Cellular NADH and NADPH Conformation as a Real-Time Fluorescence-Based Metabolic Indicator under Pressurized Conditions

Cellular conformation of reduced pyridine nucleotides NADH and NADPH sensed using autofluorescence spectroscopy is presented as a real-time metabolic indicator under pressurized conditions. The approach provides information on the role of pressure in energy metabolism and antioxidant defense with applications in agriculture and food technologies. Here, we use spectral phasor analysis on UV-excited autofluorescence from Saccharomyces cerevisiae (baker’s yeast) to assess the involvement of one or multiple NADH- or NADPH-linked pathways based on the presence of two-component spectral behavior during a metabolic response. To demonstrate metabolic monitoring under pressure, we first present the autofluorescence response to cyanide (a respiratory inhibitor) at 32 MPa. Although ambient and high-pressure responses remain similar, pressure itself also induces a response that is consistent with a change in cellular redox state and ROS production. Next, as an example of an autofluorescence response altered by pressurization, we investigate the response to ethanol at ambient, 12 MPa, and 30 MPa pressure. Ethanol (another respiratory inhibitor) and cyanide induce similar responses at ambient pressure. The onset of non-two-component spectral behavior upon pressurization suggests a change in the mechanism of ethanol action. Overall, results point to new avenues of investigation in piezophysiology by providing a way of visualizing metabolism and mitochondrial function under pressurized conditions.

Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved

The spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a > tenfold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. ± NO) and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO.

Comparative biological sensitivity and mutability of chemo-mutagens in lentil (Lens culinaris Medik)

Present investigation was carried out using three different categories of chemical mutagens viz., ethylmethane sulphonate (EMS)–an alkylating agent, hydrazine hydrate (HZ)–a base analogue and sodium azide (SA) – a respiratory inhibitor on two varieties viz., Pant L-406 and Type-8 of lentil to study the immediate biological damage induced by the mutagens and to determine the sensitivity of biological material in question. Biological damage induced in M1 generation was estimated in terms of seed germination, seedling height and pollen fertility. A dose dependent reduction with increasing concentrations of the mutagens for all these parameters was observed in both the varieties. The inhibition was more severe at the highest concentration of all the three mutagens under study. Variety Type-8 was found to be more sensitive than the var. Pant L-406 with respect to the mutagens utilized. Reduction in seed germination, seedling growth and pollen fertility in M2 generation was reasonably less as compared to M1 generation.

KlHsl1 is a component of glycerol response pathways in the milk yeast Kluyveromyces lactis

In Saccharomyces cerevisiae, HSL1 (NIK1) encodes a serine-threonine protein kinase involved in cell cycle control and morphogenesis. Deletion of its putative orthologue in Kluyveromyces lactis, KlHSL1, gives rise to sensitivity to the respiratory inhibitor antimycin A (AA). Resistance to AA on glucose (Rag+ phenotype) is associated with genes (RAG) required for glucose metabolism/glycolysis. To understand the relationship between RAG and KlHSL1, rag and Klhsl1Δ mutant strains were investigated. The analysis showed that all the mutants contained a phosphorylated form of Hog1 and displayed an inability to synthesize/accumulate glycerol as a compatible solute. In addition, rag mutants also showed alterations in both cell wall and membrane fatty acids. The pleiotropic defects of these strains indicate that a common pathway regulates glucose utilization and stress response mechanisms, suggesting impaired adaptation of the plasma membrane/cell wall during the respiratory–fermentative transition. KlHsl1 could be the link between these adaptive pathways and the morphogenetic checkpoint.

The paradoxical cyanide-stimulated respiration of Zymomonas mobilis: cyanide sensitivity of alcohol dehydrogenase (ADH II)

The respiratory inhibitor cyanide stimulates growth of the ethanologenic bacterium Zymomonas mobilis, perhaps by diverting reducing equivalents from respiration to ethanol synthesis, thereby minimizing accumulation of toxic acetaldehyde. This study sought to identify cyanide-sensitive components of respiration. In aerobically grown, permeabilized Z. mobilis cells, addition of 200 μM cyanide caused gradual inhibition of ADH II, the iron-containing alcohol dehydrogenase isoenzyme, which, in aerobic cultures, might be oxidizing ethanol and supplying NADH to the respiratory chain. In membrane preparations, NADH oxidase was inhibited more rapidly, but to a lesser extent, than ADH II. The time-course of inhibition of whole-cell respiration resembled that of NADH oxidase, yet the inhibition was almost complete, and was accompanied by an increase of intracellular NADH concentration. Cyanide did not significantly affect the activity of ADH I, the zinc-containing alcohol dehydrogenase isoenzyme. When an aerobic batch culture was grown in the presence of 200 μM cyanide, cyanide-resistant ADH II activity was observed, its appearance correlating with the onset of respiration. It is concluded that the membrane-associated respiratory chain, but not ADH II, is responsible for the whole-cell cyanide sensitivity, while the cyanide-resistant ADH II is needed for respiration in the presence of cyanide, and represents an adaptive response of Z. mobilis to cyanide, analogous to the induction of alternative terminal oxidases in other bacteria.

Response of isolated adult canine cardiac myocytes to prolonged hypoxia and reoxygenation

Isolated adult canine ventricular myocytes incubated in the absence of glucose with the respiratory inhibitor rotenone retained 67% of ATP (control, 26.0 +/- 0.9 nmol/mg protein) during 3-h incubation, yet phosphocreatine fell to 23% of initial content. Lactate production proceeded at a constant rate of 5 nmol.mg-1.min-1 in rotenone-treated glucose-free myocytes. A 36% decline in rod-shaped cells and an increase in percent 22Na permeation from 37% in aerobic cells (approximately 13 mM intracellular sodium) to 68% in rotenone-treated glucose-free myocytes paralleled the loss of ATP. Total exchangeable calcium was maintained at control aerobic levels. Exposure of canine cells to 3-h hypoxia in the absence of glucose followed by 5-min reoxygenation resulted in a 73% decrease in ATP, a rise in calcium from 3.3 +/- 0.2 to 6.6 +/- 1.6 nmol/mg, and an increase in 22Na permeation to 111%. Under these conditions the number of rod-shaped myocytes declined by 77%, with corresponding increases in viable contracted and hypercontracted myocytes. The response of canine myocytes to severe hypoxia and reaeration contrasts greatly to earlier studies using adult rat cardiac myocytes [see Hohl et al. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H1022-H1030, 1982]. Species differences with respect to basal metabolism, rates of ATP production and degradation, and regulation of cation movements are most likely responsible for the observed differences.