Priming effect of dextran sulphates on lipopolysaccharide-induced tumour necrosis factor production in mice

Abstract Dextran sulphate (DS) 500 (M.W. 500 000) is commonly used as a reticuloendothelial (RE) blocker. We found that lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF) production in sera was enhanced when mice were pretreated with DS500. When mice were pretreated with DS1000 (M.W. 1 000 000), TNF activity in sera was also significantly enhanced by the LPS injection in comparison with the saline-treated group, but not by the pretreatment with the low molecular weight of DS5 (M.W. 5 000), neutral dextran (Dex) 500, or positively-charged diethylaminoethyl dextran (DEAE-Dex) 500. The enhancement of LPS-induced TNF production occurred from 2 h after DS500 pretreatment. Pretreatment with DS500 or DS1000 significantly suppressed the carbon clearance from the blood in mice from 2 h after DS injection, but this suppression was not detected by the pretreatment with DS5, Dex500, or DEAE-Dex500. We suggest that negative-charge and high molecular weight are essential for dextran derivatives to enhance LPS-induced TNF production, and that the enhancing effect of DS is closely related to the suppression of the RE function.     

Symmetrical gaits of Cebus apella: implications for the functional significance of diagonal sequence gait in primates

Quadrupedal locomotion of primates is distinguished from the quadrupedalism of many other mammals by several features, including a diagonal sequence (DS) footfall used in symmetrical gaits. This presumably unique feature of primate locomotion has been attributed to an ancestral adaptation for cautious arboreal quadrupedalism on thin, flexible branches. However, the functional significance of DS gait remains largely hypothetical. The study presented here tests hypotheses about the functional significance of DS gait by analyzing the gait mechanics of a primate that alternates between DS and lateral sequence (LS) gaits, Cebus apella. Kinematic and kinetic data were gathered from two subjects as they moved across both terrestrial and simulated arboreal substrates. These data were used to test four hypotheses: (1) locomotion on arboreal supports is associated with increased use of DS gait, (2) DS gait is associated with lower peak vertical substrate reaction forces than LS gait, (3) DS gait is associated with greater forelimb/hind limb differentiation in force magnitudes, and (4) DS gait offers increased stability. Our results indicate that animals preferred DS gait on the arboreal substrate, and LS gait while on the ground. Peak vertical substrate reaction forces showed a tendency to be lower in DS gait, but not consistently so. Pole ("arboreal") forces were lower than ground forces in DS gait, but not in LS gait. The preferred symmetrical gait on both substrates was a grounded run or amble, with the body supported by only one limb throughout most of the stride. During periods of bilateral support, the DS gait had predominantly diagonal support couplets. This benefit for stability on an arboreal substrate is potentially outweighed by overstriding, its associated ipsilateral limb interference in DS gait and hind foot positioning in front of the hand on untested territory. DS gait also did not result in an optimal anchoring position of the hind foot under the center of mass of the body at forelimb touchdown. In sum, the results are mixed regarding the superiority of DS gait in an arboreal setting. Consequently, the notion that DS gait is an ancestral adaptation of primates, conditioned by the selection demands of an arboreal environment, remains largely hypothetical.     

Peculiarities in PMC Meiosis of Pisum sativum

Summary Nemesia strumosa plants were discovered which had styles capable of discriminating among incompatible pollen tubes from different pollinators, allowing growth of some but not others. All but 3 of 26 families tested had at least some members with discriminating styles (DS). Presence and level of DS was independent of S genotype. Plants with pseudo-self-compatiblity (PSC) levels greater than 10% had the trait, though many plants with strong DS had PSC levels less than 10%. Self pollination of highly DS plants produced mostly DS offspring, but of differing sensitivities. Some progenies from crosses between a family of highly DS plants and unrelated, probably low DS plants segregated half DS and half non-DS, while others consisted of mostly DS or mostly non-DS. The DS phenomenon is probably caused by PSC genes.Scientific Journal Series Paper No. 11,677 of the Minnesota Agricultural Experiment Station     

