[The relationship between chemerin, bone metabolism, the RANKL/RANK/OPG system, and bone mineral density in girls with anorexia nervosa].

INTRODUCTION: Based on recent studies in humans, chemerin has been classified as an adipokine that might be associated with osteoporosis and BMD. Bone loss is common in adolescents with anorexia nervosa (AN). Moreover, dysfunction in the production of chemerin has also been shown. Therefore, we carried out a comparative analysis between chemerin, bone metabolism, the RANKL/RANK/OPG system, and BMD in girls with AN. MATERIAL AND METHODS: Plasma chemerin, OC, CTx, OPG, and sRANKL were determined by ELISA in 75 girls with AN aged 12.6-17.8 years. BMD was assessed by DXA and expressed as Z-score according to the lumbar spine (s) and total body (TB) sites. According to the s-BMD- and TB-BMD Z-score, girls with AN were divided into two subgroups with parallel analyses used: normal (Z-score > -2.0) and low (Z-score ≤ -2.0) s-BMD, and normal (Z-score > -2.0) and low (Z-score ≤ -2.0) TB-BMD. RESULTS: Mean OC and the OPG/sRANKL ratio were markedly lower in the low s-BMD subgroup compared to the normal s-BMD subgroup. The s-Z-score values (both low and normal) correlated significantly and positively with the OPG/sRANKL ratio. Only in the low s-BMD subgroup did chemerin correlate significantly and positively with all nutritional indices and the OPG/sRANKL ratio. In the low TB-BMD subgroup the mean OC and the OPG/sRANKL ratio were lower than in the normal TB-BMD subgroup. The TB-Z-score values (both normal and low) correlated significantly and positively with all nutritional indices and the OPG/sRANKL ratio. The low TB-Z-score values correlated significantly and positively also with chemerin. In the low TB-BMD subgroup chemerin correlated significantly and positively with weight and BMI (expressed as absolute values), Cole index, the duration of the disease, and OPG/sRANKL ratio while its correlation with age was negative. CONCLUSIONS: Undernutrition and associated deficit of adipose tissue may result in inadequate chemerin production and skeletal disorders in girls with AN. Chemerin acts as a coordinator of the dynamic balance between bone metabolism and the OPG/RANK/RANKL system and, in turn, may contribute to the loss of bone mass in girls with AN. The cortical bone site seems to be more severely responsive to chemerin actions than the trabecular bone site.

New Variants of the Cytochrome P450 2R1 (CYP2R1) Gene in Individuals with Severe Vitamin D-Activating Enzyme 25(OH)D Deficiency.

BACKGROUND: Vitamin D is a fat-soluble cholesterol derivative found in two forms, vitamin D2, and vitamin D3. Cytochrome P450 2R1 (CYP2R1) encoded by the CYP2R1 gene is the major hydroxylase that activates vitamin D by catalyzing the formation of 25-hydroxyvitamin D (25(OH)D). METHODS: We collected 89 (100%) subjects, 46 of which (51.69%) had a documented severe deficiency of 25(OH)D (<10 ng/mL) and 43 (48.31%) in the control group with documented optimum levels of 25(OH)D (>30 ng/mL). We performed Sanger sequencing of three selected fragments of the CYP2R1 gene (Ch11: 14878000-14878499; Ch11: 14880058-14880883 and Ch11: 14885321-14886113) that affect the binding of substrates to this enzyme and analyzed the possible involvement of genetic variation in these regions with an increased risk of vitamin D deficiency in healthy Polish individuals. RESULTS: Two substitutions were found within the three fragments. Bioinformatic analysis suggested that one of these (NC_000011.10: g.14878291G>A) may influence the structure and function of CYP2R1. CONCLUSIONS: Variant NC_000011.10: g.14878291G>A may have a perturbing effect on heme binding in the active site of CYP2R1 and on the function of 25-hydroxylase and probably affects the concentration of 25(OH)D in vivo. We intend to perform functional verification in a larger patient population to confirm and extend these results.

