The highly conserved, N-terminal (RXXX)8 motif of mouse Shadoo mediates nuclear accumulation.

The prion protein (PrP)-known for its central role in transmissible spongiform encephalopathies-has been reported to possess two nuclear localization signals and localize in the nuclei of certain cells in various forms. Although these data are superficially contradictory, it is apparent that nuclear forms of the prion protein can be found in cells in either the healthy or the diseased state. Here we report that Shadoo (Sho)-a member of the prion protein superfamily-is also found in the nucleus of several neural and non-neural cell lines as visualized by using an YFP-Sho construct. This nuclear localization is mediated by the (25-61) fragment of mouse Sho encompassing an (RXXX)8 motif. Bioinformatic analysis shows that the (RXXX)n motif (n=7-8) is a highly conserved and characteristic part of mammalian Shadoo proteins. Experiments to assess if Sho enters the nucleus by facilitated transport gave no decisive results: the inhibition of active processes that require energy in the cell, abolishes nuclear but not nucleolar accumulation. However, the (RXXX)8 motif is not able to mediate the nuclear transport of large fusion constructs exceeding the size limit of the nuclear pore for passive entry. Tracing the journey of various forms of Sho from translation to the nucleus and discerning the potential nuclear function of PrP and Sho requires further studies.

Distinct hydration properties of wild-type and familial point mutant A53T of α-synuclein associated with Parkinson's disease.

The propensity of α-synuclein to form amyloid plays an important role in Parkinson's disease. Three familial mutations, A30P, E46K, and A53T, correlate with Parkinson's disease. Therefore, unraveling the structural effects of these mutations has basic implications in understanding the molecular basis of the disease. Here, we address this issue through comparing details of the hydration of wild-type α-synuclein and its A53T mutant by a combination of wide-line NMR, differential scanning calorimetry, and molecular dynamics simulations. All three approaches suggest a hydrate shell compatible with a largely disordered state of both proteins. Its fine details, however, are different, with the mutant displaying a somewhat higher level of hydration, suggesting a bias to more open structures, favorable for protein-protein interactions leading to amyloid formation. These differences disappear in the amyloid state, suggesting basically the same surface topology, irrespective of the initial monomeric state.

Epidemiological modelling of risk factors of human papilloma virus in women with positive cytology in the county of Csongrád.

This study was carried out to determine the prevalence and risk factors of genital HPV infection in women diagnosed with non-negative cytology in Southeastern Hungary. Cervical samples were collected for cytology and HPV testing from women seen at gynaecological outpatient clinics and diagnosed with non-negative cytology. The observed overall average HPV infection rate was found to be 61%. A smoking habit was the only risk factor in the logistic regression analysis that related significantly to exposure to HPV infection. Thus, prevention strategies should focus on the regular clinical cytological screening of HPV-infected patients and on the reduction of smoking.

Two mifepristone doses and two intervals of misoprostol administration for termination of early pregnancy: a randomised factorial controlled equivalence trial.

OBJECTIVE: To compare the efficacy of 100 mg and 200 mg of mifepristone and 24- and 48-hour intervals to administration of 800 microg vaginal misoprostol for termination of early pregnancy. DESIGN: Placebo-controlled, randomized, equivalence trial, stratified by centre. SETTING: 13 departments of obstetrics and gynecology in nine countries. POPULATION: 2,181 women with 63 days or less gestation requesting medical abortion. METHODS: Two-sided 95% CI for the risk differences of failure to complete abortion were calculated and compared with 5% equivalence margin between two doses of mifepristone and two intervals to misoprostol administration. Proportions of women with adverse effects were compared between the regimens using standard testes for proportions. OUTCOME MEASURES: Rates of complete abortion without surgical intervention and adverse effects associated with the regimens. RESULTS: Efficacy outcome was analysed for 2,126 women (97.5%) excluding 55 lost to follow up. Both mifepristone doses were found to be similar in efficacy. The rate of complete abortion was 92.0% for women assigned 100 mg of mifepristone and 93.2% for women assigned 200 mg of mifepristone (difference 1.2%, 95% CI: -1.0 to 3.5). Equivalence was also evident for the two intervals of administration: the rate of complete abortion was 93.5% for 24-hour interval and 91.7% for the 48-hour interval (difference -1.8%, 95% CI: -4.0 to 0.5). Interaction between doses and interval to misoprostol administration was not significant (P = 0.92). Adverse effects related to treatments did not differ between the groups. CONCLUSIONS: Both the 100 and 200 mg doses of mifepristone and the 24- and 48-hour intervals have a similar efficacy to achieve complete abortion in early pregnancy when mifepristone is followed by 800 micrograms of vaginally administered misoprostol.

Whole-Body Barometric Plethysmography Characterizes Upper Airway Obstruction in 3 Brachycephalic Breeds of Dogs.

