Variability In Enzymatic Activity Research Articles

Parallel Dielectrophoretic Capture, Isolation, and Electrical Lysis of Individual Breast Cancer Cells to Assess Variability in Enzymatic Activity.

Tumor cell heterogeneity drives disease progression and response to therapy, and therefore, there is a need for single-cell analysis methods. In this paper, we present an integrated, scalable method to analyze enzymatic activity in many individual cancer cells at once. The reported method uses dielectrophoresis (DEP) to selectively capture tumor cells at wireless electrodes aligned to an overlying array of cell-sized micropockets. Following hydrodynamic transfer of the captured cells into microfluidic chambers, the chambers are fluidically isolated and sealed with a hydrophobic ionic liquid, which possesses sufficient conductivity to allow for subsequent electrical lysis of the cells to access their contents for enzymatic assay. The wireless electrodes have an interlocking spiral design that ensures successful electrical lysis regardless of the location of the cell within the chamber. Here, breast cancer cells are assessed for β-galactosidase through its activation of a fluorogenic substrate. A key point is that the fluorogenic assay solution was optimized to allow for dielectrophoretic cell capture, thereby obviating the need for a solution exchange step. Our approach has several distinct advantages including a high rate of single-cell capture, a capture efficiency that is independent of the dimensions of the reaction chambers, no need for mechanical closure of reaction volumes, and no observed cross-talk. In this study, first, the steps of cell capture, transfer, and lysis are established on this platform in the presence of the optimized assay solution. We then quantify the increase in fluorescence intensity obtained over the duration of the enzymatic assay of individual cells. Finally, this method is applied to the analysis of β-galactosidase activity in 258 individual MDA-MB-231 breast cancer cells, revealing heterogeneity in expression of this enzyme in this cell line. We expect that the adaptability of this method will allow for expanded studies of single-cell enzymatic expression and activity. This will in turn open avenues of research into cancer cell heterogeneity in metabolism, invasiveness, and drug response. The ability to study these features of cancer at the single-cell level raises the possibility for treatment plans tailored to target the specific combinations of cell subpopulations present in tumors. Furthermore, we expect that this method can be adapted to uses outside of cancer research, such as studies of neuron metabolism, pathogenesis in bacteria, and stem cell development.

Fire blight rootstock infections causing apple tree death: A case study in high-density apple orchards with Erwinia amylovora strain characterization

A devastating outcome of fire blight in apple trees is the infection of rootstock, which leads to canker development. Fire blight cankers are infected zones of dead bark on perennial branches, trunk, or rootstock that develop after fire blight pathogen Erwinia amylovora invades wood. Cankers can girdle the trunk, branches and rootstock leading to tree death and production losses, especially significant in high-density orchards. An accurate diagnosis of trunk and rootstock blight is a top challenge for apple growers because fire blight cankers can be visually misdiagnosed with cankers caused by less frequent Oomycete and fungal pathogens (e.g., Phytophthora spp., Botryosphaeria dothidea, B. obtusa, Valsa mali). In addition, detecting E. amylovora in apple rootstocks is essential because this pathogen also causes asymptomatic infections. Accurate fire blight diagnosis is necessary to inform the complete removal of infected trees from the orchard and help replanting efforts while preventing further pathogen dissemination. To determine apple tree losses caused by fire blight rootstock infections, PCR was used to detect E. amylovora in symptomatic and asymptomatic rootstocks for two years. Rootstock canker incidence and tree death were rated in selected infection foci on seven commercial apple orchards in New York. Each infection focus consisted of central rootstock-blighted tree and the nearest surrounding edge trees showing no rootstock blight. E. amylovora strains collected from these seven orchards and other orchards in New York were characterized. In the first year, most of the orchards showed E. amylovora detection rates of 10.7 – 45.3% in asymptomatic rootstocks on the edge trees immediately surrounding visibly infected i.e. rootstock-blighted central trees. One year later, 20.8 – 56.3% cankered rootstocks were detected on the edge trees and from zero to 35.4% dead edge trees were recorded. However, the PCR from sampled edge rootstocks one year later showed no pathogen detections. E. amylovora rootstock strains showed slight variability in enzymatic activity, copper sensitivity, virulence, and exopolysaccharide production. These results elucidate the role and importance of rootstock infections for apple tree survival, the presence of latent fire blight infections, and the valuable utility of molecular detection methods to assist apple tree removal after epidemics.

