Reduced Ca2+-calmodulin-dependent protein kinase activity and expression in LV myocardium of dogs with heart failure

Studies on the status of multifunctional Ca(2+)-calmodulin (CaM)-dependent protein kinase-II (CaMKII) in failing hearts are limited and controversial. The study was performed in the left ventricular (LV) myocardium of six dogs with heart failure (HF) (LV ejection fraction, 23 +/- 2%) and six normal (NL) dogs. In the LV homogenate, CaMKII activity and its protein level were determined by using the CaMKII peptide and antibody, respectively. Furthermore, the protein level of CaM and phosphorylated phospholamban (PLB) at threonine-17 (PLB-Thr(17)) and serine-16 (PLB-Ser(16)) were also determined in the LV homogenate using a specific antibody. In addition, the level of zinc, which inhibits protein kinase A activity, was determined in the LV tissue by inductively coupled plasma mass spectrometry. CaMKII activity and phosphorylated PLB-Thr(17) and PLB-Ser(16) levels, but not CaM and Zn levels, were significantly reduced in the LV homogenate of dogs with HF compared with NL dogs. These results suggest that CaMKII activity is reduced in the failing LV myocardium, and this abnormality is associated with reduced protein expression level of the enzyme but not due to changes in CaM and zinc levels. In conclusion, reduced CaMKII activity and phosphorylated PLB level may be partly responsible for impaired sarcoplasmic reticulum function in HF.     

In planta regulation of the Arabidopsis Ca(2+)/H(+) antiporter CAX1

Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/H(+) antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca(2+)/H(+) transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.     

Sequence and N-glycan diversity analysis of immunoglobulin G from buffalo milk using RP-UHPLC MS/MS

Amino Acids - Immunoglobulin G is the abundant antibody present in the colostrum and milk of major dairy animals. In the present study, buffalo milk IgG was characterized for its amino acid...     

Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

Key message

Genetic improvements for many fiber traits are obtained by mutagenesis of elite cottons, mitigating genetic uniformity in this inbred polyploid by contributing novel alleles important to ongoing crop improvement.

Abstract

The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M₅ lines resulting from ethyl methanesulfonate (EMS) mutagenesis of two G. hirsutum breeding lines, TAM 94L-25 and Acala 1517-99, were characterized for basic components of fiber quality and selected yield components. Across all measured traits, the ranges of phenotypic values among the mutant lines were consistently larger than could be explained by chance (5.27–10.1 for TAM 94 L-25 and 5.29–7.94 standard deviations for Acala 1517-99-derived lines). Multi-year replicated studies confirmed a genetic basis for these differences, showing significant correlations between lines across years and environments. A subset of 157 lines selected for superior fiber qualities, including fiber elongation (22 lines), length (22), lint percent (17), fineness (23), Rd value (21), strength (19), uniformity (21) and multiple attributes in a selection index (26) were compared to 55 control lines in replicated trials in both Texas and Georgia. For all traits, mutant lines showing substantial and statistically significant improvements over control lines were found, in most cases from each of the two genetic backgrounds. This indicates that genetic improvements for a wide range of fiber traits may be obtained from mutagenesis of elite cottons. Indeed, lines selected for one fiber trait sometimes conferred additional attributes, suggesting pleiotropic effects of some mutations and offering multiple benefits for the incorporation of some alleles into mainstream breeding programs.     

Synthesis of novel cyclic NGR/RGD peptide analogs via on resin click chemistry

Targeted drug deliveries as well as high resolution imaging of cancerous tissues and organs via specific cancer cell markers have become important in chemotherapeutic interventions of cancer treatment. Short peptides such as RGD and NGR are showing promising results for targeted drug delivery and in vivo imaging. We have applied on resin Huisgen's 1,3-dipolar cycloaddition to synthesize new cyclic RGD and NGR peptide analogs. Preliminary binding assays of these new analogs by fluorescence polarization indicates specific binding to purified CD13 (Aminopeptidase N) and cell lysates from MCF-7 and SKOV-3 cancer cell lines.     

