Poly(I:C) treatment influences the expression of calreticulin and profilin-1 in a human HNSCC cell line: a proteomic study.

Polyinosinic:polycytidylic acid (poly (I:C)) has been formerly known to be an interferon inducer but the mechanism of its action was not revealed until the discovery of Toll-like receptors (TLRs). TLRs are members of transmembrane proteins that recognize conserved molecular motifs of viral and bacterial origin and initiate innate immune response. Recent studies have shown that they are also expressed on tumor cells, but their role in these cells is still not clear. TLR3 recognizes double-stranded RNA (poly (I:C)) and is primarily involved in the defense against viruses. TLR3 ligand binding initiates the activation of transcription factors NF-κB, IRF family members, and AP-1, which can induce wide cascading effect on the cell and consequently activate many cellular processes. Since little is known about TLR3 target genes, we have used the proteomic approach to widen the current knowledge. In this study, we have discovered 15 differentially expressed proteins, mostly connected with protein metabolic processes. Furthermore, we have confirmed by Western blot that calreticulin and profilin-1, proteins which have been shown previously to be involved in processes connected with tumor progression, are differentially expressed after poly(I:C) treatment. By using TLR3 small interfering RNA, we showed that calreticulin expression might be TLR3 dependent, unlike profilin-1.

Antitumor activity from the combined application of poly(I:C) and chemotherapeutics in human metastatic pharyngeal cell lines.

BACKGROUND: Toll-like receptor 3 (TLR3) activation in tumor cells induces apoptosis. We investigated the effect of TLR3 ligand (poly(I:C)) in combination with chemotherapeutics applied to human pharyngeal carcinoma cells as a possible antitumor therapy. METHODS: Human pharyngeal cancer cell lines were studied (FaDu and Detroit 562). Cytotoxicity assays and apoptosis assays (annexin V staining and caspase 3/7 activity measurements) were used to investigate the cytotoxic effects. By using TLR3 siRNA we confirmed that the observed effect is TLR3-dependent. RESULTS: We found that the combined application of poly(I:C) and chemotherapeutics (cisPt, HU, 5-FU and MTX) has a stronger inhibitory effect on cell growth in tumor cells expressing functional TLR3 as compared with a single treatment. This is a result of TLR3-dependent apoptosis. CONCLUSION: Our study showed that a combined application of the two agents already being used in tumor therapy could lower the necessary dosage of chemotherapeutics, leading to fewer side effects.

The dual role of TLR3 in metastatic cell line.

Toll-like receptors (TLRs) are members of transmembrane proteins that recognize conserved molecular motifs of viral and bacterial origin and initiate innate immune response. As the role of TLRs in tumors cells is still not clear, our aim was to investigate the role of TLR3 in primary tumor and metastatic cells (SW480, SW620, FaDu and Detroit 562). We have reported here on the dual role of TLR3 in pharynx metastatic cell line (Detroit 562); on one hand TLR3 activation drove cells to apoptosis while on the other its stimulation contributed to tumor progression by altering the expression of tumor promoting genes (PLAUR, RORB) and enhancing the cell migration potential. In addition, we have shown TLR3 signaling pathway is functional in another metastatic cancer cell line (SW620) suggesting TLR3 might be important in the process of tumor metastasis. Since TLR3 agonists have been used in tumor therapy with the aim to activate immune system, scientific contribution of this work is drawing attention to the importance of further work on this topic, especially pro-tumor effect of TLR3, in order to avoid possible side-effects.

Induction of CMV-1 promoter by 4-hydroxy-2-nonenal in human embryonic kidney cells.

Oxidative stress, i.e., excessive production of oxygen free radicals and reactive oxygen species, leads to lipid peroxidation and to formation of reactive aldehydes which act as second messengers of free radicals. It has previously been shown that oxidative stress may be involved in the transcriptional regulation of cytomegalovirus (CMV) immediate early promoter, involved in viral reactivation from latency. In the current study we used a plasmid containing the yellow fluorescent protein (YFP) gene under the control of CMV-1 promoter to monitor the influence of hydrogen peroxide and reactive aldehydes, 4-hydroxy-2-nonenal (HNE) and acrolein, on CMV-1 promoter activation in human embryonic kidney cells (HEK293). While acrolein was ineffective, hydrogen peroxide slightly (50 %) stimulated the CMV promoter. In contrast, HNE had a strong, up to 3-fold, enhancing effect on the CMV-1 promoter within four as well as after 24h of treatment. The most effective was the treatment with 24 microM HNE. This effect of HNE suggests that stressful conditions associated with lipid peroxidation could lead to CMV activation.

