Advance directives: the views of health care professionals.

OBJECTIVES: This study examined the views and experiences of 20 physicians and 20 nurses at a major Canadian teaching hospital regarding the use of advance directives in clinical care. DESIGN: The participants were purposively drawn from four clinical specialties: family and community medicine, oncology, intensive care and geriatrics. Detailed interviews were conducted in person. Content analysis was used to code the data, which were further analysed with both quantitative and qualitative techniques. MAIN RESULTS: Thirty-nine of the 40 participants favoured the use of advance directives in clinical care; physicians had somewhat less positive attitudes than nurses toward such directives. Advance directives were thought by participants to be helpful in resolving disagreements between patients and their families about treatment options; in making patients more comfortable, both physically and psychologically, during the process of dying; and in opening up communication and trust among patients, their families and health care professionals. Concerns about the use of advance directives focused on the lack of clarity in some patients'' instructions, the absence of legal status for directives, the possible interference with a practitioner''s clinical judgement, the adequacy and appropriateness of patients'' information about their circumstances, and the type of intervention (passive or active) requested by patients. CONCLUSIONS: New regulations and legislation are making the use of advance directives more widespread. Health care professionals should participate in the development and implementation of these directives. Continuing professional education is essential in this regard.     

Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.     

Biochemical characterization of the tobacco 42-kD protein kinase activated by osmotic stress

In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family. The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA. A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed. Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase. The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases. Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK. A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity. In phosphorylation reactions, NtOSAK exhibited a preference for Mg(2+) over Mn(2+) ions and an inability to use GTP instead of ATP as a phosphate donor. The enzyme activity was not modulated by 5'-AMP. To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.     

The copper transport protein Atox1 promotes neuronal survival

Atox1, a copper transport protein, was recently identified as a copper-dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.     

Structural organization of the human gastrointestinal glutathione peroxidase (GPX2) promoter and 3'-nontranscribed region: transcriptional response to exogenous redox agents

We describe a new family of repetitive elements, named Mimo, from the mosquito Culex pipiens. Structural characteristics of these elements fit well with those of miniature inverted-repeat transposable elements (MITEs), which are ubiquitous and highly abundant in plant genomes. The occurrence of Mimo in C. pipiens provides new evidence that MITEs are not restricted to plant genomes, but may be widespread in arthropods as well. The copy number of Mimo elements in C. pipiens (1000 copies in a 540 Mb genome) supports the hypothesis that there is a positive correlation between genome size and the magnitude of MITE proliferation. In contrast to most MITE families described so far, members of the Mimo family share a high sequence conservation, which may reflect a recent amplification history in this species. In addition, we found that Mimo elements are a frequent nest for other MITE-like elements, suggesting that multiple and successive MITE transposition events have occurred very recently in the C. pipiens genome. Despite evidence for recent mobility of these MITEs, no element has been found to encode a protein; therefore, we do not know how they have transposed and have spread in the genome. However, some sequence similarities in terminal inverted-repeats suggest a possible filiation of some of these mosquito MITEs with pogo-like DNA transposons.     

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

Background

Cytokinin is a plant hormone that plays a crucial role in several processes of plant growth and development. In recent years, major breakthroughs have been achieved in the elucidation of the metabolism, the signal perception and transduction, as well as the biological functions of cytokinin. An important activity of cytokinin is the involvement in chloroplast development and function. Although this biological function has already been known for 50 years, the exact mechanisms remain elusive.

Results

To elucidate the effects of altered endogenous cytokinin content on the structure and function of the chloroplasts, chloroplast subfractions (stroma and thylakoids) from transgenic Pssu-ipt and 35S:CKX1 tobacco (Nicotiana tabacum) plants with, respectively, elevated and reduced endogenous cytokinin content were analysed using two different 2-DE approaches. Firstly, thykaloids were analysed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (BN/SDS-PAGE). Image analysis of the gel spot pattern thus obtained from thylakoids showed no substantial differences between wild-type and transgenic tobacco plants. Secondly, a quantitative DIGE analysis of CHAPS soluble proteins derived from chloroplast subfractions indicated significant gel spot abundance differences in the stroma fraction. Upon identification by MALDI-TOF/TOF mass spectrometry, these proteins could be assigned to the Calvin-Benson cycle and photoprotective mechanisms.

Conclusion

Taken together, presented proteomic data reveal that the constitutively altered cytokinin status of transgenic plants does not result in any qualitative changes in either stroma proteins or protein complexes of thylakoid membranes of fully developed chloroplasts, while few but significant quantitative differences are observed in stroma proteins.     