Delivery of dermatan sulfate from polyelectrolyte complex-containing alginate composite microspheres for tissue regeneration

Dermatan sulfate (DS) is a glycosaminoglycan (GAG) with a great potential as a new therapeutic agent in tissue engineering. The aim of the present study was to investigate the formation of polyelectrolyte complexes (PECs) between chitosan and dermatan sulfate (CS/DS) and delivery of DS from PEC-containing alginate/chitosan/dermatan sulfate (Alg/CS/DS) microspheres for application in tissue regeneration. The CS/DS complexes were initially formed at different conditions including varying CS/DS ratio (positive/negative charge ratio), buffer, and pH. The obtained CS/DS complexes exhibited stronger electrostatic interaction, smaller complex size, and more stable colloidal structure when chitosan was in large excess (CS/DS 3:1) and prepared at pH 3.5 as compared to pH 5 using acetate buffer. The CS/DS complexes were subsequently incorporated into an alginate matrix by spray drying to form Alg/CS/DS composite microspheres with a DS encapsulation efficiency of 90-95%. The excessive CS induced a higher level of sustained DS release into Tris buffer (pH 7.4) from the microspheres formulated at pH 3.5; however, the amount of CS did not have a significant effect on the release from the microspheres formulated at pH 5. Significant cell proliferation was stimulated by the DS released from the microspheres in vitro. The present results provide a promising drug delivery strategy using PECs for sustained release of DS from microspheres intended for site-specific drug delivery and ultimately for use in tissue engineering.     

Specificity and sensitivity of S100B levels in amniotic fluid for Down syndrome diagnosis

Down syndrome (DS) is the most common chromosomal abnormality and is associated with an extra copy of the chromosome 21. Although several markers are commonly used during pregnancy for the screening of DS, the definitive diagnosis is based on karyotype after amniocentesis, which is an expensive and laborious analysis. S100B is an astrocyte protein which had its gene mapped to the long arm of chromosome 21. Previous preliminary reports have found increased levels of this protein in the amniotic fluid of DS gestations. Aiming to achieve a simpler and cheaper test then karyotype to perform prenatal diagnosis of DS, here we have extended our previous studies and evaluated the real usefulness of amniotic S100B measurement for prenatal DS diagnosis. We have measured S100B in amniotic fluid of 96 pregnancies with DS and of 50 normal pregnancies. Pregnancies with DS presented significantly higher amniotic fluid S100B levels (M = 1.16 ng/mL; IQ = 0.83/1.78) than normal pregnancies (M = 0.51 ng/mL; IQ = 0.38/0.83) (p < 0.0001). A receiver operating characteristic (ROC) curve was performed to evaluate the sensitivity and specificity of S100B for DS diagnosis, and presented an area under the curve (AUC) of 0.82, indicating that S100B could be a reliable marker of DS. Moreover, values above 1.67 ng/mL were present only in DS fetuses, representing about 30% of affected pregnancies. However, as an overlap of values was observed between normal and DS gestations, we concluded that amniotic S100B alone is not a good test to discard DS diagnosis.     

Tissue engineering for full-thickness burns: a dermal substitute from bench to bedside

Our aim was to obtain a viable and easily available dermal substitute (DS) for the definitive coverage of full-thickness burns. A DS composed of a collagen-glycosaminoglycan-chitosan dermal matrix (DM) colonized with foreskin fibroblasts (FF) is described. FF-colonized DS were compared to the DM seeded with adult dermal fibroblasts (DF). FF-colonized DS expressed more fibrillin and tropoelastin than that with DF. Reconstructed skin obtained with both FF- and DF-colonized DS similarly expressed laminin-5 and collagen VII at the dermal-epidermal junction. Both FF- and DF-colonized DS produced cutaneous wound healing mediators in a dose-dependent manner in the presence of platelet lysate. After freeze-thawing, the FF-colonized DS were recovered in culture and retained their ability to produce vascular endothelial growth factor. Grafting of DS into nude rats achieved a complete healing of a dermal-epidermal lesion with a good epidermalization.     

