Quantitative detection and comparison of sulfate glycosaminoglycans content in extracellular matrix of in vitro cultured epiphyseal, articular and rib chondrocytes

Objective To establish a method for quantitative detection of the sulfate glycosaminoglycans ( GAG) content in extracellular matrix of in vitro cultured chondrocytes so as to evaluate the biological characteristics of epiphyseal, articular and rib chondrocytes. Methods Sulfate GAG content in extracellular matrix of three chondrocytes was measured by the modified dimethylmethylene blue (DMB) method. The changes of the toluidine blue (TB) stain of chondrocytes were observed by light microscope. Results Primary chondrocytes had the highest content of sulfate GAG in the extracellular matrix, ie, epiphyseal chondrocytes reached ( 70. 12 ± 7. 72 )μg/cm2, articular chondrocytes (92.00 ± 10.15) μg/cm2 and rib chondrocytes (80.61 ± 11. 40) μg/cm2, respectively. On the third pasage chondrocytes, epiphyceal chondrocytes decreased to (53.27 ± 9. 50 ) μg/cm2, articular chondrocytes to (63.88 ± 11.92) μg/cm2 and rib chondrocytes to (58.94 ±8.21) μg/cm2, respectively. The change of TB in every passage

Bioengineered production of glycosaminoglycans and their analogues

Glycosaminoglycans(GAGs)are a class of linear polysaccharides,consisting of alternating disaccharide sequences of uronic acid and hexosamines(or galactose)with and without sulfation.They can interact with various proteins,such as growth factors,receptors and cell adhesion molecules,endowing these with various biological and pharmacological activi-ties.Such activities make GAGs useful in health care products and medicines.Currently,all GAGs,with the exception of hyaluronan,are produced by extraction from animal tissues.However,limited availability,poor control of animal tissues,impurities,viruses,prions,endotoxins,contamination and other problems have increased the interest in new approaches for GAG production.These new approaches include GAGs production by chemical synthesis,chemoenzymatic synthesis and metabolic engineering.One chemically synthesized heparin pentasaccharide,fondaparinux sodium,is in clinical use.Mostly,hyaluronan today is prepared by microbial fermentation,largely replacing hyaluronan from rooster comb.The recent gram scale chemoenzymatic synthesis of a heparin dodecasaccharide suggests its potential to replace currently used animal-sourced low molecular weight heparin(LMWH).Despite these considerable successes,such high-tech approaches still cannot meet worldwide demands for GAGs.This review gives a brief introduction on the manufacturing of unfractionated and low molecular weight heparins,the chemical synthesis and chemoenzymatic synthesis of GAGs and focuses on the progress in the bioengineered preparation of GAGs,particularly heparin.

Purification and Characterization of Hyaluronate Lyases Produced by Two Types of Bacteria

Hyaluronate lyases were obtained from two types of naturally isolated bacterial strains Paenibacillus yunnanensis and Paennarthrobacter nicotinovorans.PyHL(form P.yunnanensis)in the culture supernatant of the bacteria was purified by two steps of column chromatography.The enzyme showed the molecular mass of 74 kDa by SDS-PAGE and the maximal activity at pH 5.0,35℃.PyHL maximally degraded hyaluronate by an endo-type manner,and showed low degradation activity toward chondroitin sulfates.Dermatan sulfate was not the substrate.PnHL(from P.nicotinovorans)in the culture supernatant of the bacteria was purified by two steps of column chromatography.The enzyme showed the molecular mass of 70 kDa by SDS-PAGE and the maximal activity at pH 6.0,30℃.Genomic analysis of P.nicotinovorans on the bases of the internal amino acid sequences of PnHL.

Proteoglycans: Road Signs for Neurite Outgrowth

Proteoglycans in the central nervous system play integral roles as ＂traffic signals＂ for the direction of neurite outgrowth. This attribute of proteoglycans is a major factor in regeneration of the injured central nervous system. In this review, the structures of proteoglycans and the evidence suggesting their involvement in the response following spinal cord injury are presented. The review further describes the methods routinely used to determine the effect proteoglycans have on neurite outgrowth. The effects of proteoglycans on neurite outgrowth are not completely understood as there is disagreement on what component of the molecule is interacting with growing neurites and this ambiguity is chronicled in an historical context. Finally, the most recent findings suggesting possible receptors, interactions, and sulfation patterns that may be important in eliciting the effect of proteoglycans on neurite outgrowth are discussed. A greater understanding of the proteoglycan-neurite interaction is necessary for successfully promoting regeneration in the iniured central nervous system.