Selected E2F2 Polymorphisms in Oral and Oropharyngeal Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) are subgroups of head and neck squamous cell carcinoma. E2F Transcription Factor 2 (E2F2) could contribute to cancer development, because it plays a critical role in many cellular processes, including the cell cycle, proliferation, differentiation, DNA damage response, and cell death. In the current study, we assessed the associations of five E2F2 polymorphisms (rs6667575, rs3218121, rs3218211, rs3218148, and rs3218203) with OSCC and OPSCC and influence on the TNM staging and grading. This is the first such survey to concern the European population. The study included 94 primary tumour samples following surgical resection from patients, whereas the control group consisted of 99 healthy individuals. We tried a matching of cases and controls for age and sample size. DNA samples were genotyped by employing the 5' nuclease assay for allelic discrimination. Our results suggested that the most significant difference between the control group and the cancer group was the A/G heterozygote for rs3218121. Samples containing this genotype were mostly found in the control group. In our samples, rs6667575, rs3218121, rs3218211, and rs3218148 polymorphisms may affect the course of OSCC and OPSCC, while rs3218203 was not associated with OSCC and OPSCC. However, further studies are warranted to confirm our findings.

miREIA - an immunoassay method in assessment of microRNA levels in tumor tissue-pilot study. The impact of miR-93-5p, miR-142-5p and IFNγ on PD-L1 level in colorectal cancer.

Colorectal cancer is the second and third most common cancer in females and males, respectively. The PD-L1/PD-1 immune checkpoint is an important source of immunosuppression in the tumor microenvironment and is associated with IFNγ. Recent studies have revealed that a significant number of tumor suppressive miRNAs can regulate the expression of PD-L1.                The objective quantification of selected microRNAs using the miREIA method in CRC tissue was performed. We investigated the roles of miR-93-5p and miR-142-5p expression and the levels of IFNγ in regulating the expression of PD-L1 in tumor and margin tissues of CRC in relation to the histological grade, TNM classification, and tumor localization. 37 samples of tumor and margin tissues from CRC patients were evaluated. MiR-93-5p and miR-142-5p levels were measured by a method for quantitative measurement of human microRNA (miREIA). The concentrations of PD-L1 and IFNγ were determined by the ELISA kit. We found higher concentrations of miR-93-5p, PD-L1 and IFNγ in tumor samples compared to tumor margin samples. A significant correlation was found between PD-L1 and IFNγ in tumor and margin specimens; between miR-142-5p and miR-93-5p levels  in tumor and margin specimens. A higher level of miR-93-5p was found in tumor margin tissues on the left side of the colon. Patients with distant metastases were characterized by higher miR-93-5p concentration compared to patients without metastases. CRC is an important source of PD-L1, IFNγ and miR-93-5p expression. Understanding the mechanisms underlying intratumoral PD-L1 expression may open new opportunities for targeted immunotherapy for colorectal cancer.

VDR Gene Polymorphisms in Healthy Individuals with Family History of Premature Coronary Artery Disease.

AIM: The gene encoding the vitamin D receptor (VDR) is considered in many studies to be a good candidate responsible for susceptibility to several diseases such as coronary artery disease (CAD). Epidemiological data show that cardiovascular disease is one of the major health problems in Polish society. Basic studies show that genetic factors play a significant role in the pathogenesis of CAD. We conducted this clinical study to determine if the VDR gene polymorphisms TaqI (rs731236), ApaI (rs7975232), and FokI (rs2228570) could predispose healthy individuals to an increased risk of premature CAD (P-CAD) incidents. METHODS: We genotyped 845 subjects in a cohort consisting of 386 healthy volunteers with a documented P-CAD incident in their first-degree relatives and 459 healthy volunteers without family history (FH) of P-CAD. TaqI, ApaI, and FokI polymorphisms in VDR were genotyped using TaqMan assays and the endpoint genotyping method (qPCR). Statistical analyses were performed using the Power Analysis Software STATISTICA v.13.3. RESULTS: Although no statistical significance was found for TaqI and ApaI genotype frequencies, the AA genotype of FokI polymorphism was significantly more frequent in the study group compared to the control group (24.61% vs. 16.99%). The results of logistic regression analysis suggested a significant association between FokI polymorphism and FH of P-CAD in heathy people under the recessive model (OR: 1.26 (1.07-1.49, p = 0.007)); however, the frequency of VDR haplotypes did not differ significantly between the control and study populations. CONCLUSIONS: FokI polymorphism is may be associated with FH of P-CAD. FokI polymorphism may predispose to the development of P-CAD among healthy people over the next years.

