DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.     

Stoichiometric interpretation of Escherichia coli glucose catabolism under various oxygenation rates.

Metabolic by-product secretion is commonly observed in oxygen-limited cultures. Oxygen limitations occur because of limits in the capacity of the respiratory system or because of the oxygenation limits of the cultivation method used. The latter restriction is of considerable practical importance since it results in a critical cell concentration above which oxygenation is insufficient, leading to by-product secretion. In this study we used a flux balance approach to determine optimal metabolic performance of Escherichia coli under variable oxygen limitations. This method uses linear optimization to find optimal metabolic flux patterns with respect to cell growth. Cell growth was defined as precursor requirements on the basis of a composition analysis. A growth-associated maintenance requirement of 23 mmol of ATP per g of biomass and a non-growth-associated maintenance value of 5.87 mmol at ATP per g (dry weight)-h were incorporated on the basis of a comparison with experimental data. From computations of optimal growth increased oxygen limitations were found to result in the secretion of acetate, formate, and ethanol in that order. Consistent with the experimental data in the literature, by-product secretion rates increased linearly with the growth rate. The computed optimal growth under increasing oxygen limitation revealed four critical growth rates at which changes in the by-product secretion pattern were observed. Concomitant with by-product secretion under oxygen limitations were changes in metabolic pathway utilization. The shifts in metabolism were characterized by changes in the metabolic values (computed as shadow prices) of the various redox carriers. The redox potential was thus identified as a likely trigger that leads to metabolic shifts.2+ ă     

TDZ-induced direct shoot organogenesis and somatic embryogenesis on cotyledonary node explants of lentil (Lens culinaris Medik.)

An efficient and simple procedure for inducing high frequency direct shoot organogenesis and somatic embryogenesis in lentil from cotyledonary node explants (without both the cotyledons) in response to TDZ alone is reported. TDZ at concentration lower than 2.0 μM induced shoot organogenesis whereas at higher concentration (2.5–15 μM) it caused a shift in regeneration from shoot organogenesis to somatic embryogenesis. The cotyledonary node and seedling cultures developed only shoots even at high concentrations of BAP and TDZ, respectively. TDZ at 0.5 and 5.0 μM was found to be optimal for inducing an average of 4–5 shoots per cotyledonary node in 93 % of the cultures and 55 somatic embryos in 68 % of the cultures, respectively. The somatic embryos were germinated when transferred to lower TDZ concentration (0.5–1.0 μM). The shoots were rooted on MS basal medium containing 2.5 μM IBA. The plantlets were obtained within 8 weeks from initiation of culture and were morphologically similar to seed-raised plants. The possible role of stress in thidiazuron induced somatic embryogenesis is discussed.Key words: Thidiazuron, Lens culinaris, Somatic embryogenesis, Organogenesis     

Crystal structure of the BARD1 BRCT domains

The interaction of the breast tumor suppressor BRCA1 with the protein BARD1 results in the formation of a heterodimeric complex that has ubiquitin ligase activity and plays central roles in cell cycle checkpoint control and DNA repair. Both BRCA1 and BARD1 possess a pair of tandem BRCT domains that interact in a phosphorylation-dependent manner with target proteins. We determined the crystal structure of the human BARD1 BRCT repeats (residues 568-777) at 1.9 A resolution. The composition and structure of the BARD1 phosphoserine-binding pocket P1 are strikingly similar to those of the BRCA1 and MDC1 BRCT domains, suggesting a similar mode of interaction with the phosphate group of the ligand. By contrast, the BARD1 BRCT selectivity pocket P2 exhibits distinct structural features, including two prominent histidine residues, His685 and His686, which may be important for ligand binding. The protonation state of these histidines has a marked effect on the calculated electrostatic potential in the vicinity of P2, raising the possibility that ligand recognition may be regulated by changes in pH. Importantly, the BARD1 BRCT structure provides insights into the mechanisms by which the cancer-associated missense mutations C645R, V695L, and S761N may adversely affect the structure and function of BARD1.     