Placental accommodations for transport and metabolism during intra-uterine crowding in pigs

Litter size and birth weights are limited by uterine capacity, defined as the ability of the uterus to maintain the appropriate development of some number of conceptuses. Uterine capacity is the result of the combined effects of uterine, placental and embryo/fetal function. The number of living conceptuses that the uterus is capable of supporting is greater during early gestation compared to later gestation. Plots of log fetal weight versus log placental weight also indicate that fetal weights are less sensitive to reduced placental weight （and therefore reduced intrauterine space） in early gestation compared to late gestation. However, even in late gestation, mechanisms still exist that maintain fetal growth when the size of the placenta is reduced. One such mechanism is likely to be improved development of the folded placental-epithelial/maternal-epithelial bilayer. Fold depth, and therefore the maternal fetal interactive surface, increases as gestation advances and is greater in placenta from smal fetuses. On the fetal side of the placenta, the epithelial bilayer is embedded in stromal tissue. Glycosaminoglycans are major components of stroma, including hyaluronan and heparan sulfate. Hyaluronidases and heparanases are present within placental tissues, and likely play roles in modification of stromal components to facilitate fold development. Glycosaminoglycans are polymers of forms of glucose （glucosamine, glucuronic acid, iduronic acid） suggesting that glycosaminoglycan synthesis may compete with the glucose needs of the developing fetus. Pig conceptuses are fructogenic, such that a substantial portion of glucose transferred from mother to fetus is converted to fructose. Fructose is an intermediate product in the synthesis of glucosamine from glucose, and glucosamine is linked to regulation of trophoblast cell proliferation through regulation of mTOR. These findings suggest a link between glucose, fructose, glucosamine synthesis, GAG production, and placental morphogenesis, but the details of these interactions remain unclear. In addition, recent placental epithelial transcriptome analysis identified several glucose, amino acid, lipid, vitamin, mineral and hormone transporter mechanisms within the placenta. Further elucidation of mechanisms of placental morphogenesis and solute transport could provide clues to improving nutrient transport to the pig fetus, potentially increasing litter size and piglet birth weights.

Glycosaminoglycan remodeling during diabetes and the role of dietary factors in their modulation

Glycosaminoglycans(GAGs) play a significant role in various aspects of cell physiology.These are complex polymeric molecules characterized by disaccharides comprising of uronic acid and amino sugar.Compounded to the heterogeneity,these are variously sulfated and epimerized depending on the class of GAG.Among the various classes of GAG,namely,chondroitin/dermatan sulfate,heparin/heparan sulfate,keratan sulfate and hyaluronic acid(HA),only HA is non-sulfated.GAGs are known to undergo remodeling in various tissues during various pathophysiological conditions,diabetes mellitus being one among them.These changes will likely affect their structure thereby impinging on their functionality.Till date,diabetes has been shown to affect GAGs in organs such as kidney,liver,aorta,skin,erythrocytes,etc.to name a few,with deleterious consequences.One of the mainstays in the treatment of diabetes is though dietary means.Various dietary factors are known to play a significant role in regulating glucose homeostasis.Furthermore,in recent years,there has been a keen interest to decipher the role of dietary factors on GAG metabolism.This review focuses on the remodeling of GAGs in various organs during diabetes and their modulation by dietary factors.While effect of diabetes on GAG metabolism has been worked out quite a bit,studies on the role of dietary factors in their modulation has been few and far between.We have tried our best to give the latest reports available on this subject.

NEM® Brand Eggshell Membrane Effective in the Treatment of Pain and Stiffness Associated with Osteoarthritis of the Knee in an Italian Study Population