The levels of sICAM-1, sELAM-1, TNFα and sTNFR1 proteins in patients with colorectal adenocarcinoma in tumor and corresponding normal mucosa.

Colorectal cancer is a common malign disease of the gastrointestinal tract. The cancer survival rate depends on the stage of the disease at detection time. It is well known that several molecular mechanisms are involved in cancer and some molecules might affect or modulate cancerogenesis. The aim of the study was to assess the levels of sICAM-1, sELAM-1, TNFα and sTNFR1 protein in tumor and corresponding normal mucosa in a group of patients with colorectal adenocarcinoma and also associations of these parameters with demographic and clinical profiles of the patients. Tissue specimens were obtained during resection of neoplastic lesions. Protein levels were assayed in tissue homogenates by ELISA. The protein level of sICAM-1 in tumor was significantly increased in comparison to the corresponding normal mucosa (80.06 ng/mg vs 69.53 ng/mg, p=0.02). Furthermore, a significant positive correlation between sICAM-1 and sTNFR1 proteins levels in tumor (rs=0.58, p<0.001) and in corresponding normal mucosa (rs=0.48, p<0.001) was found. Also, significant correlations in corresponding normal mucosa were found between sELAM-1 and sICAM-1 (rs=0.58, p<0.001) and between sTNFR1 and sELAM-1 (rs=0.57, p<0.001). Significantly higher level of sTNFR1 in corresponding normal mucosa samples of patients with distant metastases was observed (p=0.04). Obtained results suggest that sICAM-1 protein could be considered as colorectal cancer marker. Furthermore, sTNFR1 also has the potential to become a good prognostic marker used during monitoring of the patients. Nevertheless, a further study in this area to confirm this correlation is required.

Selected CDKN2A and MDM2 polymorphisms in oral cavity cancer.

The head and neck squamous cell carcinoma (HNSCC) is an aggressive human malignancy diagnosed in more than 800 000 new cases worldwide, and mostly arises in the oral cavity, oropharynx, larynx, hypopharynx, and nasopharynx. The study presented here aimed to determine a possible association of rs11515 and rs3088440 gene polymorphisms in the CDKN2A gene (cyclin dependent kinase inhibitor 2A), as well as rs769412 and rs937283 in the MDM2 gene (murine double minute 2), with oral cavity cancer in a sample of Polish population; CDKN2A is crucial in regulating the cell cycle while MDM2 is an oncogene involved in regulating cell proliferation and apoptosis. The study included 95 primary tumor samples following surgical resection from patients, and the control group consisted of 100 healthy individuals. DNA samples were genotyped by employing the 5' nuclease assay for allelic discrimination using TaqMan SNP Genotyping Assays (Applied Biosystems, USA). There was no significant association between any of the polymorphism (rs11515, rs3088440, rs769412 and rs937283) and the oral cavity cancer risk. We found that the AA homozygotes for rs3088440 were significantly more frequent in the control group (OR=0.046, p<0.0001). In addition, the GG genotype of rs769412 was not found in any group. We found no influence of the examined genotypes on clinicopathological parameters, such as T, N and grading values in patients with oral cavity cancer. The results of this study indicate that none of the investigated polymorphisms were associated with the risk of oral cavity cancer in the examined sample of the Polish population.

Assessment of CMKLR1 level in colorectal cancer and its correlation with angiogenic markers.

INTRODUCTION: Colorectal cancer (CRC) is the second most common malignant neoplasm in men and third in women. It is also the third leading cause of cancer-related death, killing annually >700,000 patients in the world. The global burden of CRC is expected to increase by 60% to >2.2 million new cases and 1.1 million deaths by 2030. The pathogenesis of cancer mainly depends on angiogenesis. This process plays a key role in the growth and infiltration of tumors which is essential for distant metastases. A large number of biochemical pathways is involved in the regulation of angiogenesis. As a subject of our study, we chose chemerin/chemokine-like receptor 1 (CMKLR1) pathway which is responsible for the angiogenic processes in malignant neoplasms. AIM OF THE STUDY: To assess the CMKLR1 level and the concentrations of the two markers of angiogenesis, matrix metalloproteinase (MMP)-9 and vascular cell adhesion molecule (VCAM)-1, in tumor and margin tissues of CRC in relation to histological grade and TNM classification. MATERIALS AND METHODS: The study used 47 samples of tumor and margin tissues derived from CRC patients. To determine the concentration of CMKLR1, MMP-9, and VCAM-1, we used the commercially available enzyme-linked immunosorbent assay kit. RESULTS: We found a significantly higher concentration of CMKLR1 and MMP-9 in tumor tissue compared to margin. There was no difference in VCAM-1 concentration between tumor and margin. The margin concentration of CMKLR1 was significantly correlated with that of both MMP-9 and VCAM-1. The margin concentration of VCAM-1 was correlated with that of MMP-9. Additionally, we observed that the tumor levels of CMKLR1 and MMP-9 were positively correlated with the tumor size (T parameter). CONCLUSION: CMKLR1 activity may be associated with the angiogenic process in CRC via MMP-9 activity. Further research, involving a larger sample, may verify whether chemerin/CMKLR1 axis could be considered as a suitable target in novel molecular therapies.

