Ligands of second generation platinum analogs decrease both platinum-induced DNA cross-linking and its ability to interact with 1-beta-D-arabinofuranosyl cytosine to potentiate cytotoxic efficacy

We evaluated the cytotoxic and DNA cross-linking (CL) ability of four second generation platinum coordination complexes (TNO-6, JM-89, JM-8 and JM-9) delivered alone or in combination with 1-beta-D-arabinofuranosyl cytosine (ara-C) to human colon cancer cells (LoVo). Cell survival varied markedly as a function of the particular substitution moiety. JM-8 and JM-9 were virtually ineffective, even at concentrations as high as 50 micrograms/ml. At that concentration cis-diamminedichloroplatinum(II) (cis-DDP) killed greater than 99.99% of the cells. JM-82 was slightly more active while TNO-6 was the only derivative with appreciably higher cytotoxic activity due to an abrogation of the shoulder region of the type C survival curve. The highest CL effect was observed for cis-DDP followed closely by TNO-6. Very little CL effects were demonstrated for the other three analogs JM-82, JM-8 and JM-9 when measured 6 h after treatment. The combination of cis-DDP and ara-C augmented 10-fold the cytotoxic activity of cis-DDP alone, an effect accompanied by an almost 2-fold increase in CL; every other analog failed to interact in a potentiating manner (either cytotoxicity, or CL at 6 h) with the antimetabolite. Thus, it appears clear that the associated moieties of the Pt coordination complex play a fundamental role in reducing the interaction of the analogs with DNA (as reflected by the decreased CL and cytotoxic effects produced by each agent alone) and in totally preventing their interaction with ara-C to yield a potentiating lethal effect.     

Mechanisms of changes in human hemodynamics under the conditions of microgravity and prognosis of postflight orthostatic stability

The mechanisms of hemodynamic responses to orthostatic stresses and orthostatic stability (OS) of cosmonauts were studied before and after short-and long-term spaceflights (SFs) using orthostatic tests, as well as before, during, and after SFs using ultrasonic methods in tests with exposure to lower body negative pressure (LBNP). The capacitance and distensibility of the veins of the lower extremities were studied using occlusive air plethysmography before, during, and after SFs of different durations. A stay in microgravity has been proved to result in detraining of, mainly, the vascular mechanisms of compensating orthostatic perturbations. It has been established that the decrease in OS under the influence of microgravity is determined by a reduction of the vasoconstrictive ability of large blood vessels of the lower extremities; an increase in venous distensibility and capacitance of the legs; and an impairment of blood flow regulation, which leads to a cerebral blood flow deficit in orthostatic stresses, and of the initial individual OS before the flight. The results of preflight studies of hemodynamics by ultrasonic methods at LBNP and the data of orthostatic tests before SFs make it possible to predict the degree of decrease of OS after an SF proceeding in the normal mode. At the same time, the data of ultrasonic blood flow examination provide more a accurate estimation of OS and make it possible to assess the physiological reserves of hemodynamic regulation and to reveal the loss of regulation capacity even in cases where integrated indices (heart rate and blood pressure) are within the normal ranges.     

Peanut (Arachis hypogaea) lectin recognizes α-linked galactose, but not N-acetyl lactosamine in N-linked oligosaccharide terminals

Peanut (Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Galβ1→3GalNAc-), but not its sialylated version. However, an additional specificity towards Galβ1→4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Galβ1→4GlcNAc (LacNAc) was ineffective while terminal α-linked galactose (TAG) as well as exposed T antigen (Galβ1→3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-α-galactoside antibody.     

A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.