The immune and fibrinolytic systems in heart failure developing against a background of cardiac atherosclerosis

A study made to gain insight into the bodily immune and fibrinolytic systems in 72 patients with cardiac insufficiency developed against the background of cardial atherosclerosis showed imbalance in the ratio of cellular to humoral links of immunity and enhancement of the autoimmune processed activity, inhibition of activity of the monocytomacrophagal link. Disturbances in the fibrinolytic system were manifested by blood and urine lowering of activity of the plasminogen activator, with the antiplasmin activity being on the decrease as well, which fact is regarded as formation of a new stationary state in the system of haemocoagulation maintaining hemostasis.     

Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.     

Synthesis, structure, and antiproliferative activity of selenophenfurin, an inosine 5'-monophosphate dehydrogenase inhibitor analogue of selenazofurin

The synthesis and biological activity of selenophenfurin (5-beta-D-ribofuranosylselenophene-3-carboxamide, 1), the selenophene analogue of selenazofurin, are described. Glycosylation of ethyl selenophene-3-carboxylate (6) under stannic chloride-catalyzed conditions gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivative. Deprotected ethyl 5-beta-D-ribofuranosylselenophene-3-carboxylate (12 beta) was converted into selenophenfurin by ammonolysis. The structure of 12 beta was determined by 1H- and 13C-NMR, crystallographic, and computational studies. Selenophenfurin proved to be antiproliferative against a number of leukemia, lymphoma, and solid tumor cell lines at concentrations similar to those of selenazofurin but was more potent than the thiophene and thiazole analogues thiophenfurin and tiazofurin. Incubation of K562 cells with selenophenfurin resulted in inhibition of IMP dehydrogenase (IMPDH) (76%) and an increase in IMP pools (14.5-fold) with a concurrent decrease in GTP levels (58%). The results obtained confirm the hypothesis that the presence of heteroatoms such as S or Se in the heterocycle in position 2 with respect to the glycosidic bond is essential for both cytotoxicity and IMP dehydrogenase inhibitory activity in this type of C-nucleosides.     

Manganese transport through human erythrocyte membranes. An EPR study

Manganese uptake by human erythrocytes was investigated in the concentration range 0.5-20 mM in the suspending solution, by using the EPR technique. S shaped dependencies of manganese influx on manganese doping solution concentration for both fresh and vanadate treated erythrocytes were found, with maximum influx values of 4.1 +/- 1.9 x 10(-10) mol/m2 x s and 2.1 +/- 0.3 x 10(-9) mol/m2 x s, respectively. At low manganese concentrations (< 2 mM) the manganese permeability coefficient increases with increasing the doping concentration, the ions cooperate for achieving a transport event. For high manganese concentration (> 5 mM) the permeability coefficient decreases with increasing the doping concentration, the ions competing for the limited amount of transport system. A similar increase in manganese uptake as in vanadate treated erythrocytes was measured for 'in vitro' aged erythrocytes. These results might suggest that human erythrocytes possess an active transport mechanism by which, they oppose to manganese influx. This hypothesis is also supported by the 10-15 min time lag between the moment of doping and the start of the manganese influx into the fresh erythrocytes. The manganese uptake inhibition by nifedipine, a calcium channel blocker, for the case of vanadate treated erythrocytes, suggests that, at least partially, manganese uptake by the cells occurs via the 'calcium channels'.     

Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation

The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+] nonsense suppressor determinant. In a [PSI+] state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the [PSI+] phenotype. Sup35p is composed of the N-terminal domain (N) required for [PSI+] maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination. In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in [PSI+] cells. Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain. Similarly to Sup35p, in [PSI+] cells Sup45p was found in aggregates. The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding. The incorporation of Sup45p into the aggregates should inhibit its activity. The N domain of Sup35p, responsible for its aggregation in [PSI+] cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p. This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.     

