Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment ¹. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. Methods for fractionating leptospiral membranes ^2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids ^{6, 7}. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic β-sheets arranged in a barrel-like structure ^{8, 9}. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL54¹⁰ are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended ¹⁰. Download video file.^{(65M, mov)}     

The antioxidant properties of polypore mushroom <Emphasis Type="Italic">Daedaleopsis confragosa</Emphasis>

Daedaleopsis confragosa belong to a large and remarkable group of mushrooms called polypores. This type of mushroom could be easily said to be quite unexplored and unused when it comes to its antioxidant properties. Thus, in order to evaluate its antioxidant activity, the investigation had to include the total phenolics and flavonoide content, the content of Selenium, the content of Zinc, the scavenging capacity on DPPH^· and OH^· radicals, reducing power and capacity to remove lipid peroxidation. The investigated mushroom extract contained 54.17 mg GAE/g of total phenols and 48.46 mg CE/g of total flavonoides. Zinc and Selenium were detected and quantified by using inductively coupled plasma mass spectroscopy. The scavenging activity of the radicals was found to exhibit 50% of the inhibition value (IC₅₀ value) at the extract concentration of 0.015±0.007 mg/ml for the investigated D. confragosa extract. By using electron paramagnetic resonant spectroscopy it was found that the investigated extract does not have a significant role in the prevention of lipid peroxidation. It was effective in scavenging on ^·OH radical, RI was 56±2%.     

Present state and possibilities for improvement of cancer prevention and early detection in the Osijek-Baranya county

Cancer morbidity and mortality are on a steady increase in Croatia. Technologic possibilities for appropriate management are available for four cancer sites, i.e. cancer of the breast, cervix uteri, colorectum and prostate, and include cancer prevention and early detection in individuals yet free from manifest signs of the disease. The magnitude of the problem, the experience acquired to date, health care personnel available, and additional resources required to launch a systematic program of early detection of the disease are presented. The program should be initially launched in a county with greatest experience in early detection of cancer, where health care service is ready to immediately start its implementation. The role of family physician, gynecologic service at primary health care level, and polyclinic-consultation hospital service in program implementation is described. The following three possible options for early detection of cancer are analyzed and proposed: minimal program (early detection every 3 years), medium program (the same individuals examined every 2 years), and optimal program proposed by the American Cancer Society and other national and international organizations.     

Septic acromioclavicular arthritis in a patient with diabetes mellitus

A 44-year-old diabetic man with isolated septic arthritis of the left acromioclavicular joint (A-C) caused by Staphylococcus aureus is described. He was admitted to the Department of Rheumatology with clinical symptoms of left shoulder arthritis and fever. Laboratory findings showed leukocytosis, elevated levels of erythrocyte sedimentation rate and C-reactive protein, all indicating septic arthritis. Blood culture was positive for Staphylococcus aureus. Left A-C joint x-ray and ultrasonography, and whole body scintigraphy with 99 mTc radiolabeled autologous leukocytes pointed to septic arthritis of the A-C joint. The patient was treated for six weeks with antibiotics successfully. Infection of the A-C joint is uncommon, even in conditions such as immunodeficiency, renal dialysis and intravenous drug abuse which are associated with unusual joint infections, and can be differentiated from shoulder joint infection, by maximal tenderness over the A-C joint on examination, and findings of A-C joint widening, effusion, and bony erosions on imaging studies.     

Right atrial pacemaker lead thrombosis incidentally detected by transesophageal echocardiography

In a 62-year-old man with permanent atrial fibrillation and recurrent stroke, a large right atrial thrombus attached to a permanent pacemaker lead was incidentally identified by transesophageal echocardiography. Surgical treatment, recommended because of the large dimensions of the mass, was refused by the patient, and thrombus was successfully dissolved by anticoagulant treatment. Pathogenesis of pacemaker lead thrombosis, clinical implications, diagnostic and therapeutic options are discussed.     

Influence of the war in Croatia on the frequency of gynecological cancer in the University Hospital Osijek in the period from 1985 to 2002

