Recovery from hypoxia-induced internalization of cardiac Na+/H + exchanger 1 requires an adequate intracellular store of antioxidants

The heart is highly active metabolically but relatively underperfused and, therefore, vulnerable to ischemia. In addition to acidosis, a key component of ischemia is hypoxia that can modulate gene expression and protein function as part of an adaptive or even maladaptive response. Here, using cardiac-derived HL-1 cells, we investigate the effect of various hypoxic stimuli on the expression and activity of Na⁺/H ⁺ exchanger 1 (NHE1), a principal regulator of intracellular pH. Acute (10 min) anoxia produced a reversible decrease in the sarcolemmal NHE1 activity attributable to NHE1 internalization. Treatment with either 1% O ₂ or dimethyloxaloylglycine (DMOG; 1 mM) for 48-hr stabilized hypoxia-inducible factor 1 and reduced the sarcolemmal NHE1 activity by internalization, but without a change in total NHE1 immunoreactivity or message levels of the coding gene ( SLC9A1) determined in whole-cell lysates. Unlike the effect of DMOG, which was rapidly reversed on washout, reoxygenation after a prolonged period of hypoxia did not reverse the effects on NHE1, unless media were also supplemented with a membrane-permeant derivative of glutathione (GSH). Without a prior hypoxic episode, GSH supplementation had no effect on the NHE1 activity. Thus, posthypoxic NHE1 reinsertion can only take place if cells have a sufficient reservoir of a reducing agent. We propose that oxidative stress under prolonged hypoxia depletes intracellular GSH to an extent that curtails NHE1 reinsertion once the hypoxic stimulus is withdrawn. This effect may be cardioprotective, as rapid postischaemic restoration of the NHE1 activity is known to trigger reperfusion injury by producing an intracellular Na ⁺-overload, which is proarrhythmogenic.     

Assessment of DNA strand breakage by comet assay in diabetic patients and the role of antioxidant supplementation

Diabetes patients often show increased production of reactive oxidative species (ROS) together with vascular complications. The presence of these ROS may lead to increased DNA damage in peripheral blood lymphocytes that may be revealed by the comet assay. To test whether DNA is damaged in diabetes, peripheral blood samples were taken from 30 control individuals and 63 diabetic patients (15 insulin dependent (IDDM) and 48 non-insulin dependent (NIDDM)) and the alkaline comet assay was used to evaluate background levels of DNA damage. Significant differences were detected between control and diabetic patients in terms of frequencies of damaged cells. The extend of DNA migration was greater in NIDDM patients by comparison with IDDM patients which might indicate that IDDM patients are handling more oxidative damage on a regular basis. Smoker individuals had higher frequencies of cells with migration by comparison with the non-smokers in both groups. Also, clear differences between patients on placebo and on Vitamin E supplementation for 12 weeks were observed on the basis of the extend of DNA migration during single cell gel electrophoresis.     

Polyphenolic compounds from Geranium pratense and their free radical scavenging activities

Two new polyphenolic compounds, myricetin 3-O-(2"-O-galloyl)-beta-D-glucopyranoside and (-)-6-chloroepicatechin, were isolated from the aerial parts of Geranium pratense subsp. finitimum (Woronow) Knuth, along with three known polyphenolic compounds [quercetin 3-O-(2"-O-galloyl)-beta-D-glucopyranoside, quercetin 3-O-(2"-O-galloyl)-beta-D-galactopyranoside, methyl gallate] and tryptophan. Quercetin 3-O-beta-D-glucopyranoside, quercetin 3-O-beta-D-galactopyranoside, quercetin 3-O-(2"-O-galloyl)-beta-D-glucopyranoside and quercetin 3-O-(2"-O-galloyl)-beta-D-galactopyranoside were found to be effective against free radical induced impairment of endothelium-dependent relaxation in isolated rat aorta.     