Epigenetic Modifications in Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common human malignancy in the world, with high mortality and poor prognosis for patients. Among the risk factors are tobacco and alcohol intake, human papilloma virus, and also genetic and epigenetic modifications. Many studies show that epigenetic events play an important role in HNSCC development and progression, including DNA methylation, chromatin remodeling, histone posttranslational covalent modifications, and effects of non-coding RNA. Epigenetic modifications may influence silencing of tumor suppressor genes by promoter hypermethylation, regulate transcription by microRNAs and changes in chromatin structure, or induce genome instability through hypomethylation. Moreover, getting to better understand aberrant patterns of methylation may provide biomarkers for early detection and diagnosis, while knowledge about target genes of microRNAs may improve the therapy of HNSCC and extend overall survival. The aim of this review is to present recent studies which demonstrate the role of epigenetic regulation in the development of HNSCC.

Serum interleukin 15 in anorexia nervosa: Comparison to normal weight and obese girls.

Background: Interleukin 15 (IL-15) plays a key role in the muscle-fat interaction, reducing adipose tissue mass without changes in the lean body mass and reduction of food intake. Here we assess serum IL-15 levels in girls with anorexia nervosa (AN) relative to obese and normal weight female adolescents.Methods: Serum IL-15 concentrations were evaluated using a commercially available ELISA kit in 32 Polish girls with restrictive AN, 29 girls with obesity (O), and 21 healthy controls (C). Anthropometric measurements (weight, height, BMI) and laboratory assays (serum fasting glucose, insulin, HOMA-IR, total cholesterol, HDL, LDL, triglycerides, and C-reactive protein (CRP)) were performed.Results: Mean serum IL-15 in the AN group was significantly higher than in C, but lower than in O. In all examined girls, significant positive correlations between IL-15 and body weight, BMI, insulin, HOMA, LDL, triglycerides and CRP were noted. We also observed an inverse relationship between IL-15 and HDL.Conclusions: Our study demonstrated that serum IL-15 concentrations in adolescent girls with AN and obesity are significantly elevated in comparison to normal weight controls. However, the role of IL-15 in the pathogenesis of AN and obesity remains still unclear.

Impact of HPV infection on gene expression and methylation in oral cancer patients.

PURPOSE: The current study aimed to examine the association between head and neck squamous cell carcinoma (HNSCC) and infection with different human papillomavirus virus (HPV) subtypes, including analysis of promoter methylation of several genes (APC, CDKN2A, MGMT, CDH1 and TIMP3) and the correlation with their mRNA expression in tumours and surgical margins. METHODOLOGY: In 47 patients with a primary tumour of the oral cavity, HPV detection and identification of 33 subtypes was performed after previous DNA isolation using a GenoFlow HPV Array Test Kit. RESULTS: Fifteen patients (31.92 %) were HPV [+] and the following HPV types were detected: 16 (46.67 %), 18 (6.67 %) and 43/44 (40 %). This study is the first to describe HPV 43/44 subtypes in HNSCC in a Polish population. We noted no clinical significance of HPV [+] HNSCC compared to HPV [-], however, this differed among HPV subtypes. CDKN2A promoter methylation was more frequent in HPV-16/18 patients compared to HPV43/44 patients, but there was no difference in gene expression level between HPV [+] and [-] patients. CONCLUSION: We detected HPV infection in 31.92 % of oral cancer cases. HPV 16, along with HPV 43/44, were the most frequent subtypes. Knowledge of HPV [+] HNSCC biology may be useful in establishing the prognosis and developing novel therapies in future.