A single-center, open-label clinical study was conducted to evaluate the efficacy and safety of NEM® as a natural treatment for pain and stiffness associated with osteoarthritis of the knee in an Italian population. NEM® brand eggshell membrane is a unique dietary supplement that contains naturally occurring glycosaminoglycans and proteins essential for maintaining healthy joints and connective tissues. Twenty-five subjects received oral NEM®, 500 mg once daily for four weeks. The primary outcome measure was to evaluate the mean effectiveness of NEM® in relieving general pain associated with moderate osteoarthritis of the knee at 10 and 30 days utilizing a 10-question, abbreviated questionnaire based on the WOMAC osteoarthritis questionnaire. Supplementation with NEM® produced a significant treatment response from baseline at both 10 days and 30 days for composite pain (40.6% reduction, p p p = 0.009;59.7% reduction, p < 0.001, respectively). There were no adverse events or serious adverse events reported during the study and the treatment was reported to be well tolerated by study participants. NEM® is an effective and safe natural therapeutic option for the treatment of both pain and stiffness associated with osteoarthritis of the knee. Supplementation with NEM®, 500 mg taken once daily, significantly reduced both pain and stiffness rapidly (10 days) and this effect continued to improve through 30 days. There was also a meaningful reduction in the amount of analgesic consumed on a weekly basis, which further enhanced patients’ safety.

Biochemical composition of the glomerular extracellular matrix in patients with diabetic kidney disease

In the glomeruli,mesangial cells produce mesangial matrix while podocytes wrap glomerular capillaries with cellular extensions named foot processes and tether the glomerular basement membrane(GBM).The turnover of the mature GBM and the ability of adult podocytes to repair injured GBM are unclear.The actin cytoskeleton is a major cytoplasmic component of podocyte foot processes and links the cell to the GBM.Predominant components of the normal glomerular extracellular matrix(ECM)include glycosaminoglycans,proteoglycans,laminins,fibronectin-1,and several types of collagen.In patients with diabetes,multiorgan composition of extracellular tissues is anomalous,including the kidney,so that the constitution and arrangement of glomerular ECM is profoundly altered.In patients with diabetic kidney disease(DKD),the global quantity of glomerular ECM is increased.The level of sulfated proteoglycans is reduced while hyaluronic acid is augmented,compared to control subjects.The concentration of mesangial fibronectin-1 varies depending on the stage of DKD.Mesangial type III collagen is abundant in patients with DKD,unlike normal kidneys.The amount of type V and type VI collagens is higher in DKD and increases with the progression of the disease.The GBM contains lower amount of type IV collagen in DKD compared to normal tissue.Further,genetic variants in theα3 chain of type IV collagen may modulate susceptibility to DKD and end-stage kidney disease.Human cellular models of glomerular cells,analyses of human glomerular proteome,and improved microscopy procedures have been developed to investigate the molecular composition and organization of the human glomerular ECM.

BIOCHEMICAL STUDIES ON GLUCOAMINOGLYCANS CONTENT IN AFFECTED CARTILAGE HYALURONIC ACID LEVELS IN SERUM AND SYNOVIAL FLUID OF IDIOPATHIC FEMORAL HEAD NECROSIS

To investigate the role of cartilage alterations in the pathogenesis of the idiopathic femoral head necrosis, 3 components of glycosaminoglycans(GAG) in pathological and health cartilage, levels of hyaluronic acid(HA) in synovial fluid and serum were detected. The correlations among HA level in synovial fluid, serum and GAG content in cartilage were analyzed. The results showed that ①the three components of cartilage GAG in normal adults and children showed little difference; ②Compared with the normal autopsy specimens, steroid induced ANFH and Perthes disease specimens had significantly decreased GAG contents (P<0 025). In the former ones, sulfate radical content had no obvious reduction in Ficat Ⅱ (P>0 05), but it decreased significantly (P<0 05) in Ficat Ⅲ, while hexosamine decreased sharply in Ficat Ⅱ and had no marked alternation in Ficat Ⅲ. And hexuronic acid fell sharply during Ficat Ⅱ and Ficat Ⅲ (P<0 05). Serum level of HA did not have obvious changes until Ficat Ⅲ. HA level in synovial fluid decreased extremely in Ficat Ⅲ in contrast to Ficat Ⅱ and normal controls (P<0 05). Moreover, there was no correlation between levels of HA in serum and in synovial fluid (P>0 05); ③Children suffered from Perthes disease had a much higher level of HA in serum than the controls (P<0 01). HA level in serum led an inverse correlation with that in synovial fluid. (r=-0 663,P<0 05);④There was a positive correlation between HA level in synovial fluid and GAG content in both steroid induced ANFH and Perthes disease. GAG content was positive correlated with HA level in serum of Perthes disease, whereas, there was no correlation between them in steroid induced ANFH. The results suggest that:①GAG content in affected hip decreased significantly in the early stage. It provides clinical theory basis for auxiliary treatment to cartilage in early stage; ②Sulfate radical depletion is the most conspicuous one of GAG changes in middle and late stage. It suggests that the sulfate radical glycoaminoglycans should be replenished in these stages; ③Measurement of the serum HA level is of no value in early diagnosis of steroid induced ANFH. To some extent serum levels of HA reflect biochemical changes in cartilage in Perthes disease.

