Variable-number tandem-repeat markers for typing <Emphasis Type="Italic">Mycobacterium intracellulare</Emphasis> strains isolated in humans

Background  

Mycobacterium intracellulare, a species of the Mycobacterium avium complex, may be the cause of severe lung, lymphatic node, skin and bone/joint infections, as well as bacteriemia. The goal of this work was to identify Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) markers and to study their variability in a collection of isolates of M. intracellulare collected in humans. We studied 61 isolates collected in humans between 2001 and 2008, as well as the reference strain, M. intracellulare ATCC 13950.     

Thermodynamic and structural analysis of interactions between peptide ligands and SEB

Staphylococcal enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus and commonly associated with food poisoning. In this study, SEB‐binding peptides were identified by screening a phage displayed peptide library. The binding of peptides to SEB was tested with isothermal titration calorimetry (ITC) and of the five selected peptides, three showed affinity to SEB, with one measured to have the highest affinity constant (10⁵ M^?1). ITC revealed that the interaction of peptide ligands with SEB was driven entropically and the binding was dominated by hydrophobic interactions. Circular dichroism (CD) measurements and molecular dynamics (MD) simulations, together, give a structural insight into the interaction of peptides with SEB. While SEB binding peptides showed random coil structure before binding, after complex formation they had more ordered structures. The peptide with highest affinity to SEB showed stable conformation during MD simulation. Taken together, our approach about thermodynamic and structural characterization of peptide ligands can be used to develop aptamers, with high affinity and selectivity, for biosensor applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Estimation of the potential fertility based upon non-return rates of bulls: using polyacrylamide gel instead of cervical mucus in the sperm penetration test

In the present study, we aimed to develop a polyacrylamide gel that could be used instead of bovine cervical mucus in the cervical mucus penetration test (CMPT) to obtain coherent and replicable results in bulls. The frozen semen samples of six Holstein bulls, which were divided into two fertility groups as low and high according to their non-return rate (NRR), were used. In this study, the modified CMPT (mCMPT) was carried out within 0.25 mL transparent plastic straws with an inner diameter 1.7 mm. The penetration ability of spermatozoa to bovine cervical mucus and to polyacrylamide gels swollen with two different solutions [NaCl (G1) and PBS (G2)] was compared. For the penetration test, the straws filled with cervical mucus and both gels were dipped into thawed semen samples and incubated at 37 degrees C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at -20 degrees C. On the evaluation day, the frozen straws were cut at 1.5-1.75 cm (penetration distance range=PDR1), 3.25-3.5 cm (PDR2) and 5.0-5.25 cm (PDR3), beginning from open-end of the straws. The separated frozen parts were then immediately transferred onto special counting slides by pushing with a mandrel and left to thaw. Thawed samples were covered with cover glass and penetrated spermatozoa in these parts were counted. The relation between the results and fertility of bulls was determined. In the tests performed using mucus, the number of spermatozoa determined in the high fertility group was found to be higher at PDR3 (p<0.0001) compared to the low fertility group, while in G1 spermatozoa number was significantly higher at PDR1 and PDR3 (p<0.0001). However, in G2 medium, no significant difference was observed between either of the fertility groups with respect to spermatozoa number determined at all distance ranges. In the study, we have determined that the gel swollen with NaCl produces better results and this gel can be used instead of bovine cervical mucus for the CMPT. Therefore, we have concluded that the penetration test performed by polyacrylamide gel swollen with NaCl can be a suitable technique for estimation of the potential fertility of bull spermatozoa.     

Immunoglobulin G depletion from human serum with metal-chelated beads under magnetic field

Magnetic poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-histidine methyl ester) [mag-poly(EGDMA-MAH) beads, 50-100 microm in diameter, were produced by suspension polymerization for affinity depletion of immunoglobulin G (IgG) from human serum. Cu2+ ions were complexed directly via MAH groups (Cu2+ loading: 4.1 micromol/g). IgG depletion studies were performed by magnetically stabilized fluidized bed column. Acetate, Tris-HCl, MES and phosphate buffers all allow adsorption of similar quantities of IgG (27.3-45.6 mg/g). MOPS and HEPES allow higher adsorption quantities (79.6 mg/g and 74.1mg/g, respectively). Maximum adsorption capacities in MOPS buffer were 46.8 mg/g for mag-poly(EGDMA-MAH) and 102.1mg/g for Cu2+ chelated mag-poly(EGDMA-MAH) beads. The adsorption capacity decreased drastically from 102.1mg/g to 30.7 mg/g with the increase of the flow rate from 0.2 ml/min to 3.5 ml/min. The elution studies were performed by 1.0M NaCl. The elution results demonstrated that the adsorption of IgG to the adsorbent was reversible. To test the efficiency of IgG depletion from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. The depletion efficiency for IgG was above 99.4%. Eluted proteins include mainly IgG, and a small number of non-albumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin. When anti-HSA-sepharose adsorbent is used together with our metal-chelated mag-beads, IgG and HSA can be depleted in a single step.     

