Stereocontrolled synthesis of diene and enyne sugar-modified nucleosides and their interaction with S-adenosyl-L-homocysteine hydrolase

Conjugated diene 5-7 and enyne 8 analogs derived from adenosine and uridine were synthesized employing Pd-catalyzed cross-coupling reactions.     

Increased level of fibrinogen chains in the proteome of blood platelets in secondary progressive multiple sclerosis patients

Epidemiological studies indicate a high risk of stroke, heart failure and myocardial infarction in patients with multiple sclerosis, especially in its secondary progressive (SPMS) phase. Some ischaemic events are directly associated with abnormal platelet functions and their prothrombotic activity. Recent reports, including this study, confirm the increased activation of circulating platelets in SPMS, and also show increased platelet reactivity, among other responses, as well as strong aggregation. In this current study, we conducted a comparative analysis of the platelet proteome in SPMS patients and in healthy controls, to demonstrate the quantitative and qualitative differences likely to affect functional changes observed in SPMS. During densitometry evaluation of 2‐D fluorescence difference gel electrophoresis, we observed differences between the electrophoretic patterns of SPMS platelets and the control samples. To determine a detailed characterisation of the proteome changes in the SPMS patients’ blood platelets, in the next stage, we performed mass spectrometry of selected spots and indicated the increased presence of four proteins (fibrinogen, α‐2 macroglobulin, septin‐14 and tubulin β‐1 chain). The most important of these is the increased amount of prothrombotic protein, fibrinogen, which seems to confirm the accuracy of the imaging and potentially explains the increased risk of platelet‐origin thrombotic events. This study provides new knowledge of the potential existence of the molecular mechanisms responsible for the acceleration of the platelet pro‐coagulant function in SPMS. This can help to identify new targets for therapy, which can then be used not only in the second stage of the disease.     

A matrix-located processing peptidase of plant mitochondria

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc₁ complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the F_Ad subunit of the ATP synthetase and the tobacco F₁ subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bc₁ complex of the respiratory chain.     

Receptor for advanced glycation end products is subjected to protein ectodomain shedding by metalloproteinases

The receptor for advanced glycation end products (RAGE) is a 55-kDa type I membrane glycoprotein of the immunoglobulin superfamily. Ligand-induced up-regulation of RAGE is involved in various pathophysiological processes, including late diabetic complications and Alzheimer disease. Application of recombinant soluble RAGE has been shown to block RAGE-mediated pathophysiological conditions. After expression of full-length RAGE in HEK cells we identified a 48-kDa soluble RAGE form (sRAGE) in the culture medium. This variant of RAGE is smaller than a 51-kDa soluble version derived from alternative splicing. The release of sRAGE can be induced by the phorbol ester PMA and the calcium ionophore calcimycin via calcium-dependent protein kinase C subtypes. Hydroxamic acid-based metalloproteinase inhibitors block the release of sRAGE, and by RNA interference experiments we identified ADAM10 and MMP9 to be involved in RAGE shedding. In protein biotinylation experiments we show that membrane-anchored full-length RAGE is the precursor of sRAGE and that sRAGE is efficiently released from the cell surface. We identified cleavage of RAGE to occur close to the cell membrane. Ectodomain shedding of RAGE simultaneously generates sRAGE and a membrane-anchored C-terminal RAGE fragment (RAGE-CTF). The amount of RAGE-CTF increases when RAGE-expressing cells are treated with a gamma-secretase inhibitor, suggesting that RAGE-CTF is normally further processed by gamma-secretase. Identification of these novel mechanisms involved in regulating the availability of cell surface-located RAGE and its soluble ectodomain may influence further research in RAGE-mediated processes in cell biology and pathophysiology.     