Agonist-receptor-arrestin, an alternative ternary complex with high agonist affinity

The rapid decrease of a response to a persistent stimulus, often termed desensitization, is a widespread biological phenomenon. Signal transduction by numerous G protein-coupled receptors appears to be terminated by a strikingly uniform two-step mechanism, most extensively characterized for the beta2-adrenergic receptor (beta2AR), m2 muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model predicts that activated receptor is initially phosphorylated and then tightly binds an arrestin protein that effectively blocks further G protein interaction. Here we report that complexes of beta2AR-arrestin and m2 mAChR-arrestin have a higher affinity for agonists (but not antagonists) than do receptors not complexed with arrestin. The percentage of phosphorylated beta2AR in this high affinity state in the presence of full agonists varied with different arrestins and was enhanced by selective mutations in arrestins. The percentage of high affinity sites also was proportional to the intrinsic activity of an agonist, and the coefficient of proportionality varies for different arrestin proteins. Certain mutant arrestins can form these high affinity complexes with unphosphorylated receptors. Mutations that enhance formation of the agonist-receptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization.     

1,25-Dihydroxyvitamin D3 pretreatment limits prostaglandin biosynthesis by cytokine-stimulated adult human osteoblast-like cells

The addition of fibronectin fragments to cultured cartilage causes an initial suppression of proteoglycan synthesis, induction of matrix metalloproteinases, and resultant decrease in proteoglycan content by about 50% during the first few days in culture. Because the proteoglycan loss appears to be limited, we investigated whether the fibronectin fragments induce anabolic responses that might counter the damage. The effects of various lengths of exposure of cultured cartilage to the fibronectin fragment on proteoglycan content, proteoglycan synthesis rates, stromelysin-1 release, and tumor necrosis factor-alpha, interleukin-1alpha, and interleukin-6 release were investigated. The results showed that about 7 days of exposure of cultured cartilage to the fibronectin fragment was required for maximal cytokine release, proteoglycan depletion, and stromelysin-1 release. However, nearly maximal suppression of proteoglycan synthesis occurred within 1 day of the addition of the fibronectin fragment and, after its removal, the rates increased to supernormal levels. Decreasing exposure to 3 days caused only a small decrease in cartilage proteoglycan content, although stromelysin-1 release still occurred. Decreasing exposure to 1 day caused an immediate increase in proteoglycan synthesis and an increase to supernormal proteoglycan contents. The effect of first treating cartilage with the fibronectin fragment for various periods and then allowing a recovery was to make the cartilage more resistant to secondary exposures. This study shows that cartilage damage can be caused by short exposures to the fibronectin fragment and that exposures either optimal or suboptimal for damage additionally amplify anabolic processes to make the cartilage resistant to further damage and, thus, condition it against pending amplification of damage.     

Recreating Romance of Civil Engineering: 2001 Summer Camp at Indian Institute of Technology Kanpur, India

This paper describes an initiative to enthuse undergraduate Civil Engineering (CE) students in India about the profession. Various issues impacting CE students are addressed in a novel approach, the 2001 Summer Camp at IIT Kanpur. A select group of undergraduate students who had completed their second year of the Bachelors program in CE were chosen for the camp from different engineering colleges across the country. The objective of the camp was to provide an exposure to technology through motivation, personality development, fun-filled schedules, and excursions. For one month, through a series of activities, these students (1) interacted closely with the captains of CE industry in India to share their present concerns and future dreams in the CE profession and to understand expectations of the industry and society, (2) visited various sites with impressive engineering projects and some outstanding projects under construction, and (3) took part in diverse technical, nontechnical, and extracurricular activities, such as technical video shows, personality development workshop, debates, quizzes, sports, and entertainment. The camp proved to be an eye opener to all participants, the CE industry, and institutions imparting CE education. A questionnare administered at the end of the camp revealed that the perceptions of the students about the CE profession and their self-esteem underwent a positive change. The camp also helped bring the industry and academia in India onto a common platform.     

Phase Formation in Equiatomic High Entropy Alloys: CALPHAD Approach and Experimental Studies

CALPHAD approach has been used to predict the stable phases, their relative amounts and compositions in multicomponent equiatomic high entropy alloys. The results show a good match between the predictions and experimental results on the phase formation for two equiatomic high entropy alloys (CrCoCuNi and CrCuMnNi alloys) prepared by mechanical alloying, considering the kinetic constraints of the non-equilibrium processing route.