Reliability of fourier transform infrared spectroscopy in the characterization of human skin

Fourier transform infrared (FT-IR) spectroscopy, an organic molecule characterizing tool, is used here to differentiate young (36 ? 2.87 years) and aged (78 ? 1.25 years) skins, based on glycosaminoglycan (GAG) and protein functional groups. Female breast mas-tectomy-skin, FT-IR spectroscopy revealed intensity differences that were quantified on GAG and protein standard curves, and assigned to the corresponding functional groups. Band intensity reductions at 78- years include: 34.37% (w/w) ?1259 - 1223 cm–1, sulfate (SO42–)/sulfonate (SO3–) S=O/phosphate (PO42?) P=O stretch?;32.00% (w/w) (1383-1262 cm-1, GAG- methyl C-H/C-C-H);and 35.60% (w/w) ?1738 - 1646 cm–1, C=O stretch: N-acetylated GAG’s, Amide I, and others?. Intensity increments at 78-years are 63.32% (w/w) (1636 - 1523 cm–1, Phe/Trp/Tyr-C=C, Amide II);27.02% (w/w) [1511 - 1457 cm–1, protein ?(CH2)/ ?(CH3) stretch];and 41.90% (w/w) (1218 - 1139 cm–1, Phe/Trp/Tyr C-H/C-N/C-C6H5 vibrations). The data speak to the power of FT-IR spectroscopy as a non-invasive tool to diagnose tissue disorders such as skin, liver, kidney or any other type that would require a noninvasive tool like FT-IR, to prevent further dam-age during the diagnosis. These results also demon-strate an age-mediated decrease of skin-GAG content, and GAG-N-acetylation, in addition to protein com-position concentration increments.

糖胺聚糖降解菌株的筛选及鉴定

以土壤为材料,用透明质酸和硫酸软骨素为唯一碳源富集分离菌株,通过BSA-乙酸平板显色法及比色定糖法进行筛选。从80份土壤中筛选出13株糖胺聚糖降解活性的菌株并对其进行了16S rDNA测序鉴定。结果表明,筛选到13株糖胺聚糖降解菌株均具有透明质酸酶和硫酸软骨素酶活性;获得8株尚未报导过的产糖胺聚糖降解酶活性菌株。本研究为开发新型的糖胺聚糖降解酶提供参考。

Evaluation of Glycosaminoglycan in the Lumbar Disc Using Chemical Exchange Saturation Transfer MR at 3.0 Tesla:Reproducibility and Correlation with Disc Degeneration

Objective This study aims to explore the clinical applicability and relevance of giycosaminoglycan Chemical Exchange Saturation Transfer （gagCEST） for intervertebral disc. Methods 25 subjects ranging in age from 24 yrs to 74 yrs were enrolled, gagCEST was acquired using a single-slice TSE sequence on a 3T. Saturation used a continuous rectangular RF pulse with B1=0.8 I^T and a fixed duration time =1100 ms. Sagittal image was obtained firstly without saturation pulse, and then saturated images were acquired at 52 offsets ranging from ＋0.i25 to ＋_7 parts per million （ppm）. MR T2 relaxivity map was acquired at the identical location. Six subjects were scanned twice to assess scan-rescan reproducibility. Results GagCEST intraclass correlation coefficient （ICC） of six subjects was 0.759 for nucleus pulposus （NP） and 0.508 for annulus fibrosus （AF）. Bland-Altman plots showed NP had a mean difference of 0.10% （95% limits of agreement： -3.02% to 3.22%）; while that of AF was 0.34% （95% limits of agreement： -2.28% to 2.95%）. For the 25 subjects, gag CEST in NP decreased as disc degeneration increased, with a similar trend to T2 relaxivity. Gag CEST of AF showed a better correlation with disc degeneration than T2 relaxivity. Conclusion GagCEST in NP and AF decreased as disc degeneration increased, while gagCEST in AF showed a better correlation than T2 relaxivity.

