Inhibition of voltage­gated potassium channels affect expressions of miR-126 and miR-126* in breast cancer cell lines.

AIM: We aimed to determine the possible correlation between voltage­gated potassium channels and micro RNAs in breast cancer and metastatic breast cancer cells. METHOD: Kv1.3 and Kv10.1 channels were inhibited by specific siRNAs using a lipofectamine-based transfection in MCF-7 and MDA-MB-231 cells. After transfection, total RNA was isolated, and then miR-126 and miR-126* expressions were observed using RT-PCR. RESULTS: There was a negative correlation between Kv channels and miRNAs according to the characteristics of the breast cancer cells. The inhibition was observed not only in Kv1.3 but also in Kv10.1 in MCF-7 cells, and miR-126 and miR-126* expressions were downregulated compared to the control group (p < 0.001). The inhibition of these channels in MDA-MB-231 cells caused an upregulation of miR-126 and miR-126* expressions (p < 0.001). CONCLUSION: The miR-126 and miR-126* expressions differed according to benign and malign breast cancer cell lines. Furthermore, we found that miR-126/126* may interact with Kv1.3 and Kv10.1 voltage-gated potassium channels. Our study suggests and indicates the relationship between Kv channels and miRNAs in breast cancer cells (Tab. 1, Fig. 2, Ref. 51).

[The AOSpine Classification of Thoraco-Lumbar Spine Injuries]. / Die AOSpine-Klassifikation thorakolumbaler Wirbelsäulenverletzungen.

Optimal treatment of injuries to the thoracolumbar spine is based on a detailed analysis of instability, as indicated by injury morphology and neurological status, together with significant modifying factors. A classification system helps to structure this analysis and should also provide guidance for treatment. Existing classification systems, such as the Magerl classification, are complex and do not include the neurological status, while the TLICS system has been accused of over-simplifying the influence of fracture morphology and instability. The AOSpine classification group has developed a new classification system, based mainly upon the Magerl and TLICS classifications, and with the aim of overcoming these drawbacks. This differentiates three main types of injury: Type A lesions are compression lesions to the anterior column; Type B lesions are distraction lesions of either the anterior or the posterior column; Type C lesions are translationally unstable lesions. Type A and B lesions are split into subgroups. The neurological damage is graded in 5 steps, ranging from a transient neurological deficit to complete spinal cord injury. Additional modifiers describe disorders which affect treatment strategy, such as osteoporosis or ankylosing diseases. Evaluations of intra- and inter-observer reliability have been very promising and encourage the introduction of this AOSpine classification of thoracolumbar injuries to the German speaking community.

Micro RNA-126 coordinates cell behavior and signaling cascades according to characteristics of breast cancer cells.

BACKGROUND: Micro RNA-126 is known to enhance apoptotic processes and also plays a role in vascular growth through the regulation of vascular endothelial growth factor-mediated signaling, angiogenesis, and vascular integrity. OBJECTIVES: We aimed to determine the role of miR-126 in breast cancer cell lines with a variety of different characteristics to evaluate its interaction with certain cancer-related molecules and mechanisms. METHODS: To determine the effect of presence and absence of miR-126 in MCF-7 and MDA-MB-231 breast cancer cells, miR-126 mimics and inhibitor were transfected. miRNA and gene expressions were observed by using RT-PCR. Viability, proliferation, adhesion, invasion and lateral motility assays were performed to determine cell behavior changes. RESULTS: miR-126 is more effective on MDA-MB-231 cells on cell behavior. We observed an increase in miR-126 expression when miR-126 mimics was transfected to MCF-7 and MDA-MB-231 cells. Also, there was a decrease in miR-126 expression when MCF-7 and MDA-MB-231 cells were transfected with miR-126 inhibitor. Furthermore, presence and absence of miR-126 modulated the gene expressions of VEGF/PI3K/AKT and MAPK signaling in MCF-7 and MDA-MB-231. CONCLUSION: Our study showed that miR-126 is in a state of interaction with a multitude molecules playing a role in breast cancer. According to obtained data, we can say that miR-126 may be more effective in inhibition of metastatic breast cancer (Tab. 4, Fig. 3, Ref. 46).

Prognostic significance of sICAM-1 and sVCAM-1 molecules for cardiac surgery in pediatric patients with pulmonary hypertension.

