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).

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).

[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.

The relative levels of alpha 2-, alpha 1-, and zeta-mRNA in HB H patients with different deletional and nondeletional alpha-thalassemia determinants.

We have analyzed the alpha 2/alpha 1-, alpha/beta-, zeta/(alpha + zeta)-mRNA ratios in the retic-ulocytes of 40 patients with Hb H disease. 21 patients had deletional Hb H disease (- -/- alpha), namely combinations of one of four types of alpha-thal-1 (MED-I, MED-II, -(alpha)20.5, SEA) and one of two types of alpha-thal-2 (-3.7 or -4.2 kb); 13 had Hb H disease because of combinations of one of these alpha-thal-1 deletions with either a 5 nt deletion at the 5' splicing site of IVS-I, or a terminating codon mutation (Hb CS), or a poly(A) mutation, and six were homozygous for either a poly(A) mutation or the 5 nt deletion. Significant differences were observed between the deletional types (- -/- alpha; alpha 2/alpha 1 ratio of zero; alpha/beta ratio of approximately 1) and non-deletional types (- -/alpha T alpha; alpha 2/alpha 1 ratio of 0.05-0.3 for those with T = the 5 nt deletion or the terminating codon mutant, and approximately 1.0 for those with T = a poly(A) mutation; alpha/beta ratio in all types of approximately 0.7). Comparable data were found for the nondeletional alpha-thal-2 homozygotes. The noted differences were highly significant and the determination of the two ratios may be diagnostically of considerable value. The low alpha 2/alpha 1-mRNA ratio in the two patients with - -/alpha-5nt alpha and the one patient with alpha-5nt alpha/alpha-5nt alpha indicates the presence of minute amounts of alpha 2-mRNA; apparently splicing at the donor site is greatly impaired by this deletion but not eliminated. The high alpha 2/alpha 1-mRNA ratio in the four patients with - -/alpha PA-2 alpha and the five patients with alpha PA-1 alpha/ alpha PA-1 alpha (PA-1 and PA-2 are poly(A) mutations) is due to the presence of an elongated alpha 2-mRNA which uses an alternate location as polyadenylation site. The relative levels zeta-mRNA varied considerably; the highest levels were found in patients with the -(alpha)20.5/-alpha or - -SEA/-alpha deletional types but not in those with the -(alpha)20.5/alphaPA-2 alpha, -(alpha)20.5/alpha-5nt alpha, or - -SEA/alphaCS alpha nondeletional types. No definitive explanation can be given for these differences; perhaps certain sequences that are part of some of the alpha-thal-1 deletions are important for the suppression of the zeta-globin gene.

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.

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.

Successful bone marrow transplantation in a case of Griscelli disease which presented in accelerated phase with neurological involvement.

Griscelli disease (GD) is a rare disorder characterized by pigment dilution, immunodeficiency and occurrence of accelerated phase consisting of hemophagocytosis, pancytopenia and neurological manifestations. Allogeneic BMT in the early period is an important modality of treatment for GD. We carried out an alloBMT from an HLA-identical sibling donor on a 4-year-old girl who presented in accelerated phase with neurological manifestations including convulsions, strabismus, severe dysarthria, ataxia and clonus. She was treated with etoposide, methylprednisolone and intrathecal methotrexate for 8 weeks and underwent alloBMT after receiving a conditioning regimen including ATG (rabbit, 10 mg/kg x 5 days), Bu/Cy. 8 x 108/kg nucleated bone marrow cells were given. Engraftment occurred early and the post-BMT period was uneventful. Currently, she is at 18 months post BMT with sustained engraftment and with a normal neurological examination except for minimal clonus. Long-term follow-up will determine the prognosis regarding the neurological findings.

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.

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.

Investigation of ascorbate-Cu (II) induced cleavage of DNA by scanning tunneling microscopy.

Scanning Tunneling Microscopy (STM) was used for the investigation of oxidative DNA damage. A PCR amplified fragment of human beta-globin gene was used as a model for time dependent cleavage reaction by ascorbate and copper. Cleavage reactions were carried out in a medium containing 0.5 microgram/20 microliters DNA, 20 nM Tris-HC1 pH, 7.8 and ascorbate-Cu (II) in the final concentrations of 1 mM and 30 microM, respectively. The mixtures were incubated at 37 degrees C for 5, 15 and 30 min. For STM studies, 3 pg/5 microliters DNA samples were deposited on the gold coated mica and dried in a water flow vacuum drier. The STM was operated in air at atmospheric pressure with a tip-to-substrate bias of 100 mV and tunneling currents of < 10 pA. Etched tips of Pt/Ir wires were used in a constant current mode. The degradated DNA structure can be distinguished from the intact DNA and the sizes of the degradation products can be identified in the STM micrographs. The size of fragments decreased from approximately 3000 A to 34 A in ascorbate-Cu (II) medium, after 30 min of incubation.

