Identification of Phorbol Myristate Acetate Stimulated Kinase in Zea mays

Following DEAE-Sephacel and affinity chromatography a highly enriched lipid stimulated kinase activity could be recovered with a purification fold of 1725. The peak kinase activity fraction eluted with 0.1 mM calcium from phosphatidyl serine affinity chromatography showed a major protein of 70 kD and a minor band of 55 kD molecular weight and showed kinase activity that was stimulated by phorbol myristate acetate in the presence of phosphatidylserine and calcium. The optimum requirement was 2.5 × 10^?6 M, 1.25 × 10^?4 M, 1 × 10^?4 M, and 1.7 × 10^?6 M for phorbol myristate acetate, phosphatidyl serine, oleyl acetyl glycerol and free calcium respectively. The kinase activity was inhibited by H-7 and staurosporine. The binding of [³H]-phorbol myristate acetate was associated with purified fraction as resolved by get electrophoresis and the kinase activity was also precipitated by animal protein kinase C antibodies. The present data give strong evidence for the presence of phorbol myristate acetate stimulated kinase in plants.     

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.     

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.

     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Adverse events among medical patients after discharge from hospital

BackgroundAdverse events (AEs) are adverse outcomes caused by medical care. Several studies have indicated that a substantial number of patients experience AEs before or during hospitalization. However, few data describe AEs after hospital discharge. We determined the incidence, severity, preventability and ameliorability of AEs in patients discharged from the general internal medicine service of a Canadian hospital.MethodsAt a multisite Canadian teaching hospital, we prospectively studied patients who were consecutively discharged home or to a seniors'' residence from the general internal medicine service during a 14-week interval in 2002. We used telephone interview and chart review to identify outcomes after discharge. Two physicians independently reviewed each outcome to determine if the patient experienced an AE. The severity, preventability and ameliorability of all AEs were classified.ResultsDuring the study period, outcomes were determined for 328 of the 361 eligible patients, who averaged 71 years of age (interquartile range 54–81 years). After discharge, 76 of the 328 patients experienced at least 1 AE (overall incidence 23%, 95% confidence interval [CI] 19%–28%). The AE severity ranged from symptoms only (68% of the AEs) or symptoms associated with a nonpermanent disability (25%) to permanent disability (3%) or death (3%). The most common AEs were adverse drug events (72%), therapeutic errors (16%) and nosocomial infections (11%). Of the 76 patients, 38 had an AE that was either preventable or ameliorable (overall incidence 12%, 95% CI 9%–16%).InterpretationApproximately one-quarter of patients in our study had an AE after hospital discharge, and half of the AEs were preventable or ameliorable.The Institute of Medicine report Crossing the quality chasm identifies patient safety as a prerequisite to high-quality care.¹ The need to improve safety is highlighted by research showing that hospitalized patients have a high risk of adverse outcomes resulting from treatment. For example, the Harvard Medical Practice Study found that adverse events (AEs) occurred in 3.7% of hospitalized patients.^2,3 Similar studies have found equivalent or greater rates.^4,5,6 Other research has found that AE risk increases with design flaws in the health care system.^7,8,9,10Such flaws may particularly affect patient care immediately after hospital discharge, a period associated with discontinuities in providers and in location of care. Some authors suggest that such “gaps” are important causes of error.¹¹ It is also a time when patients frequently experience extensive changes in health¹² and therapy.¹³ Finally, communication between hospital and community physicians can be inadequate.¹⁴ For these reasons, AEs may be common after discharge. A recent study, by a group that included 1 of us, found that 19% of medical patients discharged from a single teaching hospital in the United States experienced an AE within a month.¹⁵ One-third of these AEs were preventable because they were due to an error. Another third were judged “ameliorable” because their severity could have been reduced with better monitoring or earlier response to the problem.¹⁵That study was important, but it had limitations. It was carried out in a single, very specialized institution, it relied on data available in an electronic medical record, and it had a high rate of loss to follow-up. To address these concerns, we carried out a new study to determine the risk, severity and type of AEs after discharge from 2 campuses of a Canadian teaching hospital.     

Shorter leukocyte telomere length is associated with severity of COVID-19 infection.

The infection by COVID-19 is a serious global public health problem. An efficient way to improve this disease's clinical management would be to characterize patients at higher risk of progressing to critically severe infection using prognostic biomarkers. The telomere length could be used for this purpose. Telomeres are responsible for controlling the number of maximum cell divisions. The telomere length is a biomarker of aging and several diseases. We aimed to compare leukocyte telomere length (LTL) between patients without COVID-19 and patients with different clinical severity of the infection. Were included 53 patients who underwent SARS-CoV-2 PCR divided in four groups. The first group was composed by patients with a negative diagnosis for COVID-19 (n = 12). The other three groups consisted of patients with a confirmed diagnosis of COVID-19 divided according to the severity of the disease: mild (n = 15), moderate (n = 17) and severe (n = 9). The LTL was determined by Q-PCR. The severe group had the shortest LTL, followed by the moderate group. The negative and mild groups showed no differences. There is an increase of patients with hypertension (p = 0.0099) and diabetes (p = 0.0067) in moderate and severe groups. Severe group was composed by older patients in comparison with the other three groups (p = 0.0083). Regarding sex, there was no significant difference between groups (p = 0.6279). In an ordinal regression model, only LTL and diabetes were significantly associated with disease severity. Shorter telomere length was significantly associated with the severity of COVID-19 infection, which can be useful as a biomarker or to better understand the SARS-CoV-2 pathophysiology.     

A biochemical signature for rapid recall of memory CD4 T cells

Mechanisms for the rapid recall response mediated by memory T cells remain unknown. In this study, we present a novel, multiparameter analysis of TCR-coupled signaling and function in resting and activated naive and memory CD4 T cells, revealing a biochemical basis for immunological recall. We identify a striking elevation in expression of the proximal tyrosine kinase Zap70 in resting Ag-specific and polyclonal mouse memory vs naive CD4 T cells that is stably maintained independent of protein synthesis. Elevated Zap70 protein levels control effector function as IFN-gamma production occurs exclusively from the Zap70(high) fraction of activated T cells in vitro and in vivo, and specific down-modulation of Zap70 expression in memory CD4 T cells by small interfering RNA or protein inhibition significantly reduces rapid IFN-gamma production. Downstream of Zap70, we show quantitative differences in distal phosphorylation associated with effector function in naive and memory subsets, with low accumulation of phosphorylation in memory T cells producing IFN-gamma at early time points, contrasting extensive phosphorylation associated with IFN-gamma production following sustained activation of naive T cells. Our results reveal a novel biochemical signature imparted to memory CD4 T cells enabling efficacious responses through increased Zap70 expression and reduced accumulation of downstream signaling events.