OBJECTIVE: To investigate preoperative and postoperative blood levels of soluble intercellular and vascular cell adhesion molecules (sICAM-1, sVCAM-1) in patients with and without pulmonary hypertension (PAH) due to congenital heart disease and left to right (L-R) shunt and to determine whether these molecules can be used as reliable prognostic markers of endothelial activity to predict surgical outcomes. METHODS: In this observational prospective cohort study; 42 patients, operated for L-R shunt were divided into three groups. Group 1: L-R shunt without PAH, Group 2: L-R shunt with PAH, Group 3: L-R shunt with PAH and postoperative low cardiac output syndrome (LCOS). Their sICAM-1 and sVCAM-1 levels were measured preoperatively (sICAM-0, sVCAM-0) and on the first (sICAM-1, sVCAM-1) and fifth postoperative days (sICAM-2, sVCAM-2).ROC curve for various cut-off levels of sICAM-0, sVCAM-0 in differentiating PAH patients with and without LCOS. RESULTS: In Group 3, sICAM-0 and sVCAM-2 levels were higher than Group 1 and 2. The ROC curve demonstrated a significant association between sICAM-0 in patients with L-R shunt and PAH (Group 2 and 3) and the development of LCOS (area under the curve: 0.98, p<0.01 and 0.97, p<0.01, respectively). At a sICAM-0 concentration >359 ng/mL, there was a sensitivity of 90% and specificity of 95% for identification of LCOS in patients with L-R shunt and PAH (AUC: 0.98, 95% CI: 0.95-1.02, p<0.01). CONCLUSION: High preoperative sICAM-1 molecule may be used to predict postoperative dichotomous outcome in patients with PAH associated with L-R shunt.

Prognostic significance of sICAM-1 and sVCAM-1 molecules for cardiac surgery in pediatric patients with pulmonary hypertension.

OBJECTIVE: To investigate preoperative and postoperative blood levels of soluble intercellular and vascular cell adhesion molecules (sICAM-1, sVCAM-1) in patients with and without pulmonary hypertension (PAH) due to congenital heart disease and left to right (L-R) shunt and to determine whether these molecules can be used as reliable prognostic markers of endothelial activity to predict surgical outcomes. METHODS: In this observational prospective cohort study; 42 patients, operated for L-R shunt were divided into three groups. Group 1: L-R shunt without PAH, Group 2: L-R shunt with PAH, Group 3: L-R shunt with PAH and postoperative low cardiac output syndrome (LCOS). Their sICAM-1 and sVCAM-1 levels were measured preoperatively (sICAM-0, sVCAM-0) and on the first (sICAM-1, sVCAM-1) and fifth postoperative days (sICAM-2, sVCAM-2).ROC curve for various cut-off levels of sICAM 0, sVCAM 0 in differentiating PAH patients with and without LCOS. RESULTS: In Group 3, sICAM-0 and sVCAM-2 levels were higher than Group 1 and 2. The ROC curve demonstrated a significant association between sICAM-0 in patients with L-R shunt and PAH (Group 2 and 3) and the development of LCOS (area under the curve: 0.98, p<0.01 and 0.97, p<0.01, respectively). At a sICAM 0 concentration >359 ng/mL, there was a sensitivity of 90% and specificity of 95% for identification of LCOS in patients with L-R shunt and PAH (AUC: 0.98, 95% CI: 0.95-1.02, p<0.01).CONCLUSION: High preoperative sICAM-1 molecule may be used to predict postoperative dichotomous outcome in patients with PAH associated with L-R shunt.

The effect of polymorphisms at the CD14 promoter and the TLR4 gene on asthma phenotypes in Turkish children with asthma.

BACKGROUND: Endotoxin, with its potential to enhance type 1 immunity, is a significant player in the hygiene hypothesis. The combined effects of the genetic variants of various molecules in the endotoxin response pathway on asthma related phenotypes are largely unknown. OBJECTIVE: To investigate the effects of the genetic variants of CD14 and TLR4 genes on asthma phenotypes in a large number of asthmatic children. METHODS: 613 asthmatic children were genotyped at the CD14-C159T, TLR4-A896G and TLR4-C1196T loci. IgE, eosinophil numbers and FEV1 were compared in 327 children who were not on any controller medications and were symptom free. Multivariate logistic regression was used to determine the factors associated with total IgE. RESULTS: Among children with atopic asthma, total IgE levels were significantly different among the three genotypes in the co-dominant model [CC: 435 kU/l (interquartile range: 146-820); CT: 361 (140-710); TT 204 (98-435), P = 0.035]. TT genotype was significantly and independently associated with lower IgE levels (OR: 0.5 95%; CI = 0.28-0.90, P = 0.021). Both TLR4-A896G and TLR4-C1196T polymorphisms were more frequent in the mild asthma group with atopy (P = 0.032, 0.018, respectively). The combined effects of the genetic variants in CD14 and TLR4 genes did not improve the observed associations. CONCLUSION: Our study demonstrates that the CD14-C159T promoter variant influences total IgE levels and also indicates that the T allele has a more profound effect on total IgE in children with atopic asthma. Polymorphisms in the TLR4 gene may be associated with milder forms of disease in atopic asthmatics in the population studied.