BACKGROUND: A novel test using whole-body barometric plethysmography (WBBP) was developed recently to diagnose brachycephalic obstructive airway syndrome (BOAS) in unsedated French bulldogs. HYPOTHESIS/OBJECTIVES: The hypotheses of this study were: (1) respiratory characteristics are different between healthy nonbrachycephalic dogs and brachycephalic dogs; and among pugs, French bulldogs, and bulldogs; and (2) obesity and stenotic nares are risk factors for BOAS. The main objective was to establish a diagnostic test for BOAS in these 3 breeds. ANIMALS: A total of 266 brachycephalic dogs (100 pugs, 100 French bulldogs, and 66 bulldogs) and 28 nonbrachycephalic dogs. METHODS: Prospective study. Exercise tolerance tests with respiratory functional grading, and WBBP were performed on all dogs. Data from WBBP were associated with functional grades to train quadratic discriminant analysis tools to assign dogs to BOAS+ and BOAS- groups. A BOAS index (0-100%) was calculated for each dog. Receiver operating characteristic (ROC) curves were used to evaluate classification ability. RESULTS: Minute volume was decreased significantly in asymptomatic pugs (P = .009), French bulldogs (P = .026), and bulldogs (P < .0001) when compared to nonbrachycephalic controls. Respiratory characteristics were different among breeds and affected dogs had a significant increase in trace variation. The BOAS index predicted BOAS status for each breed with 94-97% (95% confidence interval [CI], 88.9-100%) accuracy (area under the ROC curve). Both obesity (P = .04) and stenotic nares (P = .004) were significantly associated with BOAS. CONCLUSIONS AND CLINICAL IMPORTANCE: The WBBP can be used as a clinical tool to diagnose BOAS noninvasively and objectively.

One-step indirect migration inhibiton (LIF) assay.

A simple, 1-step indirect leukocyte migration inhibition (LIF) assay is described. Advantages of this method are as follows: the mitogen does not directly influence the migration of polymorphonuclear leukocytes, relatively few cells are needed, and results are obtained within 24 h. LIF is simultaneously produced by Con A-pretreated lymphocytes in the migration test system. The lymphokine-producing cells are in the test plate well, in the culture medium surrounding the agarose droplets containing migrating cells. The LIF production can be inhibited by the protein synthesis inhibitor puromycin and by prostaglandin (PGE1). In this test system the LIF production is not influenced by endogenous prostaglandins. This method can be used for complex immunopharmacological investigations because effects of agents on the random migration of polymorphonuclear leukocytes and on LIF production of lymphocytes can be tested in the same system.

Enhancement of mitogen-induced lymphocyte proliferation after preincubation is due to altered sensitivity to prostaglandins.

Twenty-four hour preincubation at 37 degrees enhances the mitogen-induced DNA synthesis of human lymphocytes. PGE1, given simultaneously with Con A at the start of lymphocyte culture, inhibits the DNA synthesis. After 24 h preincubation of cells, PGE1 fails to decrease the DNA synthesis. Similarly, preincubation abolished the effect of indomethacin increasing DNA synthesis of freshly separated lymphocytes. The intracellular cAMP level of human mononuclear leukocytes rapidly decreases during in vitro incubation at 37 degrees C. PGE1 elevates the intracellular cAMP of freshly separated lymphocytes to 45 times of its starting level. After 24 h preincubation of cells at 37 degrees C, PGE1 elevates cAMP to a lesser extent. This change of PGE1 action may explain the fact that the effect of exogenous PGE1 and endogenous prostaglandins (the production of which can be inhibited by indomethacin) diminishes after incubation of lymphocytes. It is very likely that the change of the effect of prostaglandins produced in lymphocyte cultures and the spontaneous decrease of intracellular cAMP level explains the enhancement of mitogen-induced lymphocyte proliferation after 24 h preincubation at 37 degrees C.

Inhibitory effect of tumour cells on leukocyte motility.

Hep-2 cells inhibit the random migration of human polymorphonuclear leukocytes in a tumour cell-leukocyte microenvironmental model, which is a special form of co-migration. The inhibition is indomethacin-sensitive. It is very likely that prostaglandins, produced as a consequence of interaction between tumour cells and leukocytes, are the mediators of this phenomenon. The tumour-induced inhibition of leukocyte motility may represent a tumour-protective mechanism.

Synthetic peptides comprising defined sequences of CH-2 and CH-3 domains of human IgG1 induce prostaglandin E2 production from human peripheral blood mononuclear cells.

Synthetic peptides Y48 and Y75 comprising sequences at exposed sites within the CH-2 and CH-3 domains of human IgG1 at a concentration of 10(-5) M, increase PGE2 production by human peripheral blood mononuclear cell (PBMC) cultures. An increase of leukocyte migration inhibitory factor (LMIF) production in PBMC cultures--as a result of synthetic peptide treatment--was also observed. This LMIF activity, to some extent, is attributed to the PGE2 production by the cells; the inhibition of leukocyte migration being abolished by the presence of indomethacine or antibody to PGE2.