Activity of cathepsin B,D and L in rat cerebrum after cimetidine and famotidine administration

Cathepsins are lysosomal enzymes that are used a sensitive markers in various toxicological investigations. The purpose of this study was to evaluate and compare the influence of cimetidine and famotidine on the cerebral cortex, particularly on the activity of cortical cathepsin B, D and L in the frontal lobe of rat brain. The drugs were administered intraperitoneally, twice a day, for six weeks to male Wistar rats in two doses. The initial dose was 2.85 mg/kg for cimetidine and 0.285 mg/kg for famotidine. The second dose was 10 times higher. Control animals were injected with 0.9% NaCl. Half of the animals from each of the drug-treated and control groups were sacrificed on the 42nd day of the experiment. The remaining animals were raised for another 6 weeks without any xenobiotics, and sacrificed on the 84th day. The frontal lobe of the right cerebral hemisphere was taken for biochemical investigation. The activities of free and bound fractions of cathepsin B, D and L were evaluated spectrophotometrically in cortical homogenates. The activity of bound fraction of cathepsin D and L decreased significantly in animals exposed to the higher dose of cimetidine and sacrificed on the 42nd day. Also significant elevation of the free fraction of cathepsin L was noted in the same group of rats. Cathepsin activities were normalized during the next six weeks. No behavioural changes were noted among the observed animals. Unlike cimetidine, famotidine did not change profiles of the cerebral cathepsins.     

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.     

Virus-like particles of potato leafroll virus as potential carrier system for nucleic acids

Potato leafroll virus is a member of the polerovirus genus. The isometric virion is formed by a coat protein encapsidating single-stranded, positive-sense, mono-partite genomic RNA with covalently attached viral protein at the 5' end. The coat protein of the virus exists in two forms: i) a 23 kDa protein, the product of the coat protein gene, and ii) a 78 kDa protein, the product of the coat protein gene and an additional open reading frame expressed by read-through of the coat protein gene stop codon. The aim of this work was the expression of potato leafroll virus coat protein-based proteins that would be able to assemble into virus-like particles in insect cells. These modified particles were tested for their ability to encapsidate nucleic acids. Two types of N-terminally His-tagged coat protein constructs were used for the expression in insect cells: one, encoding a 23 kDa protein with the C-terminal amino-acid sequence corresponding to the wild type coat protein and the second with additional clathrin binding domain at the C-terminus. The expression of these two proteins by a recombinant baculovirus was characterized by Western immunoblotting with antibodies directed against potato leafroll virus. The protection or putative encapsidation of nucleic acids by these two coat protein derivatives was shown by DNase I and RNase A protection assays.     

Night shift work and osteoporosis: evidence and hypothesis

Osteoporosis is an important public health problem worldwide. Among the countries with a very high population risk of fractures, there are those with the highest level of economic development. Osteoporotic fractures are the main cause of disability among elderly people, and the resultant disabilities require particularly large financial support associated not only with the direct treatment of the fracture but also with the necessity for long-term rehabilitation and care for the disabled person. Many well-established factors can have impact on bone mass and fracture risk. Recently, it has been hypothesized that working during nighttime which leads to endocrine disorders may have an indirect impact on bone physiology among night shift workers. Therefore, it can be presumed that the night shift work may contribute to the etiology of osteoporosis. The aim of our work was to make a review of the epidemiological evidence on the association between night shift work and bone mineral density or fracture risk as well as to discuss the potential biological mechanisms linking the work under this system with the development of osteoporosis. We have identified only four studies investigating the association between system of work and bone mineral density or fracture risk among workers. The findings of three out of four studies support the hypothesis. None of the studies has investigated a potential relationship between night shift work and bone turnover markers. Given that there have been no epidemiological studies in European countries that would concern working populations and the noticeable difference in the risk of osteoporosis between communities, further studies are warranted to elucidate the problem. It is presumed that further in-depth studies will not only identify the underlying factors of the disease but also contribute to developing guidelines for policy makers and employers for primary prevention of osteoporosis in workplace.     

Enzymatically stable 5' mRNA cap analogs: synthesis and binding studies with human DcpS decapping enzyme

Four novel 5' mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group. The analogs were prepared via reaction of nucleoside phosphor/phosphon-1-imidazolidates with nucleoside phosphate/phosphonate in the presence of ZnCl2. Three of the new cap analogs are completely resistant to degradation by human DcpS, the enzyme responsible for hydrolysis of free cap resulting from 3' to 5' cellular mRNA decay. One of the new analogs has very high affinity for binding to human DcpS. Two of these analogs are Anti Reverse Cap Analogs which ensures that they are incorporated into mRNA chains exclusively in the correct orientation. These new cap analogs should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes, and as a basis for further development of modified cap analogs as potential anti-cancer and anti-parasite drugs.