Serum omentin-1 and chemerin concentrations in pancreatic cancer and chronic pancreatitis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by very poor prognosis. It is caused by asymptomatic course of the disease at early stage. Symptomatic PDAC means usually advanced stage of the disease, making radical treatment impossible. Finding of biological PDAC marker could improve PDAC treatment through early diagnosis. In our study, we investigated two adipokines: omentin and chemerin concentration in PDAC, chronic pancreatitis (CP) and healthy individuals. We examined 27 PDAC patients, 10 CP patients and 36 controls. To determine concentration of adipokines we used ELISA immunoenzymatic assay. Level of both adipokines was increased when comparing control group to PDAC patients. Additionally, chemerin concentration in CP group was elevated comparing to control. To evaluate both adipokines as potential PDAC biomarkers we performed ROC analysis. Chemerin (AUC = 0.913) displayed better discriminant ability than omentin-1 (AUC = 0.73). Some authors believe that chemerin may promote tumour growth by stimulating angiogenesis and is supposed to be a factor recruiting mesenchymal stroma cells (MSC) in tumour regions. Omentin-1 can inhibit tumourigenesis by TP53 stimulation. On the other hand, according to some studies, omentin-1 may promote cancer proliferation via Akt signalling pathway. Results from our study showed significantly elevated level of chemerin and omentin-1 in PDAC patients. Therefore, we believe that both investigated adipokines may provide promising and novel pharmacological insights for oncological diagnosis in the near future.

The role of MGMT polymorphisms rs12917 and rs11016879 in head and neck cancer risk and prognosis.

Head and neck squamous cell carcinoma (HNSCC) is one of the leading cancers by incidence worldwide. The risk of these cancers is strictly associated with alkylation factors present in tobacco smoke. The crucial role in preventing DNA alkylation is played by O6-methylguanine-DNA methyltransferase (MGMT). Dysfunction or lack of MGMT is associated with an increased risk of cancer. The aim of the study was to assess the influence of MGMT polymorphisms: rs12917 and rs11016879 on HNSCC risk and course. The study consisted of 69 HNSCC patients and 242 healthy individuals. Case samples were taken from resected tumour tissue. The control group comprised samples of epithelial cells collected from mucous membranes using swabs. DNA samples were genotyped by employing the 5' nuclease assay for allelic discrimination using TaqMan SNP Genotyping Assays. The significance between distributions of genotypes and alleles was tested using Pearson's χ2 test analysis. Our results indicated that the MGMT rs12917 TT genotype increases the risk of HNSCC. The MGMT rs11016879 AG genotype and A allele were associated with increased HNSCC risk. We noted higher risk of nodal metastasis in rs11016879 AA homozygotes. Mechanisms leading to MGMT enzymatic defect are unknown and hence further studies need to be carried out. Our data suggest that the examined polymorphisms may be considered as potential prognostic factors for HNSCC risk and outcome. Further studies are necessary to verify our results.

Clinical and prognostic value of hTERT mRNA expression in patients with non-small-cell lung cancer.

Telomerase, undetectable in normal somatic cells, plays a critical role in carcinogenesis of the majority of human tumors including lung carcinoma. The aim of our study was to determine human telomerase reverse transcriptase (hTERT) mRNA expression in patients with non-small cell lung cancer (NSCLC) in order to estimate its usefulness as diagnostic and/or prognostic factor. hTERT expression was analyzed in a group of 12 females and 28 males with NSCLC using Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR method) in cancerous and non-cancerous lung tissues. Results were analyzed according to clinical data and one-, two-, and five-year survival rates. hTERT expression in the cancerous tissue was significantly higher than in the lung parenchyma free from neoplasm infiltration (p<0.05). There was no significant association between hTERT expression in the tumor tissue and age, gender, grading or clinical stage. A significant difference in hTERT expression between two types of histopathological patterns (adenocarcinoma and squamous cell carcinoma) was detected (p=0.01). No association between hTERT expression in NSCLC specimens and survival rates was found. hTERT mRNA detection by QRT-PCR in tumor and corresponding cancer-free tissues can be used as a diagnostic marker in patients with NSCLC, but seems not to be a prognostic factor.