Severe hemolytic anemia associated with Hb Volga [beta27(B9)Ala-->Asp]: GCC-->GAC at codon 27 in a Turkish family.

A boy presented at age 4 years with severe congenital hemolytic anemia characterized by highly elevated reticulocyte count (30-50%) and prominent basophilic stippling. Hb had been 4 g/dL at age 7 months. The patient was on a monthly transfusion regimen up to the age of 7 years, when he underwent splenectomy. After removal of the spleen, his Hb stabilized at 11 g/dL. No abnormal pattern was detected in hemoglobin electrophoresis at pH 9 and 6. In-vitro globin synthesis revealed the presence of an abnormal beta-chain in front of the gamma-chain. The beta(A)/beta(X) ratio was 0.77 at 30 min and 0.74 at 2 hr of incubation. Molecular analysis revealed that the patient had GCC-->GAC alteration at codon 27 (beta27(B9)Ala-->Asp) causing the abnormal hemoglobin Volga. The beta-cDNA derived from the beta-Hb Volga allele could be differentiated from HbA beta-cDNA on silver-stained gel. No imbalance in the mRNA of beta(A)/beta(Hb Volga) ratio was observed.

Efficacy of intra-articular sodium hyaluronate in the treatment of knee osteoarthritis.

To assess the efficacy of intra-articular hyaluronic acid in patients with knee osteoarthritis, sixty female patients with knee osteoarthritis were randomised to three weekly intra-articular injections of 30 mg sodium hyaluronate (Na HA) with a high molecular weight (1.0 to 2.9 million Da) or 40 mg 6-methylprednisolone acetate (6-MPA). The clinical assessments included pain at rest, at weight-bearing and on walking, Lequesne Index and active range of knee flexion. Assessments were done at baseline, at week 4, and at months 3 and 6. A significant decrease in VAS scores for pain at rest, at weight-bearing and pain on walking, and in Lequesne index was found in both groups at week 4 when compared to baseline and there was no significant differences between the two groups. However, at 3(rd) month improvement in all pain scores and Lequesne index was found in favour of hyaluronic acid. At 6(th), no significant difference was found between the treatment groups. Improvement in pain was accompanied by an increase in joint flexion at week 4 and at month 3 in both groups. Both treatments were well-tolerated. The results showed that both intra-articular hyaluronic acid and 6-MPA treatments provide clinically significant improvement and demonstrated that Na HA has a long-term beneficial effect in patients with knee osteoarthritis.

Chronic hemolytic anemia associated with glucose 6-phosphate dehydrogenase (Guadalajara)1 159 C --> T (387 Arg --> Cys) deficiency associated with Gilbert syndrome in a Turkish patient.

The case of an 8-year-old male child with severe kernicterus sequelae is presented in this paper. The child's hemoglobin value varied between 6.0 and 10.8 g/dL and his reticulocyte count ranged between 3.4 and 46.0% during the steady-state condition and hyperhemolytic crisis, respectively. A chronic hemolytic type of red cell G6PD deficiency was diagnosed. DNA studies indicate that the mutation was G6PD Guadalajara 1159 C --> T (387 Arg --> Cys) that is situated at the NADP binding site. Additionally, extra nucleotides of (TA) in the A(TA)n TAA motif of the promoter region of the uridine diphosphate-glucuronosyltransferase gene (UGT-1 A) were found to be homozygous in the patient. The coexistence of Gilbert syndrome with a chronic type of G6PD deficiency was suggested as a cause of neonatal hyperbilirubinemia leading to kernicterus.

beta-Thalassaemia intermedia in a Turkish girl: homozygosity for G-->A substitution at +22 relative to the beta-globin cap site.

