Another look at the molecular mechanism of the resistance of H5N1 influenza A virus neuraminidase (NA) to oseltamivir (OTV)

In the context of a recent pandemic threat by the worldwide spread of H5N1 avian influenza, the high resistance of H5N1 virus to the most widely used commercial drug, oseltamivir (Tamiflu), is currently an important research topic. Herein, molecular bases of the mechanism of H5N1 NA resistance to oseltamivir were elucidated using a computational approach in a systematic fashion. Using the crystal structure of the complex of H5N1 NA with OTV (PDB ID: 2hu0) as the starting point, the question, how mutations at His274 by both smaller side chain (Gly, Ser, Asn, Gln) and larger side chain (Phe, Tyr) residues influence the sensitivity of N1 to oseltamivir, was addressed and correlated with the experimental data. The smaller side chain residue mutations of His274 resulted in slightly enhanced or unchanged NA sensitivity to OTV, while His274Phe and His274Tyr reduced the susceptibility of OTV to N1. In contrast to the binding free energies, the net charges of Glu276 and Arg224, making charge-charge interactions with Glu276, were established to be more sensitive to detecting subtle conformational differences induced at the key residue Glu276 by the His274X mutations. This study provides deeper insights into the possibility of developing viable drug-resistant mutants.     

The Pleistocene subterranean volesTerricola (Rodentia) of Serbia and Montenegro

A general morphometrical analysis of the M₁ was conducted to identify the subterranean vole species found in Upper Pleistocene localities from Serbia and Montenegro, and to clarify the systematic position and the phylogenetic relationships between the different species in the Balkans. From the different localities studied, we can assign one population toMicrotus (Terricola) thomasi and the others to theM. (T.) subterraneus group. This study suggests thatM. (T.) grafi can be considered as a chronological sub-species ofM. (T.) subterraneus or as a different but phylogenetically very close species.     

HIV-infected children in Croatia--medical care versus ethical and social issues

The era of pediatric HIV-infection in Croatia began in 1987 when the first child from an HIV-infected mother was born. The authors present some medical data of children diagnosed as HIV-infected or observed because of maternal HIV-infection in Croatia until the end of 2005. Although the number of HN-infected children in Croatia is still relatively small, in the last two decades the Croatian society was confronted with many ethical and social issues regarding HIV-infection and discrimination. There were many obstacles in providing proper care and integrating HIV-infected children in the Croatian society. Stigmatisation, fear and lack of knowledge were major contributors. For the first time, the integration of an HIV-infected child with revealed HIV-status into school was described. Changes in attitudes towards HIV-infected people can only be achieved through planned permanent educational programs.     

The role of oral glucose intolerance test in reducing pregnancy complications

The influence of glucose monitoring during pregnancy on newborn body weight, and complications during pregnancy and labor was assessed. We performed a retrospective analysis of macrosomal children, fetal growth, caesarean sections, malformations, still-births and the number of oral glucose tolerance test (OGTT) carried out in a five-year period. The proportion of women participating in OGTT tests increased from 20% to 40% (p<0.05) between 2000 and 2004. Gestation diabetes mellitus (GDM) proportions among pregnant women seen at the Department of Obstetrics and Gynecology at Slavonski Brod General Hospital, Croatia increased from 1% to 6.7% (p < 0. 05) during the observed period. Proportion of births identified as macrosomal decreased from 13.3% to 12.2% (p<0.05). Additionally, infant mortality and still-births along with other fetal and maternal complications declined during the same period. These results suggest that regular measurements of glucose tolerance during pregnancy may prevent preterm birth, decrease the proportion of macrosomal newborns, lower mortality and decrease fetal and maternal complication incidence during pregnancy and delivery.     

Effect of the acute heat stress on the rat pituitary gland. Morphological and stereological study

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Morphological and stereological changes were examined in the pituitary gland of the rats exposed to heat stress (38 °C) for 60 min.     