Insulin and heparin co-immobilized 3D polyester fabrics for the cultivation of fibroblasts in low-serum media

Insulin and/or heparin immobilized/co-immobilized non-woven polyester fabric (NWPF) discs were developed for the cultivation of L929 mouse fibroblasts in low-serum media. At first, NWPF discs were hydrolyzed to obtain a carboxylic acid group-introduced matrix (NWPF-hydrolyzed). Insulin and heparin co-immobilized NWPF (NWPF-insulin-heparin) was prepared by the grafting of PEO onto NWPF-hydrolyzed disc (NWPF-PEO), followed by the reaction first with insulin and then heparin. In the presence of spacer arm, PEO, the amount of immobilized insulin molecules significantly increased from 6.96 to 84.45 microg/cm(2). The amount of heparin bound to the NWPF-PEO (5.93 microg/cm(2)) was higher than that of the insulin immobilized surface (4.59 microg/cm(2)). Insulin and heparin immobilized NWPF discs were observed with fluorescence microscopy by labeling the insulin and heparin with 8-anilino-1-naphthalene sulfonic acid (ANS) or fluorescein isothiocyanate (FITC), respectively. L929 fibroblasts were used to check the cell adhesion and cell growth capabilities of modified NWPF discs in low-serum media (containing 5% fetal bovine serum). Optical photographs showed that after 2nd day of the culture, fibroblastic cells spread along the length of modified fibers, eventually filling the interfiber space. At the end of 6-day growth period, cell yield in the presence of immobilized heparin was a little bit higher than that of the immobilized insulin. Co-immobilized (insulin/heparin) NWPF discs did not accelerate the cell growth as well as insulin or heparin immobilized discs.     

Effects of Selenium Supplementation on Antioxidant Defense and Glucose Homeostasis in Experimental Diabetes Mellitus

The objective of this study was to investigate the effects of different forms of Se supplementation on the antioxidant defense and glucose homeostasis in experimental diabetes. Sodium selenate (SS) or selenomethionine (SM) were administered (2 μmol Se kg⁻¹ day⁻¹) via orogastric route to streptozotocine (STZ)-induced diabetic rats in addition to basal diet for 12 weeks. Glucose levels in whole blood, glutathione peroxidase (GSH-Px) activity in erythrocytes, Se and fructosamine levels in plasma were evaluated monthly. Plasma Se levels increased significantly in all diabetic groups compared to basal measurements, being more prominent in SM group [p(SM₃/SM₀) = 0.018]. The increase in GSH-Px activities was significant at the end of the second month in SS [p(SS₂/SS₀) = 0.028], whereas at the end of the third month in SM the value was lower [p(SM₃/SM₀) = 0.018] and the unsupplemented diabetic control (DC) groups, p(DC₃/DC₀) = 0.012. Glucose increased significantly only in DC group. Fructosamine increased gradually in all diabetic groups, being significant in DC and SS groups. At the end of the third month, highest fructosamine levels were observed in SS group, which were significantly higher than the SM group [p(SM/SS) = 0.010]. In conclusion, Se augmented the antioxidant defense by increasing GSH-Px activity and this effect was more prominent when Se was supplemented as SM, which exerted positive effects also on glucose homeostasis.     

The protective effect of oxytocin on renal ischemia/reperfusion injury in rats

AIM: Oxytocin was previously shown to have anti-inflammatory effects in different inflammation models. The major objective of the present study was to evaluate the protective role of oxytocin (OT) in protecting the kidney against ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: Male Wistar albino rats (250-300 g) were unilaterally nephrectomized, and subjected to 45 min of renal pedicle occlusion followed by 6 h of reperfusion. OT (1 mg/kg, ip) or vehicle was administered 15 min prior to ischemia and was repeated immediately before the reperfusion period. At the end of the reperfusion period, rats were decapitated and kidney samples were taken for histological examination or determination of malondialdehyde (MDA), an end product of lipid peroxidation; glutathione (GSH), a key antioxidant; and myeloperoxidase (MPO) activity, an index of tissue neutrophil infiltration. Creatinine and urea concentrations in blood were measured for the evaluation of renal function, while TNF-alpha and lactate dehydrogenase (LDH) levels were determined to evaluate generalized tissue damage. Formation of reactive oxygen species in renal tissue samples was monitored by chemiluminescence technique using luminol and lucigenin probes. RESULTS: The results revealed that I/R injury increased (p<0.01-0.001) serum urea, creatinine, TNF-alpha and LDH levels, as well as MDA, MPO and reactive oxygen radical levels in the renal tissue, while decreasing renal GSH content. However, alterations in these biochemical and histopathological indices due to I/R injury were attenuated by OT treatment (p<0.05-0.001). CONCLUSIONS: Since OT administration improved renal function and microscopic damage, along with the alleviation of oxidant tissue responses, it appears that oxytocin protects renal tissue against I/R-induced oxidative damage.     