Compound heterozygosity for hemoglobin Knossos [alpha 2 beta 2 27 (B9) Ala-Ser] and IVS I-1 mutation.

A three-year-old female with compound heterozygosity for Hb Knossos and IVS-I-1 mutation is presented. On physical examination she had no abnormality except for pallor. Hb was 6.9 g/dl, MCV 61 fl, Hb A2 2% and Hb F 38.5%. Acrylamidegel electrophoresis at a pH of 6 revealed the presence of Hb Knossos in the child and her father. DNA studies revealed that the child was compound heterozygous for Hb Knossos and the IVS I-1 mutation. When the clinical expression of this combination in a previously reported patient with Hb Knossos/FSC8 mutation is compared, it is shown that the newly presented patient has a more severe condition, indicating that the mutations in the trans of Hb Knossos may play a role in the phenotypical expression of the disease.

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.

The treatment of lateral epicondylitis by iontophoresis of sodium salicylate and sodium diclofenac.

OBJECTIVE: To investigate the effectiveness of sodium diclofenac and sodium salicylate applied by topical iontophoresis and to compare them in the treatment of lateral epicondylitis. SUBJECTS: Forty patients with lateral epicondylitis were randomized into two groups of 20 patients who were matched for age and sex. INTERVENTIONS: The patients in one group were treated by iontophoresis of sodium diclofenac and the other group were treated by iontophoresis of sodium salicylate. Then infrared treatment was applied to patients in both groups. MAIN OUTCOME MEASURES: Pain scores obtained before and after treatment were compared. RESULTS: Pain produced by pressure on the lateral epicondyle, on resisting wrist extension, during function and spontaneous pain at rest significantly decreased in both groups after treatment (p < 0.001). When pain scores obtained after treatment were compared, greater decrease was observed in the pain produced on resisting wrist extension (p < 0.01) and by pressure on the lateral epicondyle (p < 0.05) in the group treated with sodium diclofenac than in the group treated with sodium salicylate. CONCLUSIONS: The results suggest some benefits from the process of iontophoresis and the use of infrared in the treatment of lateral epicondylitis and indicate that iontophoresis of sodium diclofenac is more effective than that of sodium salicylate.

A new Turkish type of beta-thalassaemia major with homozygosity for two non-consecutive 7.6 kb deletions of the psi beta and beta genes and an intact delta gene.

In a 2.5-month-old infant with beta-thalassaemia major, DNA analysis of the gamma-beta region revealed homozygosity for two large deletions removing the entire psi beta and beta regions including their 5' promoter regions but leaving the delta gene intact. The downstream deletion was predicted to be 7.6 kb in length extending from a point 1.5 kb on the 3' side of the delta-globin gene to about 1.8 kb on the 3' side of the beta-globin gene. The upstream deletion, which was also about 7.6 kb, extended from a point 1.5 kb on the 5' side of the psi beta-globin gene to about 4.5 kb on the 3' of the psi beta gene. The delta-globin gene was intact. From the phenotypic expression of the disease it is concluded that removal of the psi beta gene probably prevents derepression of the gamma gene that has previously been observed in the absence of the promoter region of the beta gene and the switch mechanism from gamma to beta gene expression may take place earlier than expected.

The molecular basis of Hb H disease in Turkey.

A total of 25 unrelated Hb H patients were studied at the DNA level. Ten different genotypes were found to be responsible for the disease. The most prevalent alpha-thalassemia-2 determinant was the alpha alpha/-alpha (3.7) kb deletion (56%) which was followed by a nondeletional type of alpha-thalassemia, namely the pentanucleotide deletion in the 5' first intervening sequence splice junction [alpha(-5nt) alpha] (16%). The two most frequent alpha-thalassemia-1 determinants were alpha alpha/-20.5 kb and alpha alpha/-17.5 kb (MED-I) deletions. In two patients, homozygosity for the polyadenylation signal mutation [alpha (PA-2)alpha] was found to be responsible for Hb H disease. Clinical and hematological expression seems more severe in patients with the alpha (-5nt) alpha deletion at the donor site of the first intervening sequence and the alpha(PA-2) alpha mutation in trans to an alpha-thalassemia-1 determinant. Homozygosity for the alpha (PA-2)alpha mutation was also found to be associated with severe phenotype.