Genitourinary diseases prior spontaneous abortion as a risk factor for recurrent pregnancy loss.

The etiology of recurrent spontaneous abortion (RSA) is still unexplained. Many couples do not find the cause of their RSA at all. The purpose of this research was to evaluate the association between recurrent pregnancy loss and previous (cured prior to pregnancy) acute/chronic genitourinary infections in both parents. Couples (226) having two or more (up to six) spontaneous abortions were analyzed in this retrospective case-control study. The control group consisted of 124 couples with neither miscarriages nor complicated pregnancies in their past. The data (serum immunological markers, karyotype, flow cytometry data, PHD) were collected from their medical charts. It was found that there was no statistically significant difference in average weeks of pregnancy in which the second, third and fourth abortion occurred. There was a statistically significant difference in previously experienced genitourinary infections between women from the RSA group and the control group, as well as for men from the RSA group and the control group. It can be concluded that past infections of the maternal and/or paternal genitourinary system may be the causal factor for recurrent pregnancy loss and can also pre-determine women that are of greater susceptibility to preterm pregnancy. Therefore the genetic counseling of couples should include thorough medical and family history of both partners and their first- and second-degree relatives in conjunction with typical medical examination.

Rett syndrome: from the gene to the disease.

Rett syndrome (RTT, MIM No. 312750) is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation. It is transmitted as an X-linked dominant trait, therefore almost exclusively affecting females. About 80% of RTT cases are sporadic caused by mutations in the MECP2 gene located on Xq28. The gene codes for two isoforms of the methyl-CpG-binding protein (MeCP2, MeCP2B) which are involved in transcriptional silencing through DNA methylation. The gene has 4 exons. The fourth one is the largest. Almost all mutations in MECP2 occur de novo. Although mutations are dispersed throughout the gene, about 67% of all MECP2 mutations, caused by C>T transitions at 8 CpG dinucleotides, are located in the third and fourth exon. The most common mutation is R168X. So far, there is no clear evidence on genotype-phenotype correlations. There are also reports claiming that the same mutation can provoke different phenotypes. It was shown that MeCP2 can silence certain genes. One of them, brain-derived neurotrophic factor, is essential for neural plasticity, learning and memory. This discovery revealed the role of MeCP2 in the control of neuronal activity-dependent gene regulation and suggested that the pathology of RTT may result from deregulation of this process.

Analysis of cystic fibrosis gene mutations and associated haplotypes in the Croatian population.

The aim of this study was to reveal the CFTR gene mutation status in the Croatian population as well as to establish the haplotypes associated with cystic fibrosis (CF) and those associated with specific gene mutations. A total of 48 unrelated CF patients from Croatia were examined. Among 96 tested alleles, we found nine different mutations: DeltaF508, 58.33%; G542X, 3.12%; N1303K, 2.08%; R1162X; 621 + 1G --> T; G85E; Y569C; E585X; and S466X, 1.04%. Analysis of three polymorphic loci revealed 15 different haplotypes. Two of them (21-23-13 and 21-17-13) occurred with a higher frequency (40% and 24%). Both of these haplotypes also carried a CFTR gene mutation (DeltaF508 or G542X) on 27 out of 32 chromosomes. Among 12 (of all together 29) CF alleles on which no mutations were found, we detected 10 different haplotypes. Because there are still no published data on the distribution of polymorphic loci in Croatia, nor haplotypes associated with mutations in the CFTR gene, our results greatly contribute to knowledge regarding the genetic background of CF in this region.

Biological & physiological aspects of action of insulin-like growth factor peptide family.

