Identification of a Developmentally Regulated Calcium-Binding Protein in Trypanosoma brucei1

Calcium ions mediate cellular activity by binding to specific cellular proteins. The following study systematically examines the cellular complement of calcium-binding proteins in different cell fractions and life cycle stages of Trypanosoma brucei. Using a ⁴⁵Ca-gel overlay procedure, eight calcium-binding proteins were consistently observed. The majority of proteins were cytosolic (84, 70, 64, 22, and 15 kd) while the remainder (55, 46, and 29 kd) were particulate. Although calmodulin was detected amongst the calcium-binding proteins, it did not represent the majority of calcium-binding activity. Of special interest was the 46 kd calcium-binding protein which was associated with 3-fold more calcium in cultured procyclic forms than in slender bloodstream forms. By contrast, promastigote forms of Leishmania mexicana did not contain the 46 kd calcium-binding protein. These data suggest that responsiveness to calcium signals may vary during the trypanosome life cycle as a result of changes in the cellular complement of calcium-binding proteins.     

Functional significance of phosphorylation to the human immunodeficiency virus Rev protein.

The human immunodeficiency virus Rev protein is posttranslationally modified by a serine kinase activity present in the nucleus of the cell. Site-directed mutagenesis was used to identify the site of phosphorylation. Changing of serine residues 92 and 99 dramatically reduced Rev phosphorylation, suggesting that at least one, if not both, of these residues is the one recognized by the Rev-specific serine kinase. Similarly, a truncated Rev protein lacking the 25 carboxy-terminal amino acids was not phosphorylated. By using two independent assays, both the serine mutant proteins and the truncated form of Rev were found to be fully functional. Thus, phosphorylation and the 25 carboxy-terminal amino acids appear to be dispensable for protein function.     

Ecdysterone selectively stimulates the expression of a 23000-Da heat-shock protein-β-galactosidase hybrid gene in cultured Drosophila cells

To study the regulated expression of cloned heat-shock genes in homologous cells, hybrid Drosophila heat-shock-Escherichia coli β-galactosidase genes were constructed. Segments of the ecdysterone-inducible 23,000-Da heat-shock protein (hsp23) gene and of two other hsp genes (hsp84 and 70), which are not hormone regulated, were functionally linked to the bacterial coding sequence, and the resulting hybrid genes were introduced into cultured, hormone-responsive Drosophila cells by transfection. All hybrid genes directed the synthesis of E. coli-specific β-galactosidase in heat-treated cells. hsp23 hybrid gene expression was stimulated strongly by ecdysterone, while the activities of the other hybrid genes were not affected at all by the hormone. A hybrid gene with only 147 bp of hsp23 promoter sequence could not be activated by either heat or ecdysterone treatment. Thus, far upstream sequences contain signals required for the regulated expression of the hsp23 gene in Drosophila cells.     

Genetically Diverse Low Pathogenicity Avian Influenza A Virus Subtypes Co-Circulate among Poultry in Bangladesh

Influenza virus surveillance, poultry outbreak investigations and genomic sequencing were assessed to understand the ecology and evolution of low pathogenicity avian influenza (LPAI) A viruses in Bangladesh from 2007 to 2013. We analyzed 506 avian specimens collected from poultry in live bird markets and backyard flocks to identify influenza A viruses. Virus isolation-positive specimens (n = 50) were subtyped and their coding-complete genomes were sequenced. The most frequently identified subtypes among LPAI isolates were H9N2, H11N3, H4N6, and H1N1. Less frequently detected subtypes included H1N3, H2N4, H3N2, H3N6, H3N8, H4N2, H5N2, H6N1, H6N7, and H7N9. Gene sequences were compared to publicly available sequences using phylogenetic inference approaches. Among the 14 subtypes identified, the majority of viral gene segments were most closely related to poultry or wild bird viruses commonly found in Southeast Asia, Europe, and/or northern Africa. LPAI subtypes were distributed over several geographic locations in Bangladesh, and surface and internal protein gene segments clustered phylogenetically with a diverse number of viral subtypes suggesting extensive reassortment among these LPAI viruses. H9N2 subtype viruses differed from other LPAI subtypes because genes from these viruses consistently clustered together, indicating this subtype is enzootic in Bangladesh. The H9N2 strains identified in Bangladesh were phylogenetically and antigenically related to previous human-derived H9N2 viruses detected in Bangladesh representing a potential source for human infection. In contrast, the circulating LPAI H5N2 and H7N9 viruses were both phylogenetically and antigenically unrelated to H5 viruses identified previously in humans in Bangladesh and H7N9 strains isolated from humans in China. In Bangladesh, domestic poultry sold in live bird markets carried a wide range of LPAI virus subtypes and a high diversity of genotypes. These findings, combined with the seven year timeframe of sampling, indicate a continuous circulation of these viruses in the country.     

