Protoporphyrin IX-induced structural and functional changes in human red blood cells,haemoglobin and myoglobin

Protoporphyrin IX and its derivatives are used as photosensitizers in the photodynamic therapy of cancer. Protoporphyrin IX penetrates into human red blood cells and releases oxygen from them. This leads to a change in the morphology of the cells. Spectrophotometric studies reveal that protoporphyrin IX interacts with haemoglobin and myoglobin forming ground state complexes. For both proteins, the binding affinity constant decreases, while the possible number of binding sites increases, as the aggregation state of the porphyrin is increased. The interactions lead to conformational changes of both haemoglobin and myoglobin as observed in circular dichroism studies. Upon binding with the proteins, protoporphyrin IX releases the heme-bound oxygen from the oxyproteins, which is dependent on the stoichiometric ratios of the porphyrin: protein. The peroxidase activities of haemoglobin and myoglobin are potentiated by the protein-porphyrin complexation. Possible mechanisms underlying the relation between the porphyrin-induced structural modifications of the heme proteins and alterations in their functional properties have been discussed. The findings may have a role in establishing efficacy of therapeutic uses of porphyrins as well as in elucidating their mechanisms of action as therapeutic agents.     

Analysis of glycated insulin by MALDI-TOF mass spectrometry

Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available alpha- or epsilon-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1-13 and B 22-30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.     

Effect of some nitrogenous salts on nitrogen transfer and protease activity in germinatingZea mays L. seeds

Maize seeds were allowed to germinate in the presence of different nitrogenous salts for 72 h. Changes in the ethanol soluble and insoluble nitrogen were studied in the embryo and in the endosperm. Supply of Ca(NC₃)₂ enhanced germination and protease activity in the endosperm resulting in greater solubilisation of protein to soluble nitrogen in the seeds. NH₄NO₃ and (NH₄)₂SO₄ were less effective as compared to Ca(NO₃)₂. Cycloheximide inhibited germination and protease activity. Pretreatment also resulted in increase in growth, soluble and insoluble nitrogen, and nitrate reductase activity in the primary leaves. Ca(NO₃)₂ was more effective than NH₄NO₃ and (NH₄)₂SO₄.     

Radio Frequency-Activated Nanoliposomes for Controlled Combination Drug Delivery

This work was conducted in order to design, characterize, and evaluate stable liposomes containing the hydrophobic drug raloxifene HCl (RAL) and hydrophilic doxycycline HCl (DOX), two potentially synergistic agents for treating osteoporosis and other bone lesions, in conjunction with a radio frequency-induced, hydrophobic magnetic nanoparticle-dependent triggering mechanism for drug release. Both drugs were successfully incorporated into liposomes by lipid film hydration, although combination drug loading compromised liposome stability. Liposome stability was improved by reducing the drug load and by including Pluronics® (PL) in the formulations. DOX did not appear to interact with the phospholipid membranes comprising the liposomes, and its release was maximized in the presence of radio frequency (RF) heating. In contrast, differential scanning calorimetry (DSC) and phosphorus-31 nuclear magnetic resonance (³¹P-NMR) analysis revealed that RAL developed strong interactions with the phospholipid membranes, most notably with lipid phosphate head groups, resulting in significant changes in membrane thermodynamics. Likewise, RAL release from liposomes was minimal, even in the presence of RF heating. These studies may offer useful insights into the design and optimization of multidrug containing liposomes. The effects of RAL on liposome characteristics and drug release performance underscore the importance of appropriate physical-chemical analysis in order to identify and characterize drug-lipid interactions that may profoundly affect liposome properties and performance early in the formulation development process.KEY WORDS: controlled release, drug combination, liposomes, nanoparticles     

Hydro and halo priming: influenced germination responses in wheat Var-HUW-468 under heavy metal stress

The beneficial effect of seed priming in improving critical growth stages like seed germination and early growth phases has been accepted by Plant Physiologists for many important field crops. In the present investigation, studies were made to see the effect of heavy metal stress imposed during germination using solution of HgCl₂ in four different concentrations (0.0, 0.50, 0.75 and 1.00 mM) in Petri dishes on primed and non-primed seeds of wheat. Priming has been done with distilled water (hydro), Mg(NO₃)₂ and Ca(NO₃)₂ (halo) salts. Different germination parameters, such as germination percentage, radicle and plumule lengths, seedling emergence, soluble and insoluble sugar contents and activity of α-amylase in endosperm were studied at different study periods. Primed seeds increased all the germination parameters except insoluble sugar content in respect to non-primed control in the absence of HgCl₂. However, the use of primed seeds has shown to overcome the inhibitory effects of heavy metal stress imposed in the form of HgCl₂ solution during the period of germination. Hence, the work concludes the mitigating effects of priming under heavy metal stress.     

Aluminum‐dependent dynamics of ion transport in Arabidopsis: specificity of low pH and aluminum responses

Low‐pH and Al³⁺ stresses are the major causes of poor plant growth in acidic soils. However, there is still a poor understanding of plant responses to low‐pH and Al³⁺ toxicity. Low‐pH or combined low‐pH and Al³⁺ stress was imposed in order to measure rhizosphere pH, ion fluxes, plasma membrane potential and intracellular H⁺ concentration in distal elongation and mature zones (MZs) along the longitudinal axis of Arabidopsis thaliana roots. Low‐pH stress facilitated H⁺ influx into root tissues and caused cytoplasmic acidification; by contrast, combined low‐pH/Al³⁺ treatment either decreased H⁺ influx in the distal elongation zone (DEZ) or induced H⁺ efflux in the MZ, leading to cytoplasmic alkalinization in both zones. Low‐pH stress induced an increase in rhizosphere pH in the DEZ, whereas combined low‐pH/Al³⁺ stress resulted in lower rhizosphere pH in both root zones compared with the low‐pH treatment alone. Low‐pH stress facilitated K⁺ efflux; the presence of Al³⁺ diminished K⁺ efflux or favored K⁺ influx into root tissues. In both zones, low‐pH treatment induced plasma membrane (PM) depolarization, which was significantly diminished (P≤ 0.05) when combined stresses (low‐pH/100 µM Al³⁺) were imposed. After 60 min of exposure, low pH caused PM depolarization, whereas low pH/100 µM Al³⁺ caused PM hyperpolarization. Thus, low pH and Al³⁺ toxicity differentially affect root tissues and, consequently, the rhizosphere, which might underpin the differential mechanisms of plant adaptation to these abiotic stresses.     

Contribution of sarcolemmal sodium-calcium exchange and intracellular calcium release to force development in isolated canine ventricular muscle

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.     

Role of ion channels in regulating Ca2+ homeostasis during the interplay between immune and cancer cells

Ion channels are abundantly expressed in both excitable and non-excitable cells, thereby regulating the Ca²⁺ influx and downstream signaling pathways of physiological processes. The immune system is specialized in the process of cancer cell recognition and elimination, and is regulated by different ion channels. In comparison with the immune cells, ion channels behave differently in cancer cells by making the tumor cells more hyperpolarized and influence cancer cell proliferation and metastasis. Therefore, ion channels comprise an important therapeutic target in anti-cancer treatment. In this review, we discuss the implication of ion channels in regulation of Ca²⁺ homeostasis during the crosstalk between immune and cancer cell as well as their role in cancer progression.