Variability of enzymatic activity in forest Cambisols and Brunic Arenosols of Polish lowland areas

Abstract The aim of this study was to determine the enzymatic activity (dehydrogenase and urease) in trophically diverse Brunic Arenosols and Cambisols. Efforts to establish the relationship between enzymatic activity and physico-chemical properties of various subtypes of Brunic Arenosols and Cambisols were attempted. Another aim was to determine the effect of vegetation on the properties of soil surface levels. The study was conducted on 94 plots located in nature reserves and national parks in the Polish lowland area. Dehydrogenase activity and urease showed large variations in the subtypes of the distinguished Brunic Arenosols and Cambisols. Dehydrogenases and urease activity in surface layers of fresh humus of Cambisols and Brunic Arenosols was strongly associated with the plants. This is confirmed by the relationship between the enzymatic activity and the type of accumulated humus substances, the ratio of carbon to nitrogen and humus horizons reaction.

Deterioration and Variability of Highly Purified Collagenase Blends Used in Clinical Islet Isolation

The quality and stability of enzyme blends used in islet cell processing are critical for successful human islet isolation. A wide variability in enzymatic activity among lots of Liberase HI has been reported. This study examines the interlot and intralot variability of Liberase HI and the over-time deterioration of enzyme quality based on the analysis of islet isolation outcomes. The data of 169 human isolations processed for clinical islet transplantation, using five different lots of Liberase HI, were retrospectively analyzed. Inter- and intralot variables in the islet isolation were assessed over a 15-month period. The analysis revealed significant interlot differences in the digestion time, prepurification islet counts, percent recovery, viability, and glucose stimulation insulin index. Moreover, a significant decrease in the pre- and postpurification islet yield per pancreas weight (IEQ/g) in isolations processed by two different enzyme lots used over a 15-month period was observed, suggesting a progressive deterioration of enzyme quality. Our data demonstrate a significant lot-to-lot related variability in islet isolation outcomes. In addition, the over-time decline in isolation outcomes processed using a single enzyme lot was observed even when the enzyme blends were used within the expiration dating specified by the manufacturer.

Variability of enzymatic activities in ligninolytic fungi Pleurotus ostreatus and Lentinus tigrinus after protoplasting and UV-mutagenization

Protoplast preparation and UV-irradiation of mycelial fragments were used to study the variability of production of laccase, peroxidase and manganese-dependent peroxidase (MnP) involved in lignin degradation in Pleurotus ostreatus and Lentinus tigrinus. After protoplasting, the variability of production of all enzymes increased substantially and was comparable to that of isolates after mutagenesis.

Gastric K +-stimulated adenosine triphosphatase. Demonstration of an endogenous activator

A K’stimulated, ouabain insensitive ATPase activity associated with the apical and tubulovesicular membranes of the acid secreting cells of fundic mucosa from a number of different species has recently been reported [ 1,2] and has been strongly implicated to be involved in H+ transport [3-S]. During isolation of purified microsomal membrane vesicles enriched in K’stimulated ATPase from the fundus of various species, we observed that the degree of stimulation of the enzyme by K’ in the presence of gramicidin varied considerably from one preparation to another. The ionophore, gramicidin, has been shown to be very effective for maximal activation of the microsomal K+-stimulated ATPase [2] . The variability in enzymatic activity observed in presence of gramicidin indicated that there could be some dissociable regulatory factor(s) bound to the enzyme under in vivo conditions. The degree of dissociation of the factor(s) during isolation of the purified membranes might account for the variable activity of the enzyme. In this report, we present evidence that there is a heat-labile, non-dialysable and protease-sensitive activator in the soluble supernatant fraction from rabbit fundic mucosal cells, which markedly activates the K+-stimulated ATPase and pNPPase activities of rabbit, frog, and hog gastric microsomes. Furthermore, Ca2+ at low concentrations has been shown to obliterate the activating effects of the activator(s).