Hepatocyte growth factor and epidermal growth factor activity during later stages of rat liver regeneration upon interferon α-2b influence.

INTRODUCTION: Liver regeneration is a complex, highly coordinated process which can be disturbed by the impact of the anti-proliferative interferon α activity. In the model of partial hepatectomy (PH) in the rat the expression of HGF and EGF genes and their molecules' tissue concentrations were analyzed in the later stages of liver regeneration (48-120 h). MATERIAL AND METHODS: 40 three-month-old male Wistar rats were randomized to groups of 20 animals each. The rats of the study group (IFN/H) were injected subcutaneously with IFNα-2b, while the control group was injected with 0.5 ml of 0.9% NaCl (NaCl/H). In the liver tissue samples obtained during hepatectomy and autopsy (regenerating liver mass) the expression of HGF and EGF genes was estimated with the Q-PCR method and the analysis of HGF and EGF molecule concentrations in tissue homogenates was conducted with the ELISA method. RESULTS: HGF but not EGF expression was significantly higher at 48 h after PH, while EGF expression was higher in normal than in regenerating liver tissue at 120 h. The analyses of correlations between expression of HGF and EGF in regenerating liver tissue, both normal and upon IFNα-2b influence, together with correlations between those factors genes' expression and HGF and EGF tissue concentrations in analyzed samples, showed no significant differences. CONCLUSIONS: HGF and EGF are not significantly involved in regulation of later stages of rat liver regeneration. IFNα-2b does not impact expression of their genes or the presence of these growth factor molecules in regenerating liver tissue.

Cofilin - a protein controlling dynamics of actin filaments.

Cofilins are evolutionary conserved proteins present in all Eukaryotic cells. Their primary function is dynamic reorganization of actin cytoskeleton. Two cofilin isoforms are known: cofilin 1, present in all studied non-muscle cells and in embryonic muscle cells, and cofilin 2, which dominates in mature skeletal and cardiac muscles. Polypeptide chains of both isoforms fold into a structure homological to a conservative ADF (actin depolymerizing factor) domain, which is characteristic of actin depolymerizing factor. In cofilin molecule two actin-binding sites were found. One site binds monomeric and filamentous actin, the second one interacts only with the filament. Binding of cofilin to actin filament causes a change in the orientation of subunits, which results in filament severing. This increases number of ends which can either elongate or shorten the filament, depending on the conditions. Cofilin interactions with monomeric actin decreases availability of polymerization-competent actin subunits. Cofilin activity is controlled by phosphorylation, binding membrane phospholipids, local pH and oxidative stress. Under conditions of oxidative stress oxidation of cysteine residues leads to formation of dimers, which are able to cross-link actin filaments. Stable actin-cofilin rods save cellular ATP, which is not used during active polymerization process. This facilitates faster cell recovery from the stress. The final cellular reaction on the environmental stimuli is a resultant of cofilin activity and activities of other actin-binding proteins, which function either synergistically or antagonistically. Due to the central role in the regulation of actin filaments dynamics, cofilin is involved in development of cancer, neurodegenerative diseases, congenital myopathies and cardiomyopathies.

Comparison of Selected Protein Levels in Tumour and Surgical Margin in a Group of Patients with Oral Cavity Cancer.

Oral cavity cancer belongs to head-and-neck squamous cell carcinoma group. The purpose of the study was to assess the levels of certain proteins in a tumour and surgical margin in a group of patients with oral cavity cancer. The levels of DAPK1, MGMT, CDH1, SFRP1, SFRP2, RORA, TIMP3, p16, APC and RASSF1 proteins were measured by ELISA in tissue homogenates. The protein levels of DAPK1, MGMT, CDH1, SFRP2 and RASSF1 were significantly higher in tumour tissue than in the margin, contrary to TIMP3 which was lower in the tumour itself. DAPK1 level in the tumour was significantly higher in females than in males, the MGMT and p16 levels were lower in the tumours with lymph node metastasis (N1 + N2) than in N0 samples. The CDH1 expression was higher in a group with smoking habits, whereas TIMP3 was lower in this group. Changes in the levels of proteins in tumour and surgical margin may be either reflective of tumour occurrence and development, or they might be also responsible for the progress and reoccurrence of the disease. Levels of the studied proteins might be good prognostic factors; however, further studies are required.