We provide the first description of a homozygote patient for the G-->A substitution in the 5' UTR of the beta-globin gene. The proband was a 17-year-old girl with beta-thalassaemia intermedia who had never received a blood transfusion. The physical examination revealed a well-developed women with no facial or bony abnormalities. There was mild paleness and mild splenomegaly which was 2 cm below the costal margin. The haemoglobin (Hb) was 7.6 g/dl, Hb A(2) 5.4% and Hb F 14.6% of the total Hb. The Hb A(2) of both parents was 3.5%. The Hb F level in the mother and father were 0.9, 1.2% and the mean cell volume (MCV) value was 70 and 72 fl respectively. DNA analysis of the beta-gene region of the propositus revealed homozygosity for a G-->A substitution at nucleotide +22 relative to the beta-gene cap site, within a functional downstream region that was referred to as the DCE (downstream core element). In addition to the data obtained previously from in vitro transcription assays, clinical findings and in vivo expression studies gave some valuable clues about the effect of +22 G-->A mutation on the expression of beta-gene. Phenotypic expression of this homozygous patient is highly suggestive that G-->A substitution at nt +22 confers a relatively mild (silent) beta(+)-thalassaemia phenotype.

A novel melanocortin 4 receptor (MC4R) gene mutation associated with morbid obesity.

Mutations in the melanocortin 4 receptor gene (MC4R) are the most common cause of monogenic human obesity. As part of our ongoing project entitled 'Turkish Obesity Genome Study' we determined the nucleotide sequence of the entire coding region of the MC4R gene in 40 morbidly obese subjects from independent families. Here we report a novel heterozygous mutation (N274S) in an adult female obese individual (age: 52 yrs, BMI 41.7 kg/m(2), height 158 cm, weight: 104 Kg). The sister of the index case (age: 55 yrs, height: 160 cm, weight: 110 Kg, BMI: 43 kg/m(2)) also carries the same mutation. Although both sisters were morbidly obese and hypertensive the index case had normal plasma insulin and fasting blood glucose levels whereas her sister had type 2 diabetes mellitus. No abnormalities of the reproductive function were present. Despite marked hyperphagia in childhood both sisters had a history of relatively diminished intensity of appetite after the age of 20. Of notice, index case was diagnosed to have cyclothymia whereas her sister was being treated for bipolar affective disorder. Detailed clinical evaluation revealed normal bone mineral density and serum calcium parameters as well as intact thyroid axis and hypothalamus-pituitary-adrenal axis in both patients. The human MC4-R deficient phenotype resembles the murine deficient state with regard to preserved reproductive function although hyperphagia, increased linear growth and absence of diabetes in mice are not observed in humans. Affected individuals have hyperphagia in childhood, which looses intensity later in life, and they also present with normal height and diabetes mellitus. Accumulating evidence indicate that melanocortin endocrine system or defective melanocortin signaling has inherently different characteristics in mice and humans resembling the variation observed with regard to leptin deficiency in both species.

Vitamin d-receptor gene polymorphisms and vertebral bone density in men with idiopathic hypogonadotrophic hypogonadism.

BACKGROUND: Optimal peak bone mass is closely related to sufficient and appropriately timed androgen release. However, attainment of peak bone mass in men, as in women, is under genetic control, as well as being subject to hormonal and mutational effects. With increasing recognition of osteoporosis and related fractures in men, it is of interest to consider whether there is relationship between bone density and vitamin D receptor (VDR) polymorphisms, as described in women. MATERIAL AND METHODS: To assess the influence of allelic variation in the VDR gene on vertebral bone density in men with idiopathic hypogonadrotrophic hypogonadism (IHH), 27 patients (mean age 21.4 +/- 0.4 yrs) and 25 age-and-BMI matched healthy males (mean age 21.2 +/- 0.3) were genotyped using three restriction enzymes (Apa I, Bsm I, and Taq I). Vertebral bone mineral density was measured using dual energy X-ray absorptiometry (DEXA). RESULTS: As expected, vertebral bone density was reduced significantly in patients with IHH (p < 0.001). Despite weak evidence for an association between Apa I polymorphism and vertebral bone density in the IHH group (r = 0.454, p = 0.017 and r2 = 0.20), VDR genotype was not associated with vertebral bone density in either group. When analyzing homozygous haplotypes, the probability of carrying the favorable BAt haplotype was greater in the control group (OR = 2.000 vs. 0.500). CONCLUSION: We conclude that VDR genotype has no influence on vertebral bone density in men with IHH. Thus, allelic variation in the VDR cannot help define those at increased risk for osteoporosis and related fractures among such patients.

Homozygosity for Hb E-Saskatoon [beta22(B4)Glu-->Lys] in a Turkish patient.