Lipid peroxidation and antioxidant status in blood of patients with uterine myoma, endometrial polypus, hyperplastic and malignant endometrium

Oxidative stress is considered to be involved in pathogenesis of many disorders of the female genital tract. In this study, we explored the lipid peroxidation levels and antioxidant enzyme activities in women diagnosed with different forms of uterine diseases in order to evaluate the extent of oxidative stress in blood of such patients. Blood samples of healthy subjects and gynecological patients were collected and subjected to assays for superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and lipid hydroperoxides. The results show that alterations of measured parameters vary with the enzyme type and diagnosis. However, both reduction in antioxidants and elevation of lipid peroxidation were observed in general. Lipid hydroperoxides level was negatively correlated to superoxide dismutase and glutathione peroxidase activities, as well as positively correlated to catalase activity. In addition, the lipid hydroperoxides/ glutathione peroxidase ratio was found to be increased, according to the type of uterine disease. The obtained results show that perturbation of antioxidant status is more pronounced in blood of patients with premalignant (hyperplastic) and malignant (adenocarcinoma) lesions, compared to those with benign uterine changes such as polypus and myoma.     

Rearrangement of erythrocyte band 3 molecules and reversible formation of osmotic holes under hypotonic conditions

The complex phenomenon of rearrangement of band 3 molecules after erythrocyte swelling under hypotonic condition is considered. The rearrangement includes the increase of the mobile fraction and clustering of band 3. The self-associative tendency and the action of the elastic field generated within the lipid membrane after erythrocyte swelling result in equilibration of the number of molecules per cluster and the number of clusters. The local perturbation of the elastic field induces excitation of the cluster in the nearest neighbor and changes its packing state generating changes in the free volume within the cluster. The local perturbation could result in the reversible formation of osmotic hole. We formulated a model to predict changes of the cluster packing states generated by rearrangement of band 3 molecules on two time-scales. The phenomenon is examined on the basis of two experimental sets, i.e. low (5.2 mM Na₃PO₄ solution) and high (46.0 mM Na₃PO₄ solution) hypotonicities at 21°C, from Golan and Veatch (Proc Natl Acad Sci 77(5):2537–2541, 1980). Modeling considerations suggested that lower hypotonic conditions resulted in higher values of: the driving force of agglomeration of band 3 as a measure of self-associative tendency, the specific rate of cluster breaking, the specific rate of increase of the mobile fraction of band 3, and the dispersion of cluster sizes. Lower hypotonic conditions ensure the generation of a higher average value of the free energy within the membrane after erythrocyte swelling, which enables more intensive rearrangement of band 3 molecules.     

Computational prediction of the binding site of proteinase 3 to the plasma membrane

Proteinase 3 (PR3) is a neutrophil-derived serine proteinase localized within cytoplasmic granules which can be released upon activation. PR3 is exposed at the neutrophil plasma membrane where it can mediate proinflammatory effects. Moreover, PR3 membrane expression is of special relevance in patients with Wegener's granulomatosis, a systemic vasculitis presenting anticytoplasmic neutrophil autoantibodies (ANCA) against PR3, which can bind to PR3 expressed at the surface of neutrophils and amplify their activation state. Therefore, it is of special relevance to unravel the molecular mechanisms governing its association with the membrane to be able to modulate it. To this end, we performed molecular dynamics (MD) simulations of PR3 with the implicit membrane model IMM1-GC to identify its interfacial binding site (IBS). Both the energies and structures resulting from the MD suggest that PR3 associates strongly with anionic membranes. We observe a unique IBS consisting of five basic (R177, R186A, R186B, K187, R222) and six hydrophobic (F165, F166, F224, L223, F184, W218) amino acids. The basic residues provide the driving force to orient PR3 at the membrane surface, so that the hydrophobic residues can anchor into the hydrocarbon region. Energy decomposition and in silico mutations show that only a few residues account for the membrane association. Similar calculations with HNE suggest a different membrane-binding mechanism. Our results agree with previous experimental observations and this work predicts, for the first time, the structural determinants of the binding of PR3 to membranes.     

Design, synthesis and antiproliferative activity of two new heteroannelated (-)-muricatacin mimics

Two new (-)-muricatacin mimics bearing a furano-furanone ring and an oxygen isostere in the side chain have been designed and synthesized and their in vitro antiproliferative activity was evaluated against several human tumour cell lines. Both analogues showed an increased activity against HL-60 cells with 17- and 185-fold higher potency than (-)-muricatacin. A straightforward synthesis of (-)-muricatacin is also disclosed.