Proteoglycans and their functions in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma(ESCC)is a highly malignant disease that has a poor prognosis.Its high lethality is mainly due to the lack of symptoms at early stages,which culminates in diagnosis at a late stage when the tumor has already metastasized.Unfortunately,the common cancer biomarkers have low sensitivity and specificity in esophageal cancer.Therefore,a better understanding of the molecular mechanisms underlying ESCC progression is needed to identify novel diagnostic markers and therapeutic targets for intervention.The invasion of cancer cells into the surrounding tissue is a crucial step for metastasis.During metastasis,tumor cells can interact with extracellular components and secrete proteolytic enzymes to remodel the surrounding tumor microenvironment.Proteoglycans are one of the major components of extracellular matrix.They are involved in multiple processes of cancer cell invasion and metastasis by interacting with soluble bioactive molecules,surrounding matrix,cell surface receptors,and enzymes.Apart from having diverse functions in tumor cells and their surrounding microenvironment,proteoglycans also have diagnostic and prognostic significance in cancer patients.However,the functional significance and underlying mechanisms of proteoglycans in ESCC are not well understood.This review summarizes the proteoglycans that have been studied in ESCC in order to provide a comprehensive view of the role of proteoglycans in the progression of this cancer type.A long term goal would be to exploit these molecules to provide new strategies for therapeutic intervention.

Mucin-like glycopolymer gels in electrosensory tissues generate cues which direct electrolocation in amphibians and neuronal activation in mammals

Mucin-like glycoproteins have established roles in epithelial boundary protection and lubricative roles in some tissues.This mini-review illustrates alternative functional roles which rely on keratan sulphate and sialic acid modifications to mucin glycopolymers which convey charge properties suggestive of novel electroconductive properties not previously ascribed to these polymers.Many tumour cells express mucin-like glycopolymers modified with highly sulphated keratan sulphate and sialic which can be detected using diagnostic biosensors.The mucin-like keratan sulphate glycopolymer present in the ampullae of lorenzini is a remarkable sensory polymer which elasmobranch fish(sharks,rays,skate) use to detect weak electrical fields emitted through muscular activity of prey fish.Information on the proton gradients is conveyed to neuromast cells located at the base of the ampullae and mechanotransduced to neural networks.This ampullae keratan sulphate sensory gel is the most sensitive proton gradient detection polymer known in nature.This process is known as electrolocation,and allows the visualization of prey fish under conditions of low visibility.The bony fish have similar electroreceptors located along their lateral lines which consist of neuromast cells containing sensory hairs located within a cupula which contains a sensory gel polymer which detects distortions in fluid flow in channels within the lateral lines and signals are sent back to neural networks providing information on the environment around these fish.One species of dolphin,the Guiana dolphin,has electrosensory pits in its bill with similar roles to the ampullae but which have evolved from its vibrissal system.Only two terrestrial animals can undertake electrolocation,these are the Duck-billed platypus and long and short nosed Echidna.In this case the electrosensor is a highly evolved innervated mucous gland.The platypus has 40,000 electroreceptors around its bill through which it electrolocates food species.The platypus has poor eyesight,is a nocturnal feeder and closes its eyes,nostrils and ears when it hunts,so electrolocation is an essential sensory skill.Mammals also have sensory cells containing stereocilia which are important in audition in the organ of corti of the cochlea and in olfaction in the olfactory epithelium.The rods and cones of the retina also have an internal connecting cilium with roles in the transport of phototransduced chemical signals and activation of neurotransmitter release to the optic nerve.Mucin-like glycopolymer gels surround the stereocilia of these sensory hair cells but these are relatively poorly characterized however they deserve detailed characterization since they may have important functional attributes.

Enhancement of matrix metalloproteinases 2 and 9 accompanied with neurogenesis following collagen glycosaminoglycan matrix implantation after surgical brain injury

