Flap reconstruction of the knee: A review of current concepts and a proposed algorithm.

A literature search focusing on flap knee reconstruction revealed much controversy regarding the optimal management of around the knee defects. Muscle flaps are the preferred option, mainly in infected wounds. Perforator flaps have recently been introduced in knee coverage with significant advantages due to low donor morbidity and long pedicles with wide arc of rotation. In the case of free flap the choice of recipient vessels is the key point to the reconstruction. Taking the published experience into account, a reconstructive algorithm is proposed according to the size and location of the wound, the presence of infection and/or 3-dimensional defect.

Long-term selenium supplementation of humans: selenium status and relationships between selenium concentrations in skeletal muscle and indicator materials.

Supplementation with elevated doses of l-selenomethionine (SeM) or selenium-enriched yeast that contains SeM as the main selenium species is frequently used as a protective or therapeutic measure. Information on the effects of long-term selenium supplementation on the body selenium status is, however, rather scarce. We therefore investigated fifteen male test persons who had taken selenium yeast and/or SeM supplements in medium doses of 62.5-125 microg Se/day or high doses of 200-262.5 microg Se/day for periods ranging from 1 year to 24 years. Seven non-supplemented men served as controls. As skeletal muscle is the main selenium pool, thigh muscle biopsy samples were taken. The selenium concentrations in these biopsies and in samples of the indicator materials blood, blood plasma, blood cells, head hair and toenails were determined by neutron activation analysis. Compared with the controls, the muscle selenium level was raised with additional selenium supplementation, but the relative increase in the mean muscle selenium concentration (by factors of about 1.6 and 2 for the medium and high doses, respectively) was lower than that in the selenium intake. Highly significant correlations found between the selenium concentrations in muscle and whole blood (R=0.90), red blood cells (R=0.91), blood plasma (R=0.87), head hair (R=0.89) and toenails (R=0.85) show that in humans supplemented in this way the selenium status can be assessed in a relatively easy way by analysis of the selenium retention in these indicator materials.

Observation of subtle dynamic transitions by a combination of neutron scattering, X-ray diffraction and DSC: a case study of the monoclinic L-cysteine.

The paper illustrates the benefit of combining several experimental techniques (incoherent elastic and inelastic neutron scattering, DSC, and X-ray diffraction) to study subtle dynamic transitions in a biologically important system, probing a broad time (frequency) range of the molecular motions in a wide temperature interval of 2-300K. As a case study the crystalline form (a monoclinic polymorph) of l-cysteine ((+)NH(3)-CH(CH(2)SH)-COO(-)) - an essential amino acid - has been selected. Crystals of amino acids are widely used to mimic important structural and dynamic features of peptides. The conformational lability of cysteine and the dynamics of the thiol-side chains are known to account for various phase transitions in the crystalline state and for the conformational transitions important for the biological function in the peptides. The effect of temperature on the monoclinic polymorph of l-cysteine, metastable at ambient conditions, has been studied for the first time. A dynamical transition at about 150K, marking a crossover of the molecular fluctuations between harmonic and non-harmonic dynamical regimes, was evidenced by evaluating the evolution of the mean-square displacement obtained from the elastic fixed window approach using the backscattering spectrometer IN10 located at the ILL. Although this transition does not manifest itself in the DSC, it was clearly observed by incoherent inelastic neutron scattering. By analyzing the dynamical susceptibility contribution (chi''(omega)) obtained using IN6 also at ILL we were able to evidence another relaxation process at a different time scale. The disordered soft l-cysteine structure has an excess of inelastic scattering at about 3meV, analogous to the "boson peak" observed in glass-like materials and proteins. High-precision X-ray diffraction has revealed an anomaly in the changes of selected unit cell parameters and volume at about 240K.

Metalloproteome of the prostate: carcinoma cell line DU-145 in comparison to healthy rat tissue.

AIM: To study the metalloproteome of DU-145 prostate carcinoma cells in comparison to prostate from control and selenium-deficient rats. MATERIALS AND METHODS: Total proteome of the samples was compared by two-dimensional electrophoresis (2-DE) and metalloproteome was analysed by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP/MS). Immunotests were used to quantify protein expression of superoxide dismutase, thioredoxin reductase and metallothionein. RESULTS: There was no general relation between protein expression and metal load. SEC-ICP/MS spectra for many metals varied significantly in terms of peak number and intensity between individuals of the same sample group. However, nickel and zinc peaks were consistently suppressed in DU-145 cells under selenium deficiency. Concurrent redistribution of cobalt binding to a low molecular weight fraction (presumably cobalamin) was observed. CONCLUSION: Metal load of proteins in comparison to their expression might point to yet unknown mechanisms of oncogenesis and may lead to new biomarkers of cancer.

