Erika Zilahi

A melittin-related peptide from the skin of the Japanese frog, Rana tagoi, with antimicrobial and cytolytic properties

Two peptides with antimicrobial and cytolytic properties were purified from an extract of the skin of Tagos brown frog Rana tagoi. The primary structure of one peptide (FLPILGKLLS(10)GIL.NH(2)) identifies it as a member of the temporin family, whereas the second peptide (AIGSILGALA(10)KGLPTLISWI(20)KNR.NH(2)) displays 78% sequence identity to melittin from the venom of the honeybee Apis florea. Compared with melittin, the melittin-related peptide (MRP) was equipotent in inhibiting the growth of the Gram-positive bacterium Staphylococcus aureus, 5-fold less potent against the Gram-negative bacterium Escherichia coli and against the fungal pathogen, Candida albicans. MRP was 13-fold less hemolytic than melittin against human erythrocytes and 4- and 5-fold less cytolytic against mouse EL4 T-lymphoma-derived cells and L929 fibroblasts, respectively. However, at non-cytotoxic concentrations (<or=8 microM), MRP did not protect HeLa cells from cell death produced by human rhinovirus type 2 infection.

Increased microRNA-146a/b, TRAF6 gene and decreased IRAK1 gene expressions in the peripheral mononuclear cells of patients with Sjögren's syndrome.

MicroRNA-146a (miR-146a) is a microRNA supposed to regulate innate immune, inflammatory response and antiviral pathway negatively. Recently, its potential use as a biomarker for disease diagnosis, prevention and treatment has become widely investigated. In the current study, we measured the expression of miR-146a/b, and their target genes, IRAK1, IRAK4, TRAF6 in the peripheral mononuclear cells of patients with Sjögrens syndrome (n=21) and healthy controls (n=10) by quantitative reverse transcription polymerase chain reaction. We found that both miR-146a and miR-146b, furthermore, the gene of TRAF6 were significantly overexpressed in the Sjögrens patients, whereas the expression of IRAK1 gene was significantly decreased. The expression of IRAK4 did not differ significantly. These results suggest that in the peripheral mononuclear cells of Sjögrens patients, the transcriptional repression of IRAK1 is taking place, whereas the other NF-κB pathway regulating gene, TRAF6 is overexpressed. As IRAK1 has been regarded a crucial gene in the pathogenesis of systemic lupus erythematosus, TRAF6 can be a Sjögrens syndrome specific biomarker, confirming and partly explaining the existance of different pathogenic pathways in the two diseases. These observations, however, need still wider confirmations.

Interaction of HLA-DRB1*03 and smoking for the development of anti-Jo-1 antibodies in adult idiopathic inflammatory myopathies: a European-wide case study

Objectives HLA-DRB1*03 is strongly associated with anti-Jo-1-positive idiopathic inflammatory myopathies (IIM) and there is now increasing evidence that Jo-1 antigen is preferentially expressed in lung tissue. This study examined whether smoking was associated with the development of anti-Jo-1 antibodies in HLA-DRB1*03-positive IIM. Methods IIM cases were selected with concurrent information regarding HLA-DRB1 status, smoking history and anti-Jo-1 antibody status. DNA was genotyped at DRB1 using a commercial sequence-specific oligonucleotide kit. Anti-Jo-1 antibody status was established using a line blot assay or immunoprecipitation. Results 557 Caucasian IIM patients were recruited from Hungary (181), UK (99), Sweden (94) and Czech Republic (183). Smoking frequency was increased in anti-Jo-1-positive IIM cases, and reached statistical significance in Hungarian IIM (45% Jo-1-positive vs 17% Jo-1-negative, OR 3.94, 95% CI 1.53 to 9.89, p<0.0001). A strong association between HLA-DRB1*03 and anti-Jo-1 status was observed across all four cohorts (DRB1*03 frequency: 74% Jo-1-positive vs 35% Jo-1-negative, OR 5.55, 95% CI 3.42 to 9.14, p<0.0001). The frequency of HLA-DRB1*03 was increased in smokers. The frequency of anti-Jo-1 was increased in DRB1*03-positive smokers vs DRB1*03-negative non-smokers (42% vs 8%, OR 7.75, 95% CI 4.21 to 14.28, p<0.0001) and DRB1*03-positive non-smokers (42% vs 31%, p=0.08). In DRB1*03-negative patients, anti-Jo-1 status between smokers and non-smokers was not significantly different. No significant interaction was noted between smoking and DRB1*03 status using anti-Jo-1 as the outcome measure. Conclusion Smoking appears to be associated with an increased risk of possession of anti-Jo-1 in HLA-DRB1*03-positive IIM cases. The authors hypothesise that an interaction between HLA-DRB1*03 and smoking may prime the development of anti-Jo-1 antibodies.