Surgical brain injury may result in irreversible neurological deficits. Our previous report showed that partial regeneration of a traumatic brain lesion is achieved by implantation of collagen glycosaminoglycan（CGM）. Matrix metalloproteinases（MMPs） may play an important role in neurogenesis but there is currently a lack of studies displaying the relationship between the stimulation of MMPs and neurogenesis after collagen glycosaminoglycan implantation following surgical brain trauma. The present study was carried out to further examine the expression of MMP2 and MMP9 after implantation of collagen glycosaminoglycan（CGM） following surgical brain trauma. Using the animal model of surgically induced brain lesion, we implanted CGM into the surgical trauma. Rats were thus divided into three groups：（1） sham operation group： craniotomy only;（2） lesion（L） group： craniotomy ＋ surgical trauma lesion;（3） lesion ＋ CGM（L ＋ CGM） group： CGM implanted following craniotomy and surgical trauma lesion. Cells positive for SOX2（marker of proliferating neural progenitor cells） and matrix metalloproteinases（MMP2 and MMP9） in the lesion boundary zone were assayed and analyzed by immunofluorescence and ELISA commercial kits, respectively. Our results demonstrated that following implantation of CGM after surgical brain trauma, significant increases in MMP2^＋/SOX2^＋ cells and MMP9^＋/SOX2^＋ cells were seen within the lesion boundary zone in the L ＋ CGM group. Tissue protein concentrations of MMP2 and MMP9 also increased after CGM scaffold implantation. These findings suggest that implantation of a CGM scaffold alone after surgical brain trauma can enhance the expression of MMP2 and MMP9 accompanied by neurogenesis.

Assessment of Glycosaminoglycan Content of Lumbar Intervertebral Discs in Patients with Radiculopathy

Objective: To assess glycosaminoglycan (GAG) content of lumbar intervertebral discs (IVDs) in patients with radiculopathy compared with healthy volunteers with glycosaminoglycan chemical exchange saturation transfer (gagCEST). Methods: The lumbar spines of 15 patients with radiculopathy (9 women, 6 men;mean age 45 years;range: 19 - 80 years) and 13 healthy controls (10 women, 3 men;mean age 29 years;range: 19 - 38 years) without lumbar back pain or previous spine surgery were examined at a 3 Tesla (T) magnetic resonance imaging (MRI) scanner in this prospective study. The MRI protocol included standard morphological, sagittal, and transverse T2-weighted (T2w) images of the five lumbar IVDs (L1-S1) to assess Pfirrmann score and to detect disc disorders according to the Combined Task Force classification. To analyze biochemically the lumbar IVDs, a gagCEST sequence was applied to measure the GAG content of the nucleus pulposus (NP) and annulus fibrosus (AF). Results: Patients with radiculopathy indicated significantly lower gagCEST values in NP than healthy volunteers (2.82% &plusmn;3.12% vs. 4.09% &plusmn;2.25%, P = 0.017). The GAG content of AF showed no significant difference between volunteers and patients (2.66% &plusmn;2.01% vs. 1.92% &plusmn;2.56%;P = 0.175). Conclusions. Patients with radiculopathy presented with lower GAG values than healthy volunteers in NP, indicating an association between pain and IVD degeneration. gagCEST of lumbar IVDs is a powerful, non-invasive tool to investigate early disc degeneration, which we could demonstrate in the NP in our study collective.

The effect of reduced glutathione on the chondrogenesis of human umbilical cord mesenchymal stem cells

It has been discussed whether reduced glutathione (GSH) could promote the chondrogenic differentiation ability of human umbilical cord mesenchymal stem cells (hUC-MSCs). hUC-MSCs were isolated from human umbilical cord and their specificity was identified, then induced into cartilage-like cells in chondrogenic induction medium with transforming growth factor beta 1 (TGF-β1), especially with GSH. The morphological change before and after induction was observed through inverted phase contrast microscope, Type II collagen (COL2-A1) and glycosaminoglycan (GAG) were analyzed qualitatively by Toluidine blue and immunofluorescence technique, respectively, the contents of COL2-A1 and GAG were estimated from the determination of hydroxyproline content and Alcian Blue method separately. The mRNA expressions of GAG and COL2-A1 were assayed by real-time fluorescence quantitative PCR. After continuously cultured for 21 days with GSH, Toluidine blue staining and immunofluorescence reaction were all positive in basic induction medium group (group B), basic induction medium +0.5% dimethylsulfoxide (DMSO) group (group BD) and basic induction medium +0.5% DMSO +500 μM GSH group (group BDG). Moreover, compared with group B and group BD, the contents of COL2-A1 and GAG in group BDG relatively increased and the mRNA expression level of COL2-A1 and GAG also comparatively increased (P < 0.05) and both had a significant statistical significance (P < 0.05). So GSH might promote the induction of hUC-MSCs to differentiate into cartilage-like cells.