Studies with regard to the apoptosis of testicular germ cells in rats fed a diet enriched with polyunsaturated Fatty acids.

The essential trace element selenium and polyunsaturated fatty acids (PUFA) have been used for the prevention of cancer. Both nutrients enhance the apoptosis of malignant cells and provide health benefits. However, an increased dietary intake of PUFA augments the susceptibility of lipid peroxidation and oxidative damage in many cells. So far, relatively few data are available about the interaction of selenium and PUFA in testis and thus a possible effect of both dietary components on the prevention of testicular cancer or on the apoptosis of testicular germ cells. Male germ cells in the rat contain most of the testicular phospholipid hydroperoxide glutathione peroxidase (PHGPx), mainly as the mitochondrial isoform of this selenoprotein (m-PHGPx). An experiment was therefore carried out to determine the action of fish oil, a nutrient rich in PUFA, on the testicular expression of PHGPx. Because the PHGPx formation remains nearly unchanged in the animals fed the PUFA-enriched diet, we conclude that no apoptosis of testicular germ cells is induced by an increased intake of this nutrient. The intake of fish oil in the selenium-deficient animal led to a markedly altered formation of several selenium-containing proteins, including sperm nuclei glutathione peroxidase (snGPx), also designated as the nuclear form of PHGPx (n-PHGPx), and a 10-kDa selenium-containing protein.

Effects of long-term selenium yeast supplementation on selenium status studied in the rat.

To investigate the selenium status during long-term dietary supply of selenium yeast, 30-day-old male rats were fed for 379 days a methionine-adequate low-selenium diet supplemented with 0.2mgSe/kg (selenium-adequate diet) or 1.5mgSe/kg (high-selenium diet) in the form of selenium yeast that contained 60% of the element as l-selenomethionine. Their selenium load was determined at several intervals by neutron activation analysis of the selenium concentrations in the main selenium body pools, skeletal muscle and liver. After 64 days the tissue selenium concentrations plateaued in both groups and then stayed at that level. Compared with the selenium-adequate group, elevated tissue selenium concentrations were found in the high-selenium group, but the increase by a factor of 3.5 in the muscle and by a factor of 2.3 in the liver was smaller than the 7.5-fold increase in the selenium intake. In the selenium-adequate group about 50% of the muscle selenium and 30% of the liver selenium and in the high-selenium group about 85% of the muscle selenium and 70% of the liver selenium were estimated to be present in non-selenoprotein forms. During selenium depletion the liver glutathione peroxidase activity in the high-selenium group remained unaffected for 4 weeks and then decreased more slowly than that in the selenium-adequate group. From these results it can be concluded that selenium incorporated from the selenium yeast diet into non-selenoprotein forms can serve as an endogenous selenium source to maintain selenoprotein levels in periods of insufficient selenium supply.

The brain selenoproteome: priorities in the hierarchy and different levels of selenium homeostasis in the brain of selenium-deficient rats.

The application of radionuclides for the localization of essential trace elements in vivo and the characterization of their binding proteins is a story of intermittently made improvements of the techniques used for their detection. In this study we present the use of neutron activation analysis and different autoradiographic imaging methods including real-time digital autoradiography to reveal new insights in the hierarchy of selenium homeostasis. Selenoproteins containing the essential trace element selenium play important roles in the CNS. Although the CNS does not show the highest selenium concentration in the case of selenium-sufficient supply in comparison with other organs, it shows a high priority for selenium uptake and retention in the case of dietary selenium deficiency. To characterize the hierarchy of selenium supply in the brain, in vivo radiotracer labeling with (75)Se in rats with different selenium status was combined with autoradiographic detection of (75)Se in brain tissue sections and (75)Se-labeled selenoproteins after protein separation by two-dimensional gel electrophoresis. This study demonstrates significant differences in the uptake of (75)Se into the brain of rats with different selenium status. A brain region-specific uptake pattern of the radiotracer (75)Se in selenium-deficient rats could be revealed and the CSF was identified as a key part of the brain selenium homeostasis.

Elevated metallothionein-bound cadmium concentrations in urine from bladder carcinoma patients, investigated by size exclusion chromatography-inductively coupled plasma mass spectrometry.