A 30-year-old female who is homozygous for a Hb E-like abnormal hemoglobin and her immediate relatives were studied. Clinical examination of the proband revealed no abnormality. Routine hematological analysis showed that her hemoglobin level was 12 g/dL, MCV 82 fL, MCH 28 pg, RDW 15%. DNA sequence analysis indicated the presence of a G-->A substitution at codon 22 corresponding to an abnormal hemoglobin, namely Hb E-Saskatoon [beta22(B4)Glu-->Lys (GAA-->AAA)]. Absence of any abnormalities in clinical and routine hematological investigations of the homozygous patient indicated that the phenotypical expression of the Hb E-Saskatoon is very mild. Using a reverse transcription-polymerase chain reaction technique, the alpha/beta and betaX/betaA-mRNA (X = Hb E-Saskatoon) ratios were determined. Normal alpha/beta and betaX/betaA-mRNA ratios were found in the homozygous patient and in all heterozygotes, indicating that the respective mutation did not alter the stability of the mRNA. FokI restriction enzyme analysis of the polymerase chain reaction products obtained from the genomic DNA and/or beta-globin mRNA made it possible for rapid diagnosis of Hb E-Saskatoon, and for its differentiation from Hb E [beta26(B8)Glu-->Lys (GAG-->AAG)]. Analysis of the restriction fragment length polymorphism (RFLP) in the beta-globin gene complex of the index patient and of another unrelated family with a compound heterozygosity for Hb E-Saskatoon and beta-thalassemia revealed that the Hb E-Saskatoon mutation shared a common allele.

Molecular analysis of turkish beta-thalassemia heterozygotes with normal Hb A2 levels.

Four parents of three unrelated families who are obligatory beta-thalassemia heterozygotes and two parents with Hb Knossos are presented. In these subjects, although the red blood cell counts and red cell indices were compatible with beta-thalassemia trait, the Hb A2 values were between 1.9-2.9% of the total hemoglobin. Examination of the delta-globin gene by Southern blot, restriction endonuclease analysis, and by direct sequencing of amplified DNA revealed the presence of the (delta0) -7.2 kb Corfu type deletion, the (delta+) codon 27 (G-->T) and (delta0) IVS-I-2 (T-->C) mutations in trans or in cis with a severe beta-thalassemia allele, and the (delta0) codon 59 (-A) deletion in cis with the betaKnossos allele.

A safer and relatively shorter method for Southern blot hybridization analysis.

The study presented here describes a Southern blot hybridization technique which is performed in a shorter time and safer than the classical one. In this study DNA was isolated from a small amount of blood sample and the probe for hybridization was prepared with (35)S by polymerase chain reaction.

Molecular, genetic and epidemiologic studies on selective complete C1q deficiency in Turkey.

Selective complete C1q deficiencies (SCDC1q) of the complement component C1q are rare genetic disorders with high prevalence of lupus-erythematosus-like symptoms and recurrent infections. Among the 41 published cases from 23 families, 10 derive from 6 Turkish families. One particular mutation leading to a stop codon in the C1q A gene was first identified in members of a Gypsy family from the Slovac Republic. Later the same mutation has been found in all cases in four SCDC1q families from Turkey suggesting that one particular defective allele may be present in the populations of Southeastern Europe and Turkey. This study was undertaken to investigate the frequency of C-->T mutation in exon II of C1qA gene in Turkish population by using allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Among the 1544 patients from 15 pediatric departments and an additional 89 SLE patients of various ages no C1qA gene mutation was found. There were 43 heterozygous and 4 homozygous mutations in 161 family members or relatives investigated from the 4 families known with SCDC1q. Among the 223 inhabitants who were nonrelative to the 3 SCDC1q families living in the same village were screened for mutation and one heterozygous individual was observed. Although this mutant allele appears to be at a low prevalence in the population tested, individuals with recurrent infections or symptoms of lupus erythematosus-like syndrome should be tested for this mutation to rule out this type of C1q deficiency.

Beta-thalassemia intermedia associated with homozygosity for the -87 (C-->T) mutation in a Turkish family.

We report on two siblings with beta+-thalassemia intermedia. Molecular studies of the beta-globin gene indicated that the patients are homozygous for the -87 (C-->T) mutation. This genotype has not been previously described. Homozygosity for the -87 (C-->T) mutation produces a mild form of beta+-thalassemia associated with moderate Hb F elevation (26-38%) and highly elevated Hb A2 (10-8.6%) levels, respectively. Hematological parameters of homozygous -87 (C-->G) and -87 (C-->A) mutations, and compound heterozygous patients with either C-->T, C-->G, or C-->A at -87 and one of the severe beta+- or beta0-thalassemia mutations, are given for comparison.