Structural composition and anticoagulant activity of dermatan sulfate from the skin of the electric eel, Electrophorus electricus (L.)

We determined the disaccharide composition of dermatan sulfate (DS) purified from the skin of the electric eel Electrophorus electricus. DS obtained from the electric eel was composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. The anticoagulant, antithrombotic and bleeding effects of electric eel skin DS were compared to those of porcine DS and also to those described previously for DS purified from skin of eel, Anguilla japonica. DS from electric eel is a potent anticoagulant due to a high heparin co-factor II (HC II) activity. The electric eel DS has a higher potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the porcine DS. Interestingly, it was recently demonstrated that DS obtained from skin of the eel Anguilla japonica, which possesses a disaccharide composition very similar to that of electric eel skin DS described here, did not show anticoagulant activity. Thus, the anticoagulant activity of electric eel skin DS is not merely a consequence of its charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to different arrangements of the disulfated disaccharide domain for binding to HC II within their polysaccharide chains and that it may be more efficiently arranged along the carbohydrate chain in electric eel skin DS than in the two other types of DS.     

Selection and characterization of a unique phage display-derived antibody against dermatan sulfate.

Dermatan sulfate (DS) is a member of the glycosaminoglycan (GAG) family and is primarily located in the extracellular matrix. Using a modified phage display procedure, we selected 2 different antibodies against DS of which one antibody, LKN1, was specific for DS. LKN1 was especially reactive with 4/2,4-di-O-sulfated DS, and did not react with other classes of GAGs including chondroitin sulfate and heparan sulfate. Immunohistochemical analysis of kidney, skin and tendon showed a typical fibrillar staining pattern, co-localizing with type I collagen. Staining was abolished by specific enzymatic digestion of DS. Immunoelectron microscopy confirmed the association of the DS epitope with collagen fibrils. The location of DS did not follow the main banding period of collagen, which is in line with the current concept that the core protein rather than the DS moiety of DS-proteoglycans specifically binds to collagen fibrils. This unique anti-DS antibody and the availability of its coding DNA may be instrumental in studies of the structure and function of DS.     

Cystathionine beta-synthase is enriched in the brains of Down's patients

Down's syndrome (DS) or trisomy 21 is the most common genetic cause of mental retardation, and adults with DS develop Alzheimer type of disease (AD). Cystathionine beta-synthase (CBS) is encoded on chromosome 21 and deficiency in its activity causes homocystinuria, the most common inborn error of sulfur amino acid metabolism and characterized by mental retardation and vascular disease. Here, we show that the levels of CBS in DS brains are approximately three times greater than those in the normal individuals. CBS is localized to astrocytes and those surrounding senile plaques in the brains of DS patients with AD. The over-expression of CBS may cause the developmental abnormality in cognition in DS children and that may lead to AD in DS adults.     

Brown drug substance color investigation in cell culture manufacturing using chemically defined media: A case study

Drug substance (DS) color is an important quality attribute for release, stability and comparability studies of biologics. With the increase of DS concentrations and biologics pipelines made in chemically defined media, atypical DS color other than colorless or pale yellow has been recently reported in the biopharmaceutical industry. We recently observed a brown DS color in manufacturing. Although analytical characterization data indicated that the brown color DS had no major quality issue, it is necessary to find the root cause and reduce DS color to ease placebo design for clinical use. It was demonstrated that the brown color was caused by the chemically defined basal medium containing high levels of iron and vitamin B12 (VB12) regardless of cell lines. Iron caused tryptophan oxidation in the protein to form N-formylkynurenine and kynurenine products, which likely contributed to a yellow DS color. A pink DS color was caused by the residual VB12 bound to DS. The brown color was the result of the combinatory effect of yellow and pink colors. Finally a modified basal medium was developed to produce a pale yellow DS in manufacturing.     