Cadmium is discussed as being involved in the development of transitional cell carcinoma (TCC) of the bladder and can be observed in urine of these patients. Investigations of urinary samples from bladder cancer patients and normal controls were carried out with special emphasis on metallothionein (MT)-bound cadmium. Compounds that are constituents of urine were separated in urine samples by means of size exclusion chromatography and cadmium was monitored continuously with a hyphenated inductively coupled plasma mass spectrometry (ICP-MS) system. MT-bound cadmium was quantified by peak area integration, taking into account the intensity of the rhodium signal which was added continuously before ICP-MS detection. The obtained results show that urinary cadmium is predominantly bound to the observed MT-fraction. The median of the MT-bound cadmium concentration in the control group was found to be 0.8 microgL(-1) whereas the cancer group has a median of 1.8 microgL(-1). The variance of the data in the cancer group is much higher than in the controls. However, the urinary MT-bound cadmium is significantly elevated in the cancer group; odds-ratio test: 7.11 (95% C.I.: 1.89-26.80), taking into account the total protein content. Due to the fact that only one main cadmium-containing fraction was observed, there is no necessity to separate the MT-fraction before cadmium determination in urine samples in future studies.

Biochemical analysis of selenoprotein expression in brain cell lines and in distinct brain regions.

Selenium is present in various biologically important selenoproteins. The preferential incorporation of selenium into the brain indicates its significance for this organ, but so far knowledge concerning the cerebral selenoproteome is scarce. We therefore investigated the expression of selenoproteins in various regions of the rat brain, various subcellular fractions and several brain cell lines by (75)Se-labelling, gel electrophoretic separation and autoradiography, with the (75)Se tracer as the selenoprotein marker. Quantitative evaluation of the labelled proteins in selenium-deficient rats revealed information regarding preferentially supplied selenoproteins and their distribution; 21 selenoproteins could be distinguished, among them a novel or modified 15-kDa selenoprotein enriched in the cerebellum cytosol. The selenoproteins differed in the degree of their expression among the brain regions and within a region among the subcellular fractions. Some cell-type-specific selenium-containing proteins were found in the cell lines. Differences in the distribution patterns between mono-cultured and co-cultured endothelial cells and astrocytes showed that mediators produced by other cells could affect the selenoprotein expression of a specific cell-type. This effect might play a role in the uptake and distribution of selenium in the brain but could also be of significance in the selenium metabolism of other tissues.

Activity of the glutathione peroxidase-2. Differences in the selenium-dependent expression between colon and small intestine.

BACKGROUND: Selenium (Se) is an essential element which is involved in various biological processes in nearly all tissues of animals and human, e.g. protection against oxidative stress in the cardiovascular system, and may play a role in cancer protection. It is incorporated in the proteome in the form of the genetically encoded amino acid selenocysteine, which is the characteristic component of the selenoproteins. MATERIALS AND METHODS: We investigated the expression of the selenoenzyme GPx-2 which is predominantly present in the tissues of the gastrointestinal tract such as the small intestine and therefore named gastrointestinal glutathione peroxidase. Rats were fed with a Se-adequate or Se-deficient diet and GPx-2 was assessed by means of enzyme activity with respect to the Se concentration in tissues of the colon and small intestine. Se quantification was carried out by means of graphite furnace atom absorption spectrometry and 2D-gel electrophoresis was applied to investigate the expression of the proteins of the small intestine tissue samples. RESULTS: Twenty-eight differences could be distinguished in the protein spot distribution of the 2D-gels of the homogenates. The GPx-2 activity in the Se-deficient rat colon samples was 6.8 fold lower than in the Se-adequate rats in contrast to 1.2 fold lower levels between the corresponding samples in the small intestine. CONCLUSION: This finding might explain the different susceptibility of the colon and the small intestine to cancer and support the theory of the protective effect of selenium in the gastrointestinal tract.

Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines.

The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.

Alteration of biological samples in speciation analysis of metalloproteins.

For investigations of metalloproteins by speciation analysis, the integrity of the protein-metal complexes before and during separation is crucial. Knowledge about potential alterations of the samples is thus essential to avoid misinterpretations of the analytical results. Chromatographic element profiles of different cytosolic samples from animal tissues were measured repeatedly to estimate the sample stability. The dependence of the signals on the dwell time of the sample in an autosampling device at 4 degrees C for a period of 10 h was observed. Alterations in the element content of different metal-containing fractions were quantified by means of recovery values. Some metalloprotein fractions (e.g. approximately 27-kDa arsenic, approximately 27-kDa iron and different zinc fractions) were stable or only minor alterations were observed and for their investigation an autosampling device is therefore suitable. However, most of the other metalloprotein fractions, especially nickel-containing proteins, showed major alterations: these samples should therefore be analysed immediately after preparation or directly after thawing.

The role of selenite on microglial migration.