Effect of Metformin Treatment on Lipoprotein Subfractions in Non-Diabetic Patients with Acute Myocardial Infarction: A Glycometabolic Intervention as Adjunct to Primary Coronary Intervention in ST Elevation Myocardial Infarction (GIPS-III) Trial

Objective

Metformin affects low density lipoprotein (LDL) and high density (HDL) subfractions in the context of impaired glucose tolerance, but its effects in the setting of acute myocardial infarction (MI) are unknown. We determined whether metformin administration affects lipoprotein subfractions 4 months after ST-segment elevation MI (STEMI). Second, we assessed associations of lipoprotein subfractions with left ventricular ejection fraction (LVEF) and infarct size 4 months after STEMI.

Methods

371 participants without known diabetes participating in the GIPS-III trial, a placebo controlled, double-blind randomized trial studying the effect of metformin (500 mg bid) during 4 months after primary percutaneous coronary intervention for STEMI were included of whom 317 completed follow-up (clinicaltrial.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT01217307","term_id":"NCT01217307"}}NCT01217307). Lipoprotein subfractions were measured using nuclear magnetic resonance spectroscopy at presentation, 24 hours and 4 months after STEMI. (Apo)lipoprotein measures were obtained during acute STEMI and 4 months post-STEMI. LVEF and infarct size were measured by cardiac magnetic resonance imaging.

Results

Metformin treatment slightly decreased LDL cholesterol levels (adjusted P = 0.01), whereas apoB remained unchanged. Large LDL particles and LDL size were also decreased after metformin treatment (adjusted P<0.001). After adjustment for covariates, increased small HDL particles at 24 hours after STEMI predicted higher LVEF (P = 0.005). In addition, increased medium-sized VLDL particles at the same time point predicted a smaller infarct size (P<0.001).

Conclusion

LDL cholesterol and large LDL particles were decreased during 4 months treatment with metformin started early after MI. Higher small HDL and medium VLDL particle concentrations are associated with favorable LVEF and infarct size.     