The S. pombe sep1 gene encodes a nuclear protein that is required for periodic expression of the cdc15 gene

The Schizosaccharomyces pombe sep1 gene encodes a putative transcription factor that is required for cell separation. Among the genes required for septum formation and cytokinesis in fission yeast examined to date, the only one whose mRNA fluctuates significantly during the cell cycle is cdc15. In this study we have examined cdc15 mRNA levels in sep1 mutant and null backgrounds and have found that sep1p function is required for periodic accumulation of cdc15 mRNA. We have also localised sep1p and find that it is a nuclear protein, consistent with its proposed role as a transcription factor.

Association of rheumatoid arthritis with HLA-DR1 and HLA-DR4 in Hungary.

Abstract: Susceptibility to and outcome for rheumatoid arthritis (RA) have been associated with particular HLA‐DR alleles, but these alleles vary among ethnic groups and geographic areas. The frequency of HLA‐DR1 (HLA‐DRB1*0101, DRB1*0102) and HLA‐DR4 (DRB1*0401, DRB1*0404) alleles is elevated among Caucasian patients with RA. We studied a northeastern Hungarian population of RA patients to determine the frequency of HLA‐DR1 and HLA‐DR4 phenotypes in this population and to compare it with healthy control subjects, as well as to investigate whether the presence of these alleles could be a marker for RA. We performed HLA‐DRB1 genotyping (DRB1*01‐DRB1*16) in 83 RA patients and 55 healthy controls using polymerase chain reaction with sequence‐specific primers (PCR‐SSP). In the case of HLA‐DR1‐ or HLA‐DR4‐positive patients, the DR1 and DR4 subtypes were also determined. The frequency of HLA‐DR4 alleles was significantly higher in RA patients than in controls (31.3 vs. 10.9%; P < .05). HLA‐DR1, in particular, tended to be more frequent in patients than in controls (32.5 vs. 18.1%). Among the HLA‐DR4 subtypes, DRB1*0401 and DRB1*0404 were the most common alleles found in both groups. However, no significant differences were seen in the frequency of HLA‐DRB1*0401 and HLA‐DRB1*0404 between RA patients and controls. In contrast, HLA‐DRB1*0405 and HLA‐DRB1*0408 were significantly more common in RA patients than in control subjects. Among HLA‐DR1 subtypes, the DRB1*0101 allele was most commonly detected, but HLA‐DRB1*0101 as well as DRB1*0102 and DRB1*0105 were similarly frequent in RA patients and controls. HLA‐DR12 was more common among controls than in RA patients (18.1 vs. 0%; P < .05). Our results generally agree with the findings in other Caucasian populations. Nonetheless, we found differences in the frequency of HLA‐DR1 and HLA‐DR4 subtypes among Hungarian patients compared with reports from other geographic regions (e.g., Finland and Asia). Our data suggest that in northeastern Hungary, HLA‐DR4 as well as its subtypes DRB1*0405 and DRB1*0408 may be involved in susceptibility to RA, but HLA‐DR1 may not. In addition, the presence of HLA‐DR12, at least in Hungary, may protect from this disease.

Eleven novel sep genes of Schizosaccharomyces pombe required for efficient cell separation and sexual differentiation

Genetic analysis of 20 sterile mutants prone to form hyphae revealed 11 novel ste genes (sep6 to sep16) of Schizosaccharomyces pombe. None of the mutants was completely mycelial. Most mutants formed branching hyphae and showed normal septation. Aberrant septal structures and actin distribution were seen only at 36°C. sep9‐307, sep14‐576 and sep15‐598 showed genetic interactions with sep1‐1, a mutation in a forkhead transcription factor homologue. Additional genetic interactions were detected between sep6‐194, sep15‐598 and cdc16‐116, a mutant allele of an anaphase modulator of p34cdc2. sep9‐307 and sep15‐598 caused dikaryosis in wee1− background. In mating and sporulation tests, sep6−, sep7−, sep9−, sep10−, sep11− and sep15− proved to be defective in conjugation only, whereas sep8−, sep13− and sep16− were also defective in meiosis‐sporulation. sep12− and sep14− were only partially sterile. All mutants could produce M‐factor but sep8−, sep11−, sep15− and sep16− were defective in P‐factor production. The mutations in sep8, sep11 and sep16 suppressed the pat1‐114‐driven meiosis. All mutants were sensitive to the presence of higher concentrations of chloride in the medium and to short heat shocks. The diversity of the mutant phenotypes and the pleiotropic effects of the mutations suggest that these sep genes might act in, or interact with, a multiple overlapping network of regulatory modules. Copyright

Homozygosity for the 168His variant of the minor histocompatibility antigen HA-1 is associated with reduced risk of primary Sjögren's syndrome