Oxidative brain damage, such as excitotoxicity and stroke, leads to primary neuronal destruction. The primary damage is further potentiated by macrophages and microglial cells, which are attracted and invade into the zone of damage resulting in secondary neuronal death. Since the essential trace element selenium has anti-inflammatory properties, we analyzed the effects of selenium on these inflammatory cells. Here, we show that the essential trace element selenium abrogates the stress-induced migration of microglial cells. Thus, the antimigratory effects of selenium may attenuate the secondary cell death cascade by preventing microglial invasion.

Is the distribution of selenium and zinc in the sublocations of spermatozoa regulated?

In the sperm nuclei, of mammalian species selenium has been found only in the form of sperm nuclei glutathione peroxidase (snGPx) where it is most likely bound to the chromatin of spermatozoa. Over 80% of selenium in sperm is bound to the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the midpiece of rat sperm. Zinc in sperm is mainly contained in the outer dense fiber (ODF) proteins of the flagella of mammalian spermatozoa. In the sperm nuclei, zinc is predominately located in the chromatin to the protamine proteins. In order to investigate if the insertion of zinc and selenium in sperm chromatin is regulated, the element concentrations were determined in equine spermatozoa and purified sperm nuclei. We found a significant positive correlation between the selenium concentration in equine spermatozoa and sperm nuclei. The same finding was obtained for the zinc concentration in spermatozoa and sperm nuclei. The results assume that the distribution of selenium and zinc in spermatozoa is regulated by cell signaling pathways and in this way determining the selenium and zinc amount in the chromatin of spermatozoa.

Effects of selenium diet on expression of selenoproteins in the lung of the rat.

In the present article the radiotracer techniques have been combined with biochemical separation procedures to investigate the effects of changes in the selenium status on the expression of the selenium-containing proteins in the lung and their subcellular fractions. Subcellular separation of the lung has been achieved by differential ultracentrifugation. The selenium-containing proteins in these compartments have been investigated by labeling of rats in vivo with (75)Se, gel electrophoretic separation of the proteins, and autoradiographic detection of the tracer. In the lung of the selenium-deficient animals, the selenium administered was used predominantly to restore the levels of the selenoproteins, while in the lung of the selenium-sufficient animals most of the selenium retained was incorporated into the glutathione peroxidase. Also, higher activity of this enzyme has been found in the lung of the selenium-sufficient animals. The differences in the specific incorporation of the element in the selenium deficiency into different compounds suggested that there are different metabolic pathways for selenium, strongly dependent on its status.

Studies on the selenoproteome in the cultured cells of lung and trachea by gel electrophoretic techniques.

In the present studies radiotracer techniques have been combined with biochemical separation procedures to investigate the selenium-containing proteins in the culture cells of the lung, trachea and their subcellular fractions. Subcellular separation of the lung and trachea tissues has been achieved by differential ultracentrifugation. The selenium-containing proteins in these compartments have been investigated by labeling of lung and trachea cultured cells in vitro with Se-75, gel electrophoretic separation of the proteins and autoradiographic detection of the tracer. The protein separation by gel electrophoresis using mono-dimensional (1D)- and two-dimensional (2D)-SDS-PAGE has been successfully applied for the selenium research. It has resulted in the detection of a large number of selenium-containing proteins. Two-dimensional gel electrophoresis (2-DE) was also helpful in the identification of the proteins of interest according to their molecular mass and isoelectric point. In this way more than 30 selenium-containing proteins could be distinguished in the lung and trachea samples. Some of them such as Gpx1, Trx1, SelP, SelT and Sel15 could be identified by means of immunoassays, their molecular weight and pI values and localized in the cellular compartments.

Effects of selenium deficiency on fatty acid metabolism in rats fed fish oil-enriched diets.

The hepatic fatty acid metabolism was investigated in rats stressed by selenium deficiency and enhanced fish oil intake. Changes in the composition of lipids, peroxides, and fatty acids were studied in the liver of rats fed either a Sedeficient (8 microg Se/kg) or a Se-adequate (300 microg Se/kg) diet, both rich in n-3 fatty acid-containing fish oil (100 g/kg diet) and vitamin E (146 mg alpha-tocopherol/kg diet). The two diets were identical except for their Se content. Se deficiency led to a decrease in hair coat density and quality as well as to changes in liver lipids, individual lipid fractions and phospholipid fatty acid composition of the liver. The low Se status did reduce total and reduced glutathione in the liver but did not affect the hepatic malondialdehyde level. In liver phospholipids (PL), Se deficiency significantly reduced levels of palmitic acid [16:0], fatty acids of the n-3 series such as DHA [22:6 n-3], and other long-chain polyunsaturates C-20-C-22, but increased n-6 fatty acids such as linoleic acid (LA) [18:2 n-6]. Thus, the conversion of LA to arachidonic acid was reduced and the ratio of n-6/n-3 fatty acids was increased. As in liver PL, an increase in the n-6/n-3 ratio was also observed in the mucosal total fatty acids of the small intestine. These results suggest that in rats with adequate vitamin E and enhanced fish oil intake, Se deficiency affects the lipid concentration and fatty acid composition in the liver. The changes may be related to the decreased levels of selenoenzymes with antioxidative functions. Possible effects of Se on absorption, storage and desaturation of fatty acids were also discussed.