The insulin-like growth factor (IGF) is a complex system of peptide hormones (insulin-like growth factors of type 1 and 2, IGF-1 and IGF-2), cell surface receptors (insulin receptor, IR; insulin-like growth factor receptors of type 1 and 2, IGF-R1, IGF-R2) and circulating binding proteins (insulinlike growth factor binding proteins, IGF-BP 1-6). IGF-1 and -2 are mitogens that play a role in regulating cell proliferation, differentiation and apoptosis. Their effects are mediated through the IGF-R1 which initiates signaling cascades that result in regulation of a number of biological responses. IGF-R2, together with IGF-BPs is involved in binding, internalization and degradation of IGF-2. IGF proteins regulate cell proliferation in an interconnected action via autocrine, paracrine and endocrine regulatory mechanisms. Consequently, any perturbation in each level of the IGF signaling proteins has been shown to be implicated in development and progression of numerous cancer types. The most important single components in this processes are IGF ligands as well as IGF-R1 - when disturbed they act as oncogenes. It has been shown that: (i) high serum concentrations of IGF-1 and IGF-2 are associated with an increased risk of breast, prostate, colorectal and lung cancers; and (ii) IGF-R1 is commonly disturbed in many tumours (like gastric, lung, endometrial cancer) leading to a phenotype of anchorage-independent tumour growth. In contrast, IGF-R2 is considered to act as a tumour suppressor gene; it protects the cells from neoplastic impulses. Consistent with the IGFs autocrine/paracrine regulation of tumour growth, cancer treatment strategies interfering with IGF-R1 signaling have been developed, that may be useful in future diagnostic and therapeutic strategies.

Monoamine oxidase (MAO) intron 13 polymorphism and platelet MAO-B activity in combat-related posttraumatic stress disorder.

BACKGROUND: The neurobiology of posttraumatic stress disorder (PTSD) involves alterations in multiple neuroendocrine and neurotransmitter systems. Platelet monoamine oxidase (MAO-B) has been associated with susceptibility to various psychiatric disorders, personality traits and behaviors. METHODS: Platelet MAO-B activity and MAO-B intron 13 polymorphism (a G/A substitution) were determined in male war veterans (n=106) with DSM-IV diagnosed current and chronic PTSD, divided into subgroups of PTSD patients with (n=28) or without (n=78) psychotic features, combat exposed veterans (n=41) who did not develop PTSD, and healthy control men (n=242). RESULTS: Two-way ANOVAs revealed a significant effect of diagnosis and smoking, a significant effect of smoking, no significant effect of genotype, and no significant interaction between genotype, smoking or diagnosis, on platelet MAO-B activity. One-way ANOVAs showed significantly lower platelet MAO-B activity in smokers than in nonsmokers. After controlling for smoking, veterans with psychotic PTSD had significantly higher platelet MAO-B activity than veterans with or without PTSD, or healthy subjects. LIMITATIONS: The results were obtained on peripheral biochemical marker, i.e. platelet MAO activity. CONCLUSIONS: The MAO-B intron 13 polymorphism was not functional, and did not affect platelet MAO-B activity. The allele frequencies of the MAO-B genotype were similarly distributed among healthy controls and veterans with or without PTSD and/or psychotic symptoms. The results suggest that platelet MAO-B activity, controlled for smoking status, might be used as a peripheral marker of the psychotic symptoms in PTSD.

Saccharomyces cerevisiae strain expressing a plant fatty acid desaturase produces polyunsaturated fatty acids and is susceptible to oxidative stress induced by lipid peroxidation.

Although oxygen is essential for aerobic organisms, it also forms potentially harmful reactive oxygen species. For its simplicity, easy manipulation, and cultivation conditions, yeast is used as an attractive model in oxidative stress research. However, lack of polyunsaturated fatty acids in yeast membranes makes yeast unsuitable for research in the field of lipid peroxidation. Therefore, we have constructed a yeast strain expressing a Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This yeast strain expresses the heterologous desaturase in an active form and, consequently, produces Delta9/Delta12 polyunsaturated fatty acids under inducing conditions. The functional expression of the heterologous desaturase did not affect cellular morphology or growth, indicating no general adverse effect on cellular physiology. However, the presence of polyunsaturated fatty acids changed the yeast's sensitivity to oxidative stress induced by addition of paraquat, tert-butylhydroperoxide, and hydrogen peroxide. This difference in sensitivity to the latter was followed by the formation of 4-hydroxy-2-nonenal, one of the end products of linoleic fatty acid peroxidation, which is known to play a role in cell growth control and signaling. Here we show that this yeast strain conditionally expressing the Delta12 desaturase gene provides a novel and well-defined eukaryotic model in lipid peroxidation research. Its potential to investigate the molecular basis of responses to oxidative stress, in particular the involvement of reactive aldehydes derived from fatty acid peroxidation, especially 4-hydroxy-2-nonenal, will be addressed.