The genes for the human ATP‐binding cassette (ABC) transporter ABCA7 and the minor histocompatibility antigen HA‐1 are juxtaposed in close proximity on chromosome 19p13.3. The multispan transmembrane protein ABCA7 contains an extracellular domain that is recognized by antisera from patients with Sjögrens syndrome (“Sjögren‐epitope”). Recent work from our laboratory demonstrating the involvement of ABCA7 in cellular ceramide and phosphatidylserine export suggests a role for this transporter in programmed cell death. In HA‐1, a protein of unknown function, a His/Arg polymorphism (His168Arg), which constitutes the immunologic target for HA‐1‐specific cytotoxic T cells, has been causatively linked to graft‐versus‐host disease after allogeneic stem cell transplantation. Because these findings suggest a potential implication of ABCA7 and HA‐1 in immune processes, we tested the hypothesis that allelic variants in both genes are associated with autoimmune disorders. We identified a total of 31 exonic single‐nucleotide polymorphisms (SNP) in the ABCA7/HA‐1 gene complex, nine of which represent non‐synonymous nucleotide alterations. Genotypes of ABCA7 and HA‐1 SNP were determined in three distinct Caucasian populations of patients with primary Sjögrens syndrome and ethnically matched controls. Comparison of allele frequencies between these groups revealed that the incidence of the HA‐1 168His allele is significantly lower in Sjögrens syndrome patients than in controls (p<0.003). In contrast, the frequencies of all ABCA7 allelic variants and additional HA‐1 polymorphisms were similar in patients and controls. In cohorts of patients with systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis, no significant differences in the frequencies of ABCA7 and HA‐1 allelic variants were observed relative to controls. Our results suggest that the HA‐1 168His variant is associated with reduced susceptibility to primary Sjögrens syndrome.

The Schizosaccharomyces pombe sep15 + gene encodes a protein homologous to the Med8 subunit of the Saccharomyces cerevisiae transcriptional mediator complex

Abstract We previously described the isolation of mutants defective in cell separation and the identification of 16 sep genes with complex functions. Here we report on the cloning and analysis of sep15+. The deduced amino acid sequence of the Sep15 protein shows significant homology to Med8, a component of the Saccharomyces cerevisiae transcription mediator complex. The mutation sep15-598 confers hyphal morphology and causes temperature-sensitive lethality. Disruption of sep15+ is lethal, indicating that Sep15 exerts an essential function and its role in cell separation is indirect.

Genomic expression patterns in cell separation mutants of Schizosaccharomyces pombe defective in the genes sep10 ( + ) and sep15 ( + ) coding for the Mediator subunits Med31 and Med8.

Cell division is controlled by a complex network involving regulated transcription of genes and postranslational modification of proteins. The aim of this study is to demonstrate that the Mediator complex, a general regulator of transcription, is involved in the regulation of the second phase (cell separation) of cell division of the fission yeast Schizosaccharomyces pombe. In previous studies we have found that the fission yeast cell separation genes sep10+ and sep15+ code for proteins (Med31 and Med8) associated with the Mediator complex. Here, we show by genome-wide gene expression profiling of mutants defective in these genes that both Med8 and Med31 control large, partially overlapping sets of genes scattered over the entire genome and involved in diverse biological functions. Six cell separation genes controlled by the transcription factors Sep1 and Ace2 are among the target genes. Since neither sep1+ nor ace2+ is affected in the mutant cells, we propose that the Med8 and Med31 proteins act as coactivators of the Sep1-Ace2-dependent cell separation genes. The results also indicate that the subunits of Mediator may contribute to the coordination of cellular processes by fine-tuning of the expression of larger sets of genes.

Role of Cell Shape in Determination of the Division Plane in Schizosaccharomyces pombe: Random Orientation of Septa in Spherical Cells

The establishment of growth polarity in Schizosaccharomyces pombe cells is a combined function of the cytoplasmic cytoskeleton and the shape of the cell wall inherited from the mother cell. The septum that divides the cylindrical cell into two siblings is formed midway between the growing poles and perpendicularly to the axis that connects them. Since the daughter cells also extend at their ends and form their septa at right angles to the longitudinal axis, their septal (division) planes lie parallel to those of the mother cell. To gain a better understanding of how this regularity is ensured, we investigated septation in spherical cells that do not inherit morphologically predetermined cell ends to establish poles for growth. We studied four mutants (defining four novel genes), over 95% of whose cells displayed a completely spherical morphology and a deficiency in mating and showed a random distribution of cytoplasmic microtubules, Tea1p, and F-actin, indicating that the cytoplasmic cytoskeleton was poorly polarized or apolar. Septum positioning was examined by visualizing septa and division scars by calcofluor staining and by the analysis of electron microscopic images. Freeze-substitution, freeze-etching, and scanning electron microscopy were used. We found that the elongated bipolar shape is not essential for the determination of a division plane that can separate the postmitotic nuclei. However, it seems to be necessary for the maintenance of the parallel orientation of septa over the generations. In the spherical cells, the division scars and septa usually lie at angles to each other on the cell surface. We hypothesize that the shape of the cell indirectly affects the positioning of the septum by directing the extension of the spindle.