In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.     

Search for substrate-based inhibitors fitting the S2' space of malarial aspartic protease plasmepsin II.

Plasmepsin (Plm) has been identified as an important target for the development of new antimalarial drugs, since its inhibition leads to the starvation of Plasmodium falciparum. A series of substrate-based dipeptide-type Plm II inhibitors containing the hydroxymethylcarbonyl isostere as a transition-state mimic were synthesized. The general design principle was provision of a conformationally restrained hydroxyl group (corresponding to the set residue at the P2' position in native substrates) and a bulky unit to fit the S2' pocket.     

Cellular mechanisms governing synapse formation: lessons from identified neurons in culture

The accessibility of embryonic and adult neurons within invertebrate nervous systems has made them excellent subjects for neurobiological study. The ability to readily identify individual neurons, together with their great capacity for regeneration, has been especially beneficial to investigations of synapse formation and the specificity of neuronal connectivity. Many invertebrate neurons survive for long periods following isolation into primary cell culture. In addition, they readily extend new neuritic arbors and form electrical and chemical connections at sites of contact. Thus, cell culture approaches have allowed neuroscientists greater access to, and resolution of, events underlying neurite outgrowth and synaptogenesis. Studies of identified neuromuscular synapses ofHelisoma have determined a number of signaling mechanisms involved in transsynaptic communication at sites of neuron-target contact. At these sites, both anterograde and retrograde signals regulate the transformation of growth cones into functional presynaptic terminals. We have found that specific muscle targets induce both global and local changes in neurotransmitter secretion and intracellular calcium handling. Here we review recent studies of culturedHelisoma synapses and discuss the mechanisms thought to govern chemical synapse formation in these identified neurons and those of other invertebrate species.     

A sensitive,simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O2 specificity factor

A simple approach to determine CO₂/O₂ specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO₂ fixation at varying [CO₂]/[O₂] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of ¹⁴CO₂ incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO₂]/[O₂] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO₂/O₂ specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO₂]/[O₂] ratios examined, ranging from 14 to 215 micromolar CO₂ (provided as 1–16 mM NaHCO₃) and 614 micromolar O₂ provided as 50% O₂. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid) - L large subunit of rubisco - PGA 3-phosphoglyceric acid - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - S small subunit of rubisco - XuBP d-xylulose 1,5-bisphosphate     

Maintenance of two genetic entities by habitat selection

Summary In the laboratory, the two species of copepodsLepeophtheirus thompsoni andLepeophtheirus europaensis, ectoparasites of flatfishes, can meet and mate on at least one host species. In the wild however, these two species are found isolated on their sympatric hosts. Habitat selection theoretically represents a powerful enough mechanism to explain the maintenance of genetic heterogeneity in the wide sense. In this paper, the host colonization process is studied for both parasite species. It is shown that each parasite can develop and reach adult age on each host species. However,L. thompsoni is highly selective; it almost totally refuses to colonize hosts other than its natural one.Lepeophtheirus europaensis, on the contrary, readily infests turbot and brill in single-host experiments, but strongly prefers the brill when it has a choice. It appears that these two genetic entities are sympatrically maintained due to strong habitat selection. Such a pattern could theoretically only occur in a soft-selection context (density dependence). This point is discussed with respect to the different patterns in host use found in the geographical distribution of these parasites.     

Susceptibility of brown trout to Gyrodactylus salaris (Monogenea) under experimental conditions

Brown trout ( Salmo trutta ) from anadromous River Lierelva, resident Lake Tunhovd, and resident Nordmarka stocks were exposed to Gyrodactylus salaris -infected salmon parr. The brown trout were fed pellets before the experiments, except for one group of the Nordmarka stock which was starved for 19 days before the experiments. The mean number of parasites declined directly and rapidly post infection for all groups of trout. There were no pronounced differences in resistance between the anadromous and the resident stocks. G. salaris infections tended to persist longer on starved than on fed trout of the Nordmarka stock. The maximum parasite persistence on trout was 50 days, and as parasite numbers increased on some fish parasite reproduction must have occurred on those trout. However, the limited susceptibility and marked innate resistance of trout to G. salaris establishment, development and reproduction, suggest parasite metapopulations will not survive on this species. Nevertheless, trout may still play a role in the dispersal of G. salaris within and between rivers.     

Modulated red blood cell survival by membrane protein clustering

Human and murine blood cells treated with ZnCl₂ and bis(sulfosuccinimidyl)suberate (BS³) (a cross linking agent) undergo band 3 clustering and binding of hemoglobin to red blood cell membrane proteins. These clusters induce autologous IgG binding and complement fixation, thus favouring the phagocytosis of ZnCl₂/BS³ treated cells by macrophages. The extension of red blood cell opsonization can be easily modulated by changing the ZnCl₂ concentration in the 0.1–1.0 mM range thus providing an effective way to affect blood cell recognition by macrophages. In fact, murine erythrocytes treated with increasing ZnCl₂ concentrations have proportionally reduced survivals when reinjected into the animal. Furthermore, the organ sequestration of ZnCl₂/BS³ treated cells strongly resembles the typical distribution of the senescent cells. Since the ZnCl₂/BS³ treatment can also be performed on red blood cells loaded with drugs or other substances, this procedure is an effective drug-targeting system to be used for the delivery of molecules to peritoneal, liver and spleen macrophages.     

Modulation of cisPlatin cytotoxicity by interleukin-1α and resident tumor macrophages

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1α) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1α in SCC-7 solid tumors. Neither IL-1α nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1α had no direct effect on tumor cell growthin vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC₉₀=6.0 μM), but, the addition of IL-1α (500–2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC₉₀=3.1 μM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1α. The modulation of cisPlatin cytotoxicity by IL-1α exhibited a biphasic dose response that paralleled the IL-1α dose dependent release of H₂O₂by resident tumor macrophages. Further, IL-1α modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H₂O₂ (50 μM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1α treatment for 24 hrs, before cisPlatin, produced drug resistance (IC₉₀=11.1 μM). Our study shows that IL-1α can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explanation for the synergistic antitumor activity of cisPlatin and IL-1αin vivo.     

A new survey of Brazilian marine algae for agglutinins

Aqueous protein extracts from 30 Brazilian marine algae were examined for haemagglutinating activity using native and enzyme-treated rabbit, chicken, sheep and human erythrocytes. Most extracts agglutinated at least one of the blood cells used. Sheep and rabbit erythrocytes were more suitable for detection of the agglutinating activity. The minimum protein concentration necessary to produce positive agglutination was usually lower with enzyme-treated erythrocytes than native ones. The five algal protein extracts showing the greatest haemagglutination titre were tested for sugar-binding specificity. Only the activity present in the green alga Cauler pacupressoides was inhibited by simple sugars and not by the glycoproteins tested. The activity of the other four extracts was inhibited by at least one of the glycoproteins utilised. This revised version was published online in August 2006 with corrections to the Cover Date.