Regulation of actin filament turnover by cofilin-1 and cytoplasmic tropomyosin isoforms.

Tropomyosin and cofilin are actin-binding proteins which control dynamics of actin assembly and disassembly. Tropomyosin isoforms can either inhibit or enhance cofilin activity, but the mechanism of this diverse regulation is not well understood. In this work mechanisms of actin dynamics regulation by four cytoskeletal tropomyosin isoforms and cofilin-1 were studied with the use of biochemical and fluorescent microscopy assays. The recombinant tropomyosin isoforms were products of two genes: TPM1 (Tpm1.6 and Tpm1.8) and TPM3 (Tpm3.2 and Tpm3.4). Tpm1.6/1.8 bound to F-actin with higher apparent binding constants and lower cooperativities than Tpm3.2/3.4. In consequence, subsaturating concentrations of cofilin-1 removed 50% of Tpm3.2/3.4 from F-actin. By contrast, 2 and 5.5 molar excess of cofilin-1 over actin was required to dissociate 50% of Tpm1.6/1.8. All tropomyosins inhibited the rate of spontaneous polymerization of actin, which was reversed by cofilin-1. Products of TPM1 favored longer filaments and protected them from cofilin-induced depolymerization. This was in contrast to the isoforms derived from TPM3, which facilitated depolymerization. Tpm3.4 was the only isoform, which increased frequency of the filament severing by cofilin-1. Tpm1.6/1.8 inhibited, but Tpm3.2/3.4 enhanced cofilin-induced conformational changes leading to accelerated release of rhodamine-phalloidin from the filament. We concluded that the effects were executed through different actin affinities of tropomyosin isoforms and cooperativities of tropomyosin and cofilin-1 binding. The results obtained in vitro were in good agreement with localization of tropomyosin isoforms in stable or highly dynamic filaments demonstrated before in various cells.

TGF-ß1, bone metabolism, osteoprotegerin, and soluble receptor activator of nuclear factor-kB ligand in girls with anorexia nervosa.

INTRODUCTION: Numerous investigations, and especially in vitro studies, indicate that TGF-ß1 may act as an important regulator of bone remodelling. Thus, it could be expected that disturbances of this cytokine production observed by several researchers might play a role in the mechanism leading to the development of osteoporosis in girls with anorexia nervosa (AN). The aim of the study was to determine whether 1) girls with AN exhibited a relationship between TGF-ß1 and bone metabolism (as assessed based on serum OC and CTx concentrations) and 2) whether OPG and sRANKL might modify the possible relationship between TGF-ß1 and bone metabolism. MATERIAL AND METHODS: Serum concentrations of TGF-ß, OC, CTx, OPG, and its soluble ligand sRANKL were determined by ELISA in 60 girls with AN and in 20 healthy controls (C). All study participants were aged 13 to 17 years. RESULTS: Body weight, BMI, BMI-SDS and the Cole index, serum TGF-ß1, OC, CTx, and the OPG/sRANKL ratio were significantly reduced, while OPG and sRANKL levels were significantly increased, in girls with AN compared to healthy participants. BMI and the Cole index correlated negatively and significantly with serum CTx and OPG (AN group) or CTx only (groups C and C + AN). Girls with AN showed a positive and significant correlation between the Cole index and serum TGF-ß1. The combination group (C + AN) showed a positive and significant correlation between BMI, the Cole index, and the OPG/sRANKL ratio and TGF-ß1 concentration, while TGF-ß1 correlated positively and significantly with OC concentrations and the OPG/sRANKL ratio. The Cole index and BMI were identified to be significant and independent predictors of CTx (C, AN, and C+AN groups) and OPG (AN group); the Cole index, BMI, and TGF-ß1 independently predicted the OPG/sRANKL ratio (C, AN, and C + AN groups); TGF-ß1 was found to be an independent predictor of OC (C + AN group). CONCLUSIONS: Changes in bone markers, OPG, and/or OPG/sRANKL ratio observed in girls with AN are associated with changes in serum TGF-ß1 concentrations. TGF-ß1 suppression in girls with AN might lead to disturbances in the relationship between bone metabolism and the OPG/sRANKL system, which, in turn, might compromise the mechanism compensating for bone remodelling disturbances. (Endokrynol Pol 2016; 67 (5): 493-500).