The chemokines CXCL8 and CXCL12:molecular and functional properties,role in disease and efforts towards pharmacological intervention

Chemokines are an indispensable component of our immune system through the regulation of directional migration and activation of leukocytes.CxCL8 is the most potent human neutrophil-attracting chemokine and plays crucial roles in the response to infection and tissue injury.CXCL8 activity inherently depends on interaction with the human CXC chemokine receptors CXCR1 and CXCR2,the atypical chemokine receptor ACKR1,and glycosaminoglycans.Furthermore,(hetero)dimerization and tight regulation of transcription and translation,as well as post-translational modifications further fine-tune the spatial and temporal activity of CXCL8 in the context of inflammatory diseases and cancer.The CxCL8 interaction with receptors and glycosaminoglycans is therefore a promising target for therapy,as illustrated by multiple ongoing clinical trials.CXCL8-mediated neutrophil mobilization to blood is directly opposed by CXCL12,which retains leukocytes in bone marrow.CXCL12 is primarily a homeostatic chemokine that induces migration and activation of hematopoietic progenitor cells,endothelial cells,and several leukocytes through interaction with CXCR4,ACKR1,and ACKR3.Thereby,it is an essential player in the regulation of embryogenesis,hematopoiesis,and angiogenesis.However,CXCL12 can also exert inflammatory functions,as illustrated by its pivotal role in a growing list of pathologies and its synergy with CXCL8 and other chemokines to induce leukocyte chemotaxis.Here,we review the plethora of information on the CXCL8 structure,interaction with receptors and glycosaminoglycans,different levels of activity regulation,role in homeostasis and disease,and therapeutic prospects.Finally,we discuss recent research on CXCL12 biochemistry and biology and its role in pathology and pharmacology.

Extraction and quantification of sulfated glycosaminoglycan content in five different aquatic species of Malaysia

Objective:To extract,characterize and quantify glycosaminoglycans(GAGs)from the body of cuttlefish,tennis-ball sea cucumber,shrimp,seabass and fresh water fish Nile tilapia.Methods:The extracted crude powder was evaluated for the content of GAGs.The qualitative analysis of sulfated pattern and other important functional groups related with GAGs were explained in the form of Fourier transform infra-red spectroscopy data.Proteins and nucleic acid in the crude extract were determined by the ultraviolet spectrophotometer,while the quantification of total sulfated GAGs and estimation of N-sulfated and O-sulfated GAGs in the crude mixture were performed by using Blyscan kit.Results:The sulfated pattern and other important functional groups related with GAGs were intercepted in Fourier transform infrared analysis.Blyscan quantification method reported that a rare variety of sea cucumber(tennis-ball sea cucumber)emerged as a rich source of GAGs with high values of both N-sulfated and O-sulfated GAGs in comparison to its other counterparts.Conclusions:Findings in this study point out the potential of tennis-ball sea cucumber,a rare variety of sea cucumber to act as an alternative source for GAG extraction for commercial purpose.

Genetic Analysis of 17 Children with Hunter Syndrome:Identification and Functional Characterization of Four Novel Mutations in the Iduronate-2-Sulfatase Gene

Mucopolysaccharidosis type II （MPS II） is a rare X-linked disorder caused by alterations in the iduronate-2-sulfatase （IDS） gene. In this study, IDS activity in peripheral mononuclear blood monocytes （PMBCs） was measured with a fluorimetric enzyme assay. Urinary glycosaminoglycans （GAGs） were quantified using a colorimetric assay. All IDS exons and intronic flanks were bidirectionally sequenced. A total of 15 mutations （all exonic region） were found in 17 MPS II patients. In this cohort of MPS II patients, all alterations in the IDS gene were caused by point nucleotide substitutions or small deletions. Mutations p.Arg88His and p.Arg172＊ occurred twice. All mu- tations were inherited except for p.Gly489Alafs＊7, a germline mutation. We found four new mutations （p.Ser142Phe, p.Arg233Gly, p.Glu430＊, and p.Ile360Tyrfs＊31）. In Epstein-Barr virus （EBV）-immortalized PMBCs derived from the MPS II patients, no IDS protein was detected in case of the p.Ser142Phe and p.Ile360Tyrfs＊31 mutants. For p.Arg233Gly and p.Glu430＊, we observed a residual expression of IDS. The p.Arg233Gly and p.Glu430＊ mutants had a residuary enzymatic activity that was lowered by 14.3 and 76-fold, respectively, compared with healthy controls. This observation may help explain the mild disease phenotype in MPS II patients who had these two mutations whereas the p.Ser142Phe and p.Ile360Tyrfs＊31 mutations caused the severe disease manifestation.