The Allelochemical MDCA Inhibits Lignification and Affects Auxin Homeostasis

The phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA) is a plant-derived compound first extracted from roots of Asparagus officinalis and further characterized as an allelochemical. Later on, MDCA was identified as an efficient inhibitor of 4-COUMARATE-CoA LIGASE (4CL), a key enzyme of the general phenylpropanoid pathway. By blocking 4CL, MDCA affects the biosynthesis of many important metabolites, which might explain its phytotoxicity. To decipher the molecular basis of the allelochemical activity of MDCA, we evaluated the effect of this compound on Arabidopsis thaliana seedlings. Metabolic profiling revealed that MDCA is converted in planta into piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H), the enzyme directly upstream of 4CL. The inhibition of C4H was also reflected in the phenolic profile of MDCA-treated plants. Treatment of in vitro grown plants resulted in an inhibition of primary root growth and a proliferation of lateral and adventitious roots. These observed growth defects were not the consequence of lignin perturbation, but rather the result of disturbing auxin homeostasis. Based on DII-VENUS quantification and direct measurement of cellular auxin transport, we concluded that MDCA disturbs auxin gradients by interfering with auxin efflux. In addition, mass spectrometry was used to show that MDCA triggers auxin biosynthesis, conjugation, and catabolism. A similar shift in auxin homeostasis was found in the c4h mutant ref3-2, indicating that MDCA triggers a cross talk between the phenylpropanoid and auxin biosynthetic pathways independent from the observed auxin efflux inhibition. Altogether, our data provide, to our knowledge, a novel molecular explanation for the phytotoxic properties of MDCA.Plants growing in a tight community are in continuous competition for space, light, water, and nutrients. Potential survival strategies include optimizing plant architecture and maximizing growth rate, allowing the plant to capture light and receive nutrients and water more efficiently, while placing neighboring plants in an unfavorable position (Einhellig, 1995; Weir et al., 2004). Besides developmental shifts, plants release an array of secondary metabolites (allelochemicals) into the rhizosphere to negatively affect the growth and reproduction of neighboring, competitor plants (Putnam, 1988; Bertin et al., 2003). Despite a lot of research effort having been devoted to allelopathic chemical warfare over the past decades, it remains a difficult study object due to the complexity of plant-plant interactions (Zeng, 2014). Nevertheless, the significance of allelochemicals in structuring plant communities and preserving biodiversity has been fully recognized by the scientific community. Moreover, allelochemicals show the potential to be used as an environmentally friendly alternative for weed control to improve agricultural productivity (Zeng, 2014).Strictly speaking, the term “allelochemical” refers to a compound produced and released by one organism to affect the growth and development of susceptible species (Weir et al., 2004). In practice, compounds derived from plant extracts or exudates are often cataloged as allelochemicals based on their inhibitory effect on seed germination and/or growth of other plant species in an artificial setup. Despite their importance, the molecular mode of action of a given allelochemical compound has rarely been studied in detail; however, toxicity is relatively easily demonstrated, identifying its molecular target is far more challenging. An interesting example is the phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA), which was isolated from lyophilized root tissues of Asparagus [Asparagus officinalis L.; Hartung et al. (1990)]. It was suggested to be an allelochemical based on its inhibitory effect on root and shoot growth of Lepidium sativum (Hartung et al., 1990). Independent studies revealed that MDCA acts as an efficient competitive inhibitor of 4-COUMARATE-CoA LIGASE (4CL), the enzyme converting hydroxycinnamates to their corresponding CoA-esters (Knobloch and Hahlbrock, 1977; Chakraborty et al., 2009). This conversion is an early step in the general phenylpropanoid pathway leading to a wide array of metabolites, including coumarins, stilbenes, salicylic acid, flavonoids, and monolignols (Vogt, 2010). Given that inhibition of 4CL in this metabolic pathway will have far-reaching effects on plant growth and development (Voelker et al., 2010), it is tempting to link the proposed phytotoxicity of MDCA to this metabolic block.Here, we evaluate whether the phytotoxicity of MDCA is a direct consequence of the inhibition of 4CL or if MDCA targets also other biological processes in Arabidopsis (Arabidopsis thaliana). We found that MDCA causes strong developmental defects in Arabidopsis seedlings at early developmental stages. Convincing evidence was found that MDCA affects the homeostasis of the plant signaling compound auxin. Our results provide an alternative explanation for the molecular mechanism underlying the phytotoxic properties of MDCA, and suggest that these multiple modes of action make it an attractive candidate as an environmental agrochemical or synergist.     

The effects of knee joint kinematics on anterior cruciate ligament injury and articular cartilage damage

This study determined which knee joint motions lead to anterior cruciate ligament (ACL) rupture with the knee at 25° of flexion. The knee was subjected to internal and external rotations, as well as varus and valgus motions. A failure locus representing the relationship between these motions and ACL rupture was established using finite element simulations. This study also considered possible concomitant injuries to the tibial articular cartilage prior to ACL injury. The posterolateral bundle of the ACL demonstrated higher rupture susceptibility than the anteromedial bundle. The average varus angular displacement required for ACL failure was 46.6% lower compared to the average valgus angular displacement. Femoral external rotation decreased the frontal plane angle required for ACL failure by 27.5% compared to internal rotation. Tibial articular cartilage damage initiated prior to ACL failure in all valgus simulations. The results from this investigation agreed well with other experimental and analytical investigations. This study provides a greater understanding of the various knee joint motion combinations leading to ACL injury and articular cartilage damage.     

Slow inactivation in human cardiac sodium channels.

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.     

A rapid dot immunoassay for detecting the Brazilian Purpuric Fever clone of Haemophilus influenzae biogroup aegyptius with a “flow through” device

Brazilian purpuric fever (BPF) is a highly fatal pediatric disease that may follow an episode of purulent conjunctivitis caused by a virulent clone of Haemophilus influenzae biogroup aegyptius (Hae). Oral rifampin prophylaxis, by eliminating carriage of the BPF clone in children with conjunctivitis, may prevent onset of the systemic disease. A test to detect the BPF clone directly from eye swabs could indentify those in need of prophylaxis. This is a preliminary report of a rapid dot immunoassay performed on a flow-through cartridge that was developed for use under field conditions. The test is based upon recognition of a unique epitope of the 25-kDa pilin protein on the surface of BPF clone cells by a monoclonal antibody. With 36 laboratory-maintained cultures of Hae (15 clone isolates and 21 others), sensitivity of the assay was 67% and specificity was 95%. When fimbrial-enriched (25-kDa+) phenotypes of five false-negative clone strains were prepared for use as test antigens, sensitivity rose to 100%. Evaluation of the immunoassay under field conditions is necessary to prove its efficacy.