[A new method of molecular testing in the differential diagnosis of hereditary hemochromatosis]. / Uj, molekuláris genetikai módszer az öröklödö haemochromatosis differenciáldiagnosztikájában.

Hereditary hemochromatosis is an autosomal, recessive disorder of the iron metabolism. The hemochromatosis gene (HFE) was previously located on chromosome 6 and recently identified by positional cloning. A point mutation, C282Y, was found to be present in the HFE gene in homozygous form in 64 to 100% of patients with established hemochromatosis. The relationship of a second polymorphic variant of the HFE gene, H63D to the formation of iron overload is debated. Although hemochromatosis is one of the most common inherited disorders among Caucasians, in the absence of specific signs it is rarely diagnosed. In order to obtain comparable epidemiological data for Hungary, we tested 1271 and 277 randomly selected, unrelated, healthy subjects for C282Y and H63D respectively. In addition C282Y testing was carried out in 58 patients suffering from liver cirrhosis, and in 191 individuals with suspected hemochromatosis. For C282Y and H63D mutation analyses polymerase chain reaction technique followed by Rsa I and Bcl I restriction enzyme digestion was used. We developed an alternative method for the detection of C282Y based on an amplification-generated Kpn I restriction site. The allele frequencies were 3.8% and 12.3% for C282Y and H63D respectively in the normal Hungarian population. There was no significant difference in C282Y allele frequencies between liver disease patients (1.7%) and the normal population. We identified 15 homozygous and 25 heterozygous individuals among 191 individuals with suspected hemochromatosis. The C282Y and the H63D allele frequencies in the normal Hungarian population were found to be similar to the allele frequencies observed in other European populations, indicating that there is a large number of individuals susceptible for iron overload in Hungary (1:700). Mutation analysis is a novel, non-invasive method in the diagnostics of hereditary hemochromatosis, which increasingly becomes part of the routine clinical work.

[Detection of human papillomavirus infection by the nucleic acid hybridization method (a multicenter study)]. / Human papillomavírus-fertözés kimutatás nukleinsav hibridizációs módszerrel (multicentrikus tanulmány).

The human papillomaviruses (HPV) are regarded as one of the important agents of cervical carcinoma. A multicentre study was organized to determine the prevalence of HPV in the fertile female population in Hungary. Parallel with the clinical sample collection, a questionnaire interview was performed to acquire data on the life style, socioeconomic status, sexual practice, etc. 1200 women were examined colposcopically and cervix samples were collected for cytology and the detection of HPV DNA. 17.4% of the samples were HPV-infected. 3.9% of the patients had acquired low-risk, and 10.1% 10.2% high-risk HPV types; 3.4% of the women were at the same time infected with both low-risk and high-risk HPV types. Simultaneously performance of cytology and the HPV hybrid capture assay contribute to recognise and treat the precancerous status and risk factors.

Effect of prostaglandins on polymorphonuclear leukocyte motility.

The effect of prostaglandins (PGE1, E2, F2 alpha on LIF production, LIF activity, and on the random migration of polymorphonuclear leukocytes was investigated. Concanavalin A-induced LIF production was tested in an indirect LIF assay using agarose microdroplet technique; PGE1 and PGE2 inhibited the LIF production. PGE1, PGE2, and F2 alpha in physiological concentration, decreased the random migration of leukocytes, they also interfered with the LIF activity of supernatants, decreasing their inhibitory effect on cell migration; but the opposite explanation that LIF-treated PMNs escaped the inhibitory effect of PGs, cannot be ruled out. Macrophages produce supernatant factor(s) in the course of phagocytosis which inhibit the motility of polymorphonuclear leukocytes. The active component of supernatant seems to be PG, since its production can be blocked by indomethacin. These data indicate that prostaglandins may have different sites of attack in the local regulation of leukocyte motility.

Dissociation of the mitogenic and rosette-forming effects of PHA to stimulators of cyclic AMP levels.

The mitogenic and the spontaneous E-rosette stimulating effects of phytohaemagglutinin (PHA) were studied by examining their responses to an elevation of intracellular cAMP induced by pretreatment with aminophylline, adrenaline or PGE1 or by adding exogenous cAMP. Elevation of the intracellular cAMP levels of lymphocytes was found inhibitory to the mitogenic effect of PHA, while leaving the increased early and stable spontaneous rosette formation unaffected. It also failed to depress the PHA-induced slight increase in the proportion of total spontaneous E-rosette-forming lymphocytes. The responses of the two PHA effects under study to the elevation of the cAMP level are thus dissociated, suggesting that the two effects represent two different manifestations of lymphocyte activation, though these manifestations may not be independent of each other. It has been confirmed that PHA produces a shift primarily within the T-cell population toward the active and the stable E-rosette-forming subpopulation, while the increase in the total E-rosette-forming lymphocyte population is but slight. This may possibly involve the activation of the T-derived "null" cells unable to bind sheep red blood cells without PHA treatment.