Luteinizing hormone releasing hormone increases proliferation of meningioma cells in vitro

The fact that meningioma shows at least a 2:1 predilection for women over men is considered to be due to endocrinological and paracrine regulation of the development of this tumour. The presence of receptors for the luteinizing hormone releasing hormone (LHRH) in gynaecological cancer permits the use of LHRH agonistic or antagonistic analogues with a direct effect or by the gonado-pituitary axis suppression in the treatment of these tumours. Therefore, the effect of LHRH on meningioma cells is tested in this study. Meningioma cells from three female patients were cultured and LHRH (50 ng/ml) was added to the growth medium daily, for fourteen days. At the end of this period the cells were counted by means of a Coulter Counter. The stimulating effects of LHRH on the increase of the amount of cells in the meningioma monolayer culture were 146% (p < 0.01), 134% (p < 0.05) and 141% (p < 0.05) of the control, respectively, for the three patients.     

Enzymatic determination of zinc in vegetables using apocarbonic anhydrase

A new enzymatic method has been developed to determine trace amounts of Zn2+ in vegetables. The basis of the method is that apocarbonic anhydrase regains its activity in proportion to the concentration of Zn2+ present in solution. Bovine carbonic anhydrase was purified from erythrocyte haemolysate by affinity chromatography and the bound Zn2+ removed by dialysis of purified enzyme against a solution of pyridine-2, 6-dicarboxylic acid. Pure (100%) apoenzyme was obtained. The concentration of Zn2+ in vegetable samples was determined using the enzymatic method and by atomic absorption spectroscopy. Determinations made using the two methods were not significantly different one from another.     

Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.     

Three acylated flavone glycosides from Sideritis ozturkii Aytac & Aksoy

From the aerial parts of Sideritis ozturkii, three new flavonoids, chrysoeriol 7-O-[2'-O-caffeoyl-O-acetyl-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside], chrysoeriol 7-O[2'-O-caffeoyl-beta-D-glucopyranosyl-(1 -->2)-beta-D-glucopyranoside] and chrysoeriol 7-O[2'-O-p-coumaroyl-6'-beta-O-acetyl-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside] named as ozturkosides A, B and C, respectively, were isolated, along with three known phenylethanoid glycosides, verbascoside, leucoseptoside A, martynoside and five known diterpenoids, 7-epicandicandiol, linearol, sidol, sideroxol, epoxyisolinearol. The structures were elucidated mainly by spectroscopic methods.     

Reconstruction of orbital floor fracture using solvent-preserved bone graft

The orbital floor is one of the most frequently damaged parts of the maxillofacial skeleton during facial trauma. Unfavorable aesthetic and functional outcomes are frequent when it is treated inadequately. The treatment consists of spanning the floor defect with a material that can provide structural support and restore the orbital volume. This material should also be biocompatible with the surrounding tissues and easily reshaped to fit the orbital floor. Although various autografts or synthetic materials have been used, there is still no consensus on the ideal reconstruction method of orbital floor defects. This study evaluated the applicability of solvent-preserved cadaveric cranial bone graft and its preliminary results in the reconstruction of the orbital floor fractures. Twenty-five orbital floor fractures of 21 patients who underwent surgical repair with cadaveric bone graft during a 2-year period were included in this study. Pure blowout fractures were determined in nine patients, whereas 12 patients had other accompanying maxillofacial fractures. Of the 21 patients, 14 had clinically evident diplopia (66.7 percent), 12 of them had enophthalmos (57.1 percent), and two of them had gaze restriction preoperatively. Reconstruction of the floor of the orbit was performed following either the subciliary or the transconjunctival approach. A cranial allograft was placed over the defect after sufficient exposure. The mean follow-up period was 9 months. Postoperative diplopia, enophthalmos, eye motility, cosmetic appearance, and complications were documented. None of the patients had any evidence of diplopia, limited eye movement, inflammatory reactions in soft tissues, infection, or graft extrusion in the postoperative period. Providing sufficient orbital volume, no graft resorption was detected in computed tomography scan controls. None of the implants required removal for any reason. Enophthalmos was seen in one patient, and temporary scleral show lasting up to 3 to 6 weeks was detected in another three patients. Satisfactory cosmetic results were obtained in all patients. This study showed that solvent-preserved bone, which is a nonsynthetic, human-originated, processed bioimplant, can be safely used in orbital floor repair and can be considered as another reliable treatment alternative.