Goldenseal (<Emphasis Type="Italic">Hydrastis canadensis</Emphasis> L.): <Emphasis Type="Italic">In vitro</Emphasis> regeneration for germplasm conservation and elimination of heavy metal contamination

Summary Hydrastis canadensis L. (Goldenseal) is an endangered medicinal plant used in the treatment of many ailments, such as gastrointestinal disturbances, urinary disorders, hemorrhage, skin, mouth and eye infections, and inflammation. Commercial preparations of wild-harvested goldenseal were found to contain heavy metal contaminants including aluminum (848 μgg⁻¹), cadmium (0.4μgg⁻¹), lead (18.7μgg⁻¹), and mercury (0.1 μgg⁻¹). As well, goldenseal is an endangered species listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix II. Therefore, the practice of wild-harvest is actually decimating natural populations of goldenseal and endangering its genetic diversity. In vitro propagation protocol by tissue culture was developed for producing high-quality tissues of goldenseal. Significantly more de novo regeneration was induced on stem explants of 3-mo.-old plants cultured on a medium containing 10 μM 6-benzylaminopurine (BA) (22 regenerants per explant) than any other treatment. Subculture of the regenerants on a medium devoid of growth regulators resulted in the development of complete plants that were acclimatized and thrived in standard greenhouse conditions. The plants regenerated in vitro contained the lowest levels of heavy metals. The findings of this study provide the first evidence that heavy metal contaminants bioaccumulate in goldenseal tissues and also provide a method for germplasm conservation, mass multiplication, and production of goldenseal tissues free from abiotic contamination.     

Breakfast cereals and risk of type 2 diabetes in the Physicians' Health Study I

Objective: To examine the association between breakfast cereal consumption and the risk of type 2 diabetes (DM). Research Methods and Procedures: We analyzed prospectively data from 21,152 male participants of the Physicians’ Health Study I. Consumption of breakfast cereals was estimated using an abbreviated food questionnaire, and incident DM was ascertained through yearly follow‐up questionnaires. Results: The average age was 53.6 ± 9.4 years (range, 39.7 to 85.9) during the initial assessment of cereal intake (1981 to 1983). During a mean follow‐up of 19.1 years, 1958 cases of DM occurred. The crude incidence rates of DM were 57.7, 53.8, 43.5, and 35.4 cases/10,000 person‐years for people reporting breakfast cereal intake of 0, ≤1, 2 to 6, and ≥7 servings/wk, respectively. In a Cox regression model adjusting for age, cigarette smoking, BMI, physical activity, vegetable consumption, and alcohol intake, hazard ratios (95% confidence interval) for DM were 1.0 (reference), 0.89 (0.79 to 1.00), 0.76 (0.67 to 0.86), and 0.63 (0.55 to 0.72) from the lowest to the highest category of cereal consumption, respectively (p for trend < 0.0001). In secondary analyses, the inverse association between cereal intake and DM was stronger with whole‐grain than refined cereals. Discussion: These results suggest that intake of breakfast cereals might confer a lower risk of DM. Consumption of whole‐grain products may help lower the risk of DM.     

Analysis of the zinc finger domain of TnpA, a DNA targeting protein encoded by mobilizable transposon Tn4555

The mobilizable transposon Tn4555, found in Bacteroides spp., is an important antibiotic resistance element encoding a broad spectrum beta-lactamase. Tn4555 is mobilized by conjugative transposons such as CTn341 which can transfer the transposon to a wide range of bacterial species where it integrates into preferred sites on the host chromosome. Selection of the preferred target sites is mediated by a DNA-binding protein TnpA which has a prominent zinc finger motif at the N-terminus of the protein. In this report the zinc finger motif was disrupted by site directed mutagenesis in which two cysteine residues were changed to serine residues. Elemental analysis indicated that the wild-type protein but not the mutated protein was able to coordinate zinc at a molar ration of 1/1. DNA binding electrophoretic mobility shift assays showed that the ability to bind the target site DNA was not significantly affected by the mutation but there was about a 50% decrease in the ability to bind single stranded DNA. Consistent with these results, electrophoretic mobility shift assays incorporating zinc chelators did not have a significant on affect the binding of DNA target. In vivo, the zinc finger mutation completely prevented transposition/integration as measured in a conjugation assay. This was in contrast to results in which a TnpA knockout was still able to insert into host genomes but there was no preferred target site selection. The phenotype of the zinc finger mutation was not effectively rescued by providing wild-type TnpA in trans. Taken together these results indicated that the zinc finger is not required for DNA binding activity of TnpA but that it does have an important role in transposition and it may mediate protein/protein interactions with integrase or other Tn4555 proteins to facilitate insertion into the preferred sites.