Chain-terminating natural mutations affect the function of activating KIR receptors 3DS1 and 2DS3

To determine the nucleotide polymorphism of activating killer-cell immunoglobulin-like receptors (aKIR) 3DS1 and 2DS3, we developed a novel direct-sequencing method and analyzed DNA samples of 175 KIR3DS1(+) individuals and 72 KIR2DS3(+) individuals from the white population. The putative ligand-binding extracellular immunoglobulin (Ig)-like domains of these aKIR receptors are highly conserved, a scenario contrary to inhibitory KIRs that recognize polymorphic human leukocyte antigen (HLA) class I molecules. Null alleles 3DS1*049N and 2DS3*003N that do not express cell-surface receptors were discovered, and they occur commonly in whites (3DS1*049N = 2%; 2DS3*003N = 0.8%). Sequence-specific polymerase chain reaction (PCR) detecting these null alleles is negative with DNA from nonwhite subjects, suggesting that these null alleles are specific to whites and probably originated after the colonization of modern humans in Europe.     

Down's syndrome: a pathology involving the lack of balance of reactive oxygen species

Mongolism, or Down's syndrome (DS) is an inborn pathology due to trisomy 21. Apart from mental deficiency and anatomical abnormalities, premature aging is characteristic for this disease. This paper mainly reviews data on the generation of reactive oxygen species (ROS) and antioxidative defense mechanisms in DS, and on the sensitivity of DS cells to model sources of ROS. Changes in the antioxidative defense of DS cells are due mainly to the location of the gene for (Cu,Zn)-superoxide dismutase on chromosome 21. It is suggested that a disturbance of balance of ROS may be a key point in the pathogenesis of DS.     

Comparison of molecular linkage maps and QTLs for morphological traits in two reciprocal backcross populations of rice

Comparison of maps and QTLs between populations may provide us with a better understanding of molecular maps and the inheritance of traits. We developed and used two reciprocal BC1F1 populations, IP/DS//IP and IP/DS//DS, for QTL analysis. DS (Dasanbyeo) is a Korean tongil-type cultivar (derived from an indica x japonica cross and similar to indica in its genetic make-up) and IP (Ilpumbyeo) is a Korean japonica cultivar. We constructed two molecular linkage maps corresponding to each backcross population using 196 markers for each map. The length of each chromosome was longer in the IP/DS//IP population than in the IP/DS//DS population, indicating that more recombinants were produced in the IP/DS//IP population. Distorted segregation was observed for 44 and 19 marker loci for the IP/DS//IP and IP/DS//DS populations, respectively; these were mostly skewed in favor of the indica alleles. A total of 36 main effect QTLs (M-QTLs) and 15 digenic epistatic interactions (E-QTLs) were detected for the seven traits investigated. The phenotypic variation explained (PVE) by M-QTLs ranged from 3.4% to 88.2%. Total PVE of the M-QTLs for each trait was significantly higher than that of the E-QTLs. The total number of M-QTLs identified in the IP/DS//IP population was higher than in the IP/DS//DS population. However, the total PVE by the M-QTLs and E-QTLs together for each trait was similar in the two populations, suggesting that the two BC1F1 populations are equally useful for QTL analysis. Maps and QTLs in the two populations were compared. Eleven new QTLs were identified for SN, SF, GL, and GW in this study, and they will be valuable in marker-assisted selection, particularly for improving grain traits in tongil-type varieties.     