Immunomodulatory Expression of Cathelicidins Peptides in Pulp Inflammation and Regeneration: An Update

The role of inflammatory mediators in dental pulp is unique. The local environment of pulp responds to any changes in the physiology that are highly fundamental, like odontoblast cell differentiation and other secretory activity. The aim of this review is to assess the role of cathelicidins based on their capacity to heal wounds, their immunomodulatory potential, and their ability to stimulate cytokine production and stimulate immune-inflammatory response in pulp and periapex. Accessible electronic databases were searched to find studies reporting the role of cathelicidins in pulpal inflammation and regeneration published between September 2010 and September 2020. The search was performed using the following databases: Medline, Scopus, Web of Science, SciELO and PubMed. The electronic search was performed using the combination of keywords “cathelicidins” and “dental pulp inflammation”. On the basis of previous studies, it can be inferred that LL-37 plays an important role in odontoblastic cell differentiation and stimulation of antimicrobial peptides. Furthermore, based on these outcomes, it can be concluded that LL-37 plays an important role in reparative dentin formation and provides signaling for defense by activating the innate immune system.     

Ligand-independent negative chronotropic responses of rat and mouse right atria to beta-adrenoceptor antagonists

Negative chronotropic effects of beta-adrenoceptor (betaAR) antagonists on right atria from reserpine-treated rats and mice were determined as a test of their inverse agonist activities. BetaAR antagonist ICI-118,551 and nonselective betaAR antagonists alprenolol, propranolol, and timolol produced negative chronotropic effects. In contrast, nonselective betaAR antagonists pindolol and nadolol as well as beta1AR-selective antagonists atenolol, acebutolol, and metoprolol did not cause a significant decrease in atrial rates. The neutral antagonist pindolol but not the inverse agonist alprenolol inhibited the negative chronotropic activities of ICI-118,551. Isoprenaline, salbutamol, and noradrenaline produced positive chronotropic effects; the chronotropic effects of isoprenaline and salbutamol but not of noradrenaline were antagonized by ICI-118,551. It is concluded that both beta1AR and beta2AR mediate positive chronotropic effects of catecholamines on rat and mouse atria but only beta2AR are constitutively active.     

Nanoscale organization of multiple GPI-anchored proteins in living cell membranes

Cholesterol and sphingolipid-enriched "rafts" have long been proposed as platforms for the sorting of specific membrane components including glycosyl-phosphatidylinositol-anchored proteins (GPI-APs), however, their existence and physical properties have been controversial. Here, we investigate the size of lipid-dependent organization of GPI-APs in live cells, using homo and hetero-FRET-based experiments, combined with theoretical modeling. These studies reveal an unexpected organization wherein cell surface GPI-APs are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters. These clusters are composed of at most four molecules and accommodate diverse GPI-AP species; crosslinking GPI-APs segregates them from preexisting GPI-AP clusters and prevents endocytosis of the crosslinked species via a GPI-AP-selective pinocytic pathway. In conjunction with an analysis of the statistical distribution of the clusters, these observations suggest a mechanism for functional lipid-dependent clustering of GPI-APs.     

Mechanisms underlying ischemic diastolic dysfunction: relation between rigor,calcium homeostasis,and relaxation rate

Increased diastolic chamber stiffness (upward arrow DCS) during ischemia may result from increased diastolic calcium, rigor, or reduced velocity of relaxation. We tested these potential mechanisms during severe ischemia in isolated red blood cell-perfused isovolumic rabbit hearts. Ischemia (coronary flow reduced 83%) reduced left ventricular (LV) contractility by 70%, which then remained stable. DCS progressively increased. When LV end-diastolic pressure had increased 5 mmHg, myofilament calcium responsiveness was altered with 50 mmol/l NH(4)Cl or 10 mmol/l butanedione monoxime. These affected contractility (i.e., a calcium-mediated force) but not upward arrow DCS. Second, quick length changes reversed upward arrow DCS, supporting a rigor mechanism. Third, ischemia increased the time constant of isovolumic pressure decline from 47 +/- 3 to 58 +/- 3 ms (P < 0.02) but concomitantly abbreviated the contraction-relaxation cycle, i.e., pressure dissipation occurred earlier without diastolic tetanization. Finally, to assess any link between rate of relaxation and upward arrow DCS, hearts were exposed to 10 mmol/l calcium. Calcium doubled contractility and accelerated relaxation velocity, but without affecting upward arrow DCS. Thus upward arrow DCS developed during ischemia despite severely reduced contractility via a rigor (and not calcium mediated) mechanism. Calcium resequestration capacity was preserved, and reduced relaxation velocity was not linked to upward arrow DCS.