The aim of this study is to estimate the influence of war in Croatia on the frequency of gynecological cancer (cancer of corpus and cervix uteri and ovary) in the Clinical Hospital Osijek, particularly the relation between the pre-war and post-war period. We analyzed 1455 patients with corpus uteri and cervix uteri cancer and ovarian cancer treated in the Clinical Hospital Osijek in the period 1985-2002 (group I). Patients from Osjecko-Baranjska County were analyzed separately--1273 women, (group II) and in the group III there were 182 patients from other counties. The analyzed period was divided into: pre-war 1985-1990, war 1991-1993 and post-war period 1997-2002. In all three groups the number of patients treated for gynecological cancer was significantly larger in the post-war period (group I, N = 611, group II, N = 498, group III, N = 113) than in the pre-war period (group I, N = 457, group II, N = 433, group III, N = 24). The analysis of cancer frequency in relation to the site shows that a total number of patients treated for cervical cancer was larger in the post-war (N = 229) than in the pre-war period (N = 214), but the difference wasn't significant. However, the number of patients from Osjecko-baranjska County treated for cervical cancer was larger in the pre-war (N = 207) than in the post-war period (N = 178) but still, the difference wasn't significant. The number of patients treated for corpus uteri cancer and ovarian cancer was significantly larger for the I group in the post-war (N = 225 and N = 157 respectively) than in the pre-war period (N = 136, and N = 107 respectively). In the group II the number of patients treated for corpus uteri cancer and ovarian cancer was larger in the post-war (N = 196 and N = 124 respectively) than in the pre-war period (N = 130 and N = 96 respectively) but the difference was significant only for corpus uteri cancer. Significantly more women were treated for gynecological cancer in the post-war than in the pre-war period. However, the war had probably an indirect influence on the increased number of patients treated for gynecological cancer mainly because many more women arrived from other counties.     

Inhibiting properties of stable nitroxyl radicals in reactions of linoleic acid and linoleyl alcohol oxidation catalyzed by 5-lipoxygenase

The inhibiting effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and its 4-substituted derivatives in reactions of linoleyl acid or linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase were investigated. Inhibiting properties of stable nitroxyl radicals in presence of lubrol and SDS were reduced at the transition from TEMPO to 4-hydroxy-TEMPO or 4-amino-TEMPO and increased at use of adamantane-1-carboxylic or 3-methyladamantane-1-carboxylic acid 1-oxyl-2,2,6,6-tetramethylpiperidine-4-yl esters. Enzyme activity at saturating concentrations of inhibitor was not suppressed completely, and decreased up to the certain level determined by the substrate nature. The dependence of partial inhibition efficiency on rotational correlation time of stable nitroxides in model micellar systems were analysed. It was supposed that 5-lipoxygenase inhibition includes the interaction of hydrophobic nitroxide with radical intermediate formed in enzymatic process.     

Synthesis and anti-aromatase activity of some new steroidal D-lactones

Starting from D-seco derivatives of 5-androstene 1-3, the D-homo lactones, 4 and 5, were synthesized. By the Oppenauer oxidation and/or by dehydration of 4 and 5 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil), the corresponding D-lactones 6-12 were obtained. The structures of 6 and 10 were unambiguously proved by the appropriate X-ray structural analysis. Anti-aromatase assay showed that tested compounds possess inhibition potency, however, two to four times smaller (IC50 from 0.2 to 0.7 microM, respectively) in comparison to aminoglutethimide (AG).     

Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation

Sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and certain signaling molecules segregate from bulk membrane lipids into lateral domains termed lipid rafts, which are often isolated based on their insolubility in cold nonionic detergents. During immunohistological studies of gangliosides, major sphingolipids of the brain, we found that cold Triton X-100 solubility is bidirectional, leading to histological redistribution from gray to white matter. When brain sections were treated with > or =0.25% Triton X-100 at 4 degrees C, ganglioside GD1a, which is normally enriched in gray matter and depleted in white matter, redistributed into white matter tracts. Incubation of brain sections from knockout mice lacking GD1a with wild-type sections in the presence of cold Triton X-100 resulted in GD1a redistribution from wild-type gray matter to knockout white matter. GM1, which is normally enriched in white matter, remained in white matter after cold detergent treatment and did not migrate to knockout mouse brain sections. However, when gray matter gangliosides were enzymatically converted into GM1 in situ, the newly formed GM1 transmigrated to knockout mouse brain sections in the presence of cold detergent. When purified GD1a was added to knockout mouse brain sections in the presence of cold Triton X-100, it preferentially incorporated into white matter tracts. These data demonstrate that brain white matter is a sink for gangliosides, which redistribute from gray matter in the presence of low concentrations of cold Triton X-100. A GPI-anchored protein, Thy-1, also transmigrated from wild-type to Thy-1 knockout mouse brain sections in the presence of detergent at 4 degrees C, although less efficiently than did gangliosides. These data raise technical challenges for using nonionic detergents in certain histological protocols and for isolation of lipid rafts from brain tissue.     

Protocerebral mediodorsal A2' neurosecretory neurons in late pupae of yellow mealworm (Tenebrio molitor) after exposure to a static magnetic field

The activity of large dorsomedial protocerebral A2' neurosecretory neurons were investigated in late pupae of Tenebrio molitor L, which were exposed to a static magnetic field of 320 mT. Experimental groups were C: the control group which was kept at 5 meters from the magnet; CMF: pupae which were reared in control conditions and sacrificed on the eighth day of pupal stage (parents were kept in a magnetic field); and MF: pupae kept in a permanent magnetic field for eight days. Our results indicate the effects of a static magnetic field on the cytological characteristics and activity of large A2' neurosecretory neurons of Tenebrio molitor pupae.