Selenium deficiency abrogates inflammation-dependent plasma cell tumors in mice.

The role of the micronutrient, selenium, in human cancers associated with chronic inflammations and persistent infections is poorly understood. Peritoneal plasmacytomas (PCTs) in strain BALB/c (C), the premier experimental model of inflammation-dependent plasma cell transformation in mice, may afford an opportunity to gain additional insights into the significance of selenium in neoplastic development. Here, we report that selenium-depleted C mice (n = 32) maintained on a torula-based low-selenium diet (5-8 micro g of selenium/kg) were totally refractory to pristane induction of PCT. In contrast, 11 of 26 (42.3%) control mice maintained on a selenium adequate torula diet (300 micro g of selenium/kg) and 15 of 40 (37.5%) control mice fed standard Purina chow (440 micro g of selenium/kg) developed PCT by 275 days postpristane. Abrogation of PCT was caused in part by the striking inhibition of the formation of the inflammatory tissue in which PCT develop (pristane granuloma). This was associated with the reduced responsiveness of selenium-deficient inflammatory cells (monocytes and neutrophils) to chemoattractants, such as thioredoxin and chemokines. Selenium-deficient C mice exhibited little evidence of disturbed redox homeostasis and increased mutant frequency of a transgenic lacZ reporter gene in vivo. These findings implicate selenium, via the selenoproteins, in the promotion of inflammation-induced PCT and suggest that small drug inhibitors of selenoproteins might be useful for preventing human cancers linked with chronic inflammations and persistent infections.

Selenium deficiency increases susceptibility to glutamate-induced excitotoxicity.

Excitotoxic brain lesions, such as stroke and epilepsy, lead to increasing destruction of neurons hours after the insult. The deadly cascade of events involves detrimental actions by free radicals and the activation of proapoptotic transcription factors, which finally result in neuronal destruction. Here, we provide direct evidence that the nutritionally essential trace element selenium has a pivotal role in neuronal susceptibility to excitotoxic lesions. First, we observed in neuronal cell cultures that addition of selenium in the form of selenite within the physiological range protects against excitotoxic insults and even attenuates primary damage. The neuroprotective effect of selenium is not directly mediated via antioxidative effects of selenite but requires de novo protein synthesis. Gel shift analysis demonstrates that this effect is connected to the inhibition of glutamate-induced NF-kappaB and AP-1 activation. Furthermore, we provide evidence that selenium deficiency in vivo results in a massive increase in susceptibility to kainate-induced seizures and cell loss. These findings indicate the importance of selenium for prevention and therapy of excitotoxic brain damage.

Distribution of an 18 kDa-selenoprotein in several tissues of the rat.

By combining methods for trace element analysis, tracer techniques and various biochemical and electrophoretical procedures, information on the characteristics of an 18 kDa-selenoprotein was obtained. By labeling of rats in vivo with [75Se]-selenite and gel electrophoretic separation of the proteins in tissues and subcellular fractions, a larger number of selenium-containing proteins could be distinguished. In most of the tissues investigated a labeled 18 kDa-band was present. After co-electrophoresis of the 18 kDa-bands from kidney, liver and brain we found that they all migrated in the same way. Using ultracentrifugational fractionation the 18 kDa-band was localized in the mitochondrial and microsomal membranes. Two-dimensional electrophoresis showed that it consists of a single selenium-containing protein with an isoelectric point of about 4.9-5.0. By means of proteolytic cleavage of the 18 kDa-protein and separation of its peptides by tricine-SDS-PAGE six selenium-containing peptides with molecular masses of 17, 16, 14, 12, 10, and 8 kDa were detected. After electrophoretic separation of the mitochondrial and/or microsomal proteins and acid hydrolysis of the electroeluted protein its amino acid composition was analyzed by RP-HPLC. In this way it was shown that selenium is present in the 18 kDa-protein in form of selenocysteine which is a characteristic of a genetically encoded selenoprotein.