Radiation sensitivity of Down's syndrome fibroblasts might be due to overexpressed Cu/Zn-superoxide dismutase (EC 1.15.1.1)

Trisomy 21 (Down's syndrome, DS) is the most frequent chromosomal aberration. Triplication of a small region of chromosome 21, the fragment 21q22 is sufficient to cause the DS phenotype including immunodeficiency, premature aging, neurodegenerations, mental retardation and an increased risk of leukemia. Chromosomal aberrations caused by X-ray irradiation were observed in DS lymphocytes and DS fibroblasts, but the correlation to cell death or repair deficiency was not clear. We approached this problem and report here on a profound X-ray repair deficiency of DS cells. With a colorimetric viability assay we observed an UV sensitivity of DS fibroblasts at doses beyond 14 Jm-2 but no significant X-ray sensitivity. By the nucleoid sedimentation technique, a deficient restoration of nucleoids in DS cells after X-ray irradiation was demonstrated. The same features apply for cells, which contain an overexpressed Cu/Zn-superoxide dismutase (SOD-1) gene. Radiation sensitivity of DS cells and SOD-1 overexpressing cells resemble those of ataxia telangiectasia (AT) fibroblasts. Additionally, DS and AT cells exert lack of inhibition of DNA synthesis after X-ray irradiation.     

Proton magnetic resonance spectroscopy in 22q11 deletion syndrome

Objective

People with velo-cardio-facial syndrome or 22q11 deletion syndrome (22q11DS) have behavioral, cognitive and psychiatric problems. Approximately 30% of affected individuals develop schizophrenia-like psychosis. Glutamate dysfunction is thought to play a crucial role in schizophrenia. However, it is unknown if and how the glutamate system is altered in 22q11DS. People with 22q11DS are vulnerable for haploinsufficiency of PRODH, a gene that codes for an enzyme converting proline into glutamate. Therefore, it can be hypothesized that glutamatergic abnormalities may be present in 22q11DS.

Method

We employed proton magnetic resonance spectroscopy (¹H-MRS) to quantify glutamate and other neurometabolites in the dorsolateral prefrontal cortex (DLPFC) and hippocampus of 22 adults with 22q11DS (22q11DS SCZ+) and without (22q11DS SCZ−) schizophrenia and 23 age-matched healthy controls. Also, plasma proline levels were determined in the 22q11DS group.

Results

We found significantly increased concentrations of glutamate and myo-inositol in the hippocampal region of 22q11DS SCZ+ compared to 22q11DS SCZ−. There were no significant differences in levels of plasma proline between 22q11DS SCZ+ and 22q11DS SCZ−. There was no relationship between plasma proline and cerebral glutamate in 22q11DS.

Conclusion

This is the first in vivo ¹H-MRS study in 22q11DS. Our results suggest vulnerability of the hippocampus in the psychopathology of 22q11DS SCZ+. Altered hippocampal glutamate and myo-inositol metabolism may partially explain the psychotic symptoms and cognitive impairments seen in this group of patients.     

Dextran sulphate enhancement of lipopolysaccharide-induced tumour necrosis factor-α production by murine peritoneal macrophages: Correlation with macrophage blockade

Abstract Proteose peptone-induced murine peritoneal macrophages (Mø) were preincubated with 100–800 μg/ml of dextran sulphate (DS) 500 ( M _r 500 000) or DS1000 ( M _r 1 000 000). After 2–24 h of the preincubation, the Mø were stimulated with 1 μg/ml of lipopolysaccharide (LPS) in vitro for 18 h in DS-free culture medium. The culture supernatants were then collected for TNF assay. The LPS-induced TNF activity of Mø supernatant preincubated with DS500 or DS1000 for 6 h was enhanced by up to about ten-fold compared with those preincubated without DS. This enhancing effect was not observed when Mø were preincubated with 100–800 μg/ml of low molecular weight DS5 ( M _r 5000) or neutral dextran (Dex) 500 ( M _r 500 000). The enhancement of LPS-induced TNF-α production from Mø was observed after 2 or 4 h of incubation with DS1000 or DS500, respectively. The phagocytic activity of Mø was determined in vitro by the ingestion index and phagocytic capacity using Saccharomyces cerevisiae . Treatment with DS500 or DS1000 significantly suppressed the phagocytic activity from 2 h after the incubation, but this suppression was not observed in Mø incubated with DS5 or Dex500. Our experiments indicate that DS500 and DS1000 act directly on Mø and enhance LPS-induced TNF-α production from Mø, and that the enhancement is closely related to the suppression of Mø phagocytic function.     

