DAT loss and the decrease of VMAT2 mRNA in the substantia nigra. In nonlesioned mice, 17beta-oestradiol, oestriol or oestrone had no effect on all the dopaminergic markers investigated. In conclusion, a weak or a lack of effect of oestriol and oestrone was observed compared to 17beta-oestradiol in MPTP mice and none of these steroids had an effect in nonlesioned mice. A DAT and VMAT2 specific binding decrease after MPTP in the striatum and substantia nigra, as well as a decrease of substantia nigra VMAT2 mRNA, was observed and could be prevented by oestradiol.

The presence and leachability of antimony in different wastes and waste handling facilities in Norway.

The environmental behaviour of antimony (Sb) is gathering attention due to its increasingly extensive use in various products, particularly in plastics. Because of this it may be expected that plastic waste is an emission source for Sb in the environment. This study presents a comprehensive field investigation of Sb concentrations in diverse types of waste from waste handling facilities in Norway. The wastes included waste electrical and electronic equipment (WEEE), glass, vehicle fluff, combustibles, bottom ash, fly ash and digested sludge. The highest solid Sb concentrations were found in WEEE and vehicle plastic (from 1238 to 1715 mg kg(-1)) and vehicle fluff (from 34 to 4565 mg kg(-1)). The type of acid used to digest the diverse solid waste materials was also tested. It was found that HNO3:HCl extraction gave substantially lower, non-quantitative yields compared to HNO3:HF. The highest water-leachable concentration for wastes when mixed with water at a 1 : 10 ratio were observed for plastic (from 0.6 to 2.0 mg kg(-1)) and bottom ash (from 0.4 to 0.8 mg kg(-1)). For all of the considered waste fractions, Sb(v) was the dominant species in the leachates, even though Sb(iii) as Sb2O3 is mainly used in plastics and other products, indicating rapid oxidation in water. This study also presents for the first time a comparison of Sb concentrations in leachate at waste handling facilities using both active grab samples and DGT passive samples. Grab samples target the total suspended Sb, whereas DGT targets the sum of free- and other chemically labile species. The grab sample concentrations (from 0.5 to 50 µg L(-1)) were lower than the predicted no-effect concentration (PNEC) of 113 µg L(-1). The DGT concentrations were substantially lower (from 0.05 to 9.93 µg L(-1)) than the grab samples, indicating much of the Sb is present in a non-available colloidal form. In addition, air samples were taken from the chimney and areas within combustible waste incinerators, as well as from the vent of WEEE sorting facility. The WEEE vent had the highest Sb concentration (from <100 to 2200 ng m(-3)), which were orders of magnitude higher than the air surrounding the combustible shredder (from 25 to 217 ng m(-3)), and the incinerator chimney (from <30 to 100 ng m(-3)). From these results, it seems evident that Sb from waste is not an environmental concern in Norway, and that Sb is mostly readily recovered from plastic and bottom ash.

Effect of HIV constructs containing protease-reverse transcriptase fusion proteins on viral replication.

OBJECTIVE: To determine whether disruption of the cleavage site between protease and reverse transcriptase (RT) or the HIV-1 frameshift site could yield trans-dominant negative HIV-1 variants that interfere with wild-type viral replication. DESIGN: Residues at the cleavage site between the HIV-1 protease and RT coding regions were mutagenized to produce a protease-RT (PR-RT) fusion protein that was expressed in the context of a full-length provirus. The PR-RT cleavage site mutation was also combined with a read-through mutation at the frameshift site in order to overexpress the mutant Gag-Pol polyproteins. METHODS: COS-7 cells were transiently transfected with the mutant constructs to produce viruses harbouring the PR-RT fusion protein. In addition, we performed cotransfection studies in various cell types to analyze the inhibition of wild-type replication by the mutant constructs. RESULTS: Immunoblot analysis revealed that this novel mutation prevented cleavage between the two proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. While both the protease and RT components of this fusion protein remained functionally active, viruses containing the cleavage site mutation were less infectious in tissue culture than wild-type viruses produced by COS-7 cells. This defect was further pronounced when the cleavage site mutation between protease and RT was overexpressed as a consequence of an additional mutation that interfered with frameshifting. Cotransfection of COS-7 cells with the mutant constructs and wild-type HIV-1 interfered with the replication of the latter and reduced the infectiousness of the virus particles produced. CONCLUSIONS: HIV-1 constructs harbouring a cleavage site mutation between protease and RT can probably act as trans-dominant negative mutants to interfere with wild-type viral replication.