Cytogenetic analyses of eight species in the genus <Emphasis Type="Italic">Leptodactylus</Emphasis> Fitzinger, 1843 (Amphibia,Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations

Background

The karyotypes of Leptodactylus species usually consist of 22 bi-armed chromosomes, but morphological variations in some chromosomes and even differences in the 2n have been reported. To better understand the mechanisms responsible for these differences, eight species were analysed using classical and molecular cytogenetic techniques, including replication banding with BrdU incorporation.

Results

Distinct chromosome numbers were found: 2n = 22 in Leptodactylus chaquensis, L. labyrinthicus, L. pentadactylus, L. petersii, L. podicipinus, and L. rhodomystax; 2n = 20 in Leptodactylus sp. (aff. podicipinus); and 2n = 24 in L. marmoratus. Among the species with 2n = 22, only three had the same basic karyotype. Leptodactylus pentadactylus presented multiple translocations, L. petersii displayed chromosome morphological discrepancy, and L. podicipinus had four pairs of telocentric chromosomes. Replication banding was crucial for characterising this variability and for explaining the reduced 2n in Leptodactylus sp. (aff. podicipinus). Leptodactylus marmoratus had few chromosomes with a similar banding patterns to the 2n = 22 karyotypes. The majority of the species presented a single NOR-bearing pair, which was confirmed using Ag-impregnation and FISH with an rDNA probe. In general, the NOR-bearing chromosomes corresponded to chromosome 8, but NORs were found on chromosome 3 or 4 in some species. Leptodactylus marmoratus had NORs on chromosome pairs 6 and 8. The data from C-banding, fluorochrome staining, and FISH using the telomeric probe helped in characterising the repetitive sequences. Even though hybridisation did occur on the chromosome ends, telomere-like repetitive sequences outside of the telomere region were identified. Metaphase I cells from L. pentadactylus confirmed its complex karyotype constitution because 12 chromosomes appeared as ring-shaped chain in addition to five bivalents.

Conclusions

Species of Leptodactylus exhibited both major and minor karyotypic differences which were identified by classical and molecular cytogenetic techniques. Replication banding, which is a unique procedure that has been used to obtain longitudinal multiple band patterns in amphibian chromosomes, allowed us to outline the general mechanisms responsible for these karyotype differences. The findings also suggested that L. marmoratus, which was formerly included in the genus Adenomera, may have undergone great chromosomal repatterning.

     

Disruption of the ugt1 locus in mice resembles human Crigler-Najjar type I disease

The 9 UDP-glucuronosyltranferases (UGTs) encoded by the UGT1 locus in humans are key enzymes in the metabolism of most drugs as well as endogenous substances such as bile acids, fatty acids, steroids, hormones, neurotransmitters, and bilirubin. Severe unconjugated hyperbilirubinemia in humans that suffer from Crigler-Najjar type I disease results from lesions in the UGT1A1 gene and is often fatal. To examine the physiological importance of the Ugt1 locus in mice, this locus was rendered non-functional by interrupting exon 4 to create Ugt1(-/-) mice. Because UGT1A1 in humans is responsible for 100% of the conjugated bilirubin, it followed that newborn Ugt1(-/-) mice developed serum levels of unconjugated bilirubin that were 40-60 times higher than Ugt1(+/-) or wild-type mice. The result of extreme unconjugated bilirubin in Ugt1(-/-) mice, comparable to the induced levels noted in patients with Crigler-Najjar type 1 disease, is fatal in neonatal Ugt1(-/-) mice within 2 weeks following birth. The extreme jaundice is present as a phenotype in skin color after 8 h. Neonatal Ugt1(-/-) mice exhibit no detectable UGT1A-specific RNA, which corresponds to a complete absence of UGT1A proteins in liver microsomes. Conserved glucuronidation activity attributed to the Ugt1 locus can be defined in Ugt1(-/-) mice, because UGT2-dependent glucuronidation activity is unaffected. Remarkably, the loss of UGT1A functionality in liver results in significant alterations in cellular metabolism as investigated through changes in gene expression. Thus, the loss of UGT1A function in Ugt1(-/-) mice leads to a metabolic syndrome that can serve as a model to further investigate the toxicities associated with unconjugated bilirubin and the impact of this disease in humans.     

The emerging role of the physician in genetic counselling and testing for heritable breast,ovarian and colon cancer.

As a genetic testing for susceptibility to breast, ovarian and colon cancer becomes more readily available, physicians are faced with an increasing demand for information about inherited cancer risk. Because advances in treatment have not kept pace with advances in predictive testing, the provision of genetic counselling and testing marks a departure from the traditional role of the physician. A systematic framework is needed within which the physician''s emerging role in predictive testing for heritable cancer can be delineated. The development of such a framework will require collaboration among professionals in a range of scientific disciplines, as well as the suspension of traditional assumptions about the physicians role.