Role of glutathione reductase system in disulfiram conversion to diethyldithiocarbamate.

Experiments were carried out to establish the role of glutathione reductase (GR), if any, in the metabolic conversion of disulfiram (DS) to diethyldithiocarbamate (DDC). It was observed that, under standard assay conditions, whereas DS was incorporated as a substrate instead of oxidised glutathione (GSSG), the enzymes from both human liver extract and yeast sources failed to reduce the parent compound, implying that glutathione reductase perse do not reduce disulfiram. However, the incorporation of disulfiram into an assay system comprising of GSSG, NADPH and reductase resulted in DS reduction to DDC. Further, the observation, that the GR assay system devoid of either GSSG or NADPH was found to lack DS reducing ability, implies that GSH as a reaction product of GR system is responsible for the reduction of DS to DDC. The results of in-vitro experiments indicated that GSH perse could reduce DS to DDC nonenzymatically, with a stoichiometric relationship of 2:1. Thus it is inferred that GR perse do not reduce DS, whereas GSH, as an intermediary metabolite of GR system, brings about non-enzymatic reduction of DS via a sulfhydral group exchange reaction.     

Defective Mitochondrial Function In Vivo in Skeletal Muscle in Adults with Down’s Syndrome: A 31P-MRS Study

Down’s syndrome (DS) is a developmental disorder associated with intellectual disability (ID). We have previously shown that people with DS engage in very low levels of exercise compared to people with ID not due to DS. Many aspects of the DS phenotype, such as dementia, low activity levels and poor muscle tone, are shared with disorders of mitochondrial origin, and mitochondrial dysfunction has been demonstrated in cultured DS tissue. We undertook a phosphorus magnetic resonance spectroscopy (³¹P-MRS) study in the quadriceps muscle of 14 people with DS and 11 non-DS ID controls to investigate the post-exercise resynthesis kinetics of phosphocreatine (PCr), which relies on mitochondrial respiratory function and yields a measure of muscle mitochondrial function in vivo. We found that the PCr recovery rate constant was significantly decreased in adults with DS compared to non-DS ID controls (1.7±0.1 min⁻¹ vs 2.1±0.1 min⁻¹ respectively) who were matched for physical activity levels, indicating that muscle mitochondrial function in vivo is impaired in DS. This is the first study to investigate mitochondrial function in vivo in DS using ³¹P-MRS. Our study is consistent with previous in vitro studies, supporting a theory of a global mitochondrial defect in DS.     

Gait modification when decreasing double support percentage

Much is still unknown about walking stability, including which aspects of gait contribute to higher stability. Walking stability appears to be related to walking speed, although the exact relationship is unclear. As walking speed decreases, the double support (DS) period of gait increases both in time and as a percentage of the gait cycle. Because humans have more control over their center of mass movement during DS, increasing DS duration may alter stability. This study examined how human gait is affected by changing DS percentage independent of walking speed. Sixteen young, healthy adults walked on a treadmill at a single speed for six one-minute trials. These trials included normal gait as well as longer- and shorter-than-normal DS percentage gaits. Subjects were consistently able to decrease DS percentage but had difficulty increasing DS percentage. In some cases, subjects altered their cadence when changing DS percentage, particularly when attempting to increase DS percentage. The changes to gait when decreasing DS percentage were similar to changes when increasing walking speed but occurred mainly during the swing period. These changes include increased hip and knee flexion during the swing period, increased swing foot height, and larger magnitude peaks in ground reaction forces. The changes in gait when attempting to increase DS percentage trended toward changes when decreasing walking speed. Altering DS percentage induced gait changes that were similar to, yet clearly distinct from, gait changes due to walking speed. Further, the difficulty of increasing DS percentage when walking at a constant speed suggests that people walk more slowly when they want to increase time spent in DS.