Differential responses of antioxidative enzymes and lipid peroxidation to salt stress in salt-tolerant Plantago maritima and salt-sensitive Plantago media

The changes in plant growth, relative water content (RWC), stomatal conductance, lipid peroxidation and antioxidant system in relation to the tolerance to salt stress were investigated in salt-tolerant Plantago maritima and salt-sensitive Plantago media. The 60 days old P. maritima and P. media seedlings were subjected to 0, 100 and 200 mM NaCl for 7 days. Reduction in shoot length was higher in P. media than in P. maritima after exposure to 200 mM NaCl, but 100 mM NaCl treatment did not show any effect on shoot length of P. maritima. Shoot dry weight decreased in P. media and did not change in P. maritima. Two hundred millimolar NaCl treatment had no effect on leaf RWC in P. maritima, but it was reduced in P. media. Salt stress caused reduction in stomatal conductance being more pronounced in P. media than in P. maritima. Activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2) decreased in P. media with increasing salinity. Ascorbate peroxidase (APX; EC 1.11.1.11) activity in leaves of P. media was increased and showed no change under 100 and 200 mM NaCl, respectively. However, activities of CAT, APX and GR increased under 200 mM NaCl while their activities did not change under 100 mM NaCl in P. maritima. SOD activity in leaves of P. maritima increased with increasing salinity. Concomitant with this, four SOD activity bands were identified in leaves of P. maritima, two bands only were observed in P. media. Peroxidase (POX; EC 1.11.1.7) activity increased under both salt concentrations in P. maritima, but only under 200 mM NaCl in P. media. Confirming this, five POX activity bands were identified in leaves of P. maritima, but only two bands were determined in P. media. Malondialdehyde levels in the leaves increased under salt stress in P. media but showed no change and decreased in P. maritima at 100 and 200 mM NaCl, respectively. These results suggest that the salt-tolerant P. maritima showed a better protection mechanism against oxidative damage caused by salt stress by its higher induced activities of antioxidant enzymes than the salt-sensitive P. media.     

Homozygous mutations in fibroblast growth factor 3 are associated with a new form of syndromic deafness characterized by inner ear agenesis, microtia, and microdontia

We identified nine individuals from three unrelated Turkish families with a unique autosomal recessive syndrome characterized by type I microtia, microdontia, and profound congenital deafness associated with a complete absence of inner ear structures (Michel aplasia). We later demonstrated three different homozygous mutations (p.S156P, p.R104X, and p.V206SfsX117) in the fibroblast growth factor 3 (FGF3) gene in affected members of these families, cosegregating with the autosomal recessive transmission as a completely penetrant phenotype. These findings demonstrate the involvement of FGF3 mutations in a human malformation syndrome for the first time and contribute to our understanding of the role this gene plays in embryonic development. Of particular interest is that the development of the inner ear is completely disturbed at a very early stage--or the otic vesicle is not induced at all--in all of the affected individuals who carried two mutant FGF3 alleles.     

A microscopic study of kidney tissue in Familial Mediterranean Fever patients

Familial Mediterranean Fever (FMF) is an autosomal recessive hereditary disease leading mostly to renal failure and nephrotic syndrome. The ultrastructure of kidney has not been fully investigated in FMF associated renal disease. The aim of this study is to provide further evidence on the ultrastructure of kidney in patients with FMF who suffer from renal disease. Renal biopsies obtained from two patients who were diagnosed with FMF renal disease complications were examined. Examination of renal tissue by light and electron microscopy identified degenerations both in tubules and the filtration barrier. Foot processes were partly effaced. Amorphous material was found in thickened glomerular basement membranes. Fibrous material deposits in thick Bowman's capsule wall were also seen. Finally, degeneration in the form of folding of plasma membrane and vacuolization as well as fusion in mitochondria cristae, was observed. Accumulation of tissue remnants in the lumen was also found in tubules.