The lack of association between monoamine oxidase (MAO) intron 13 polymorphism and platelet MAO-B activity among men.

Monoamine oxidase (MAO), a mitochondrial flavine containing enzyme, exists in two isoenzymes, MAO-A and MAO-B. Platelets contain MAO-B subtype, proposed to be a biomarker for different personality characteristics and vulnerability for substance abuse. The most common polymorphism of MAO-B gene, a single base change (A or G) occurs in intron 13. It has been proposed to be a functional polymorphism, controlling the activity of MAO-B in platelets. The aim of the study was to determine the association between platelet MAO-B activity and MAO-B intron 13 polymorphism in 225 racially and ethnically uniform healthy Caucasian men of the Croatian origin. Our results showed that platelet MAO-B activity did not differ between subjects subdivided into those with <> or <>. This polymorphism of the MAO-B gene did not control the activity of the MAO-B in platelets. Platelet MAO-B activity was associated only with the smoking status, and it was significantly decreased in smokers when compared to nonsmokers. No significant association was found between MAO-B polymorphism and smoking status. In healthy individuals of the Croatian origin, the studied MAO-B polymorphism showed a lack of functional importance in regulating MAO-B activity in platelets. Since different populations may vary in the association between functional polymorphism and the MAO-B activity, and the genotype of transcription factor AP-2 beta was reported to be associated with altered platelet MAO-B activity, and with specific personality traits, further studies on different populations should be conducted to elucidate the molecular mechanism/s regulating platelet MAO-B activity.

The MECP2 gene mutation screening in Rett syndrome patients from Croatia.

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder almost exclusively affecting females and is usually sporadic. Mutations in MECP2 gene have been found in more than 80% of females with typical features of RTT. In this study, we analyzed 15 sporadic cases of RTT. In 7 of 15 patients (47%), we detected pathogenic mutations in the coding parts of MECP2 fourth exon. We found two missense (T158M, R133C), two nonsense (R168X, R270X), two frameshift mutations (P217fs and a double deletion of 28-bp at 1132-1159 and 10-bp at 1167-1176), and one in-frame deletion (L383_E392del10). To our knowledge, the last two mutations have not been reported yet. We also detected one previously described polymorphism (S194S). In conclusion, these results show that the fourth exon should be the first one analyzed because it harbors most of the known mutations. Moreover, mutation-negative cases should be further analyzed for gross rearrangements. This is the first study of its kind in Croatia and it enabled us to give the patients an early confirmation of RTT diagnosis.

Prevalence of the 35delG mutation in the GJB2 gene of patients with nonsyndromic hearing loss from Croatia.

The aim of this study was to investigate the allelic frequency of 35delG mutation in patients with recessive, nonsyndromic hearing loss (NSHL) compared to normal hearing individuals in the Croatian population. For this purpose, we analyzed 27 unrelated individuals with nonsyndromic hearing loss and 342 healthy individuals. The method we used is based on the principle of polymerase chain reaction (PCR)-mediated, site-directed mutagenesis, followed by a BsiYI digestion. Among patients with NSHL, the 35delG mutation was found on 51.85% alleles. Carrier frequency among healthy control individuals was 1 in 68.4 (1.5%). The patients, found to be wild-type, either in heterozygous or homozygous form, were further tested by direct sequencing. Among them, two different mutations were observed, W24X and 313del14. Relatively high prevalence of 35delG mutation among patients with NSHL indicate that it is an important cause of NSHL in Croatia. Early diagnosis by identification of the 35delG mutation would greatly improve genetic counseling, as well as treatment and management of deafness in Croatia.