Sialic acid determinations of serum and urine specimens in calcium-containing urinary stone formers

PURPOSE: The aim of the present study was to compare sialic acid concentrations of serum and urine specimens in both calcium (Ca)-containing urinary stone formers and non-stone formers. Moreover, we studied inhibitory activity of sialic acid upon the calcium oxalate (CaOx) crystal aggregation and growth. MATERIALS AND METHODS: Sialic acid determinations were done on fresh serum and urine samples of 35 Ca-containing urinary stone formers (stone formers group) and 20 non-stone formers (patient controls group). Inhibitory activity of sialic acid upon the CaOx crystal aggregation and growth was studied by using in vitro assay method of seed crystal system. RESULTS: Serum sialic acid concentrations were found to be similar in the two groups. Urinary sialic acid concentrations were significantly lower in the urine specimens of stone formers than in their patient controls. Sialic acid showed a dose dependent inhibitory activity upon the CaOx crystal aggregation and growth into seed crystal method. CONCLUSION: It is suggested that urinary sialic acid may play some role during the phase of stone formation from the results of the present study, because sialic acid shows marked inhibitory activity upon the CaOx crystal aggregation and growth at concentrations higher than 100 mg/dl.     

Hyperuricosuria and urolithiasis

Hyperuricosuria with or without hypercalciuria amounted to about 23% of the possible cause of urolithiasis in my clinical experience. Approximately three forth of urolithiasis caused by hyperuricosuria was calcium oxalate stones and the rest was uric acid stones. Uric acid is one of the composition of urinary stones itself, but it has an activity of calcium oxalate stone formation. The hypotheses why the uric acid induced calcium oxalate stones were introduced. The preventive effect of allopurinol on the recurrent calcium oxalate stone formers was proved in our previous study which may revealed the urinary uric acid promoted calcium oxalate stone formation or masked the inhibitory activity of urinary macromolecular inhibitors. On the basis of above statement, I emphasize the importance of the treatment of hyperuricosuria in preventing urinary stones.     

A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Glycosaminoglycan binding properties of annexin IV, V, and VI

We have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679-7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space.     

Urinary calcium oxalate saturation in healthy infants and children

PURPOSE: A number of factors influence the development of renal calculi, the most essential of which is the supersaturation of urine with lithogenic substances. Calcium oxalate stones occur most frequently in adult and pediatric patients with urolithiasis. Therefore, we established normal age and sex related data for urinary calcium oxalate saturation in infancy and childhood to allow a more specific prediction of the risk of (recurrent) stone disease. MATERIALS AND METHODS: We collected 24-hour urine samples from 473 healthy infants and children without a history of renal stones. Urinary lithogenic and stone inhibitory substances were measured, and the urinary calcium oxalate saturation was calculated using a computer program. RESULTS: Mean urinary calcium oxalate saturation was always higher in boys than in girls, which was significant in infancy (5.22 versus 2.03, p < 0.05) and at ages 7 to 9 years (8.84 versus 5.47, p < 0.05). The saturation first increased (p < 0.05) until age 7 to 9 years in boys and girls, and remained at high levels at ages 10 to 12 years (7.03 versus 5.49, p < 0.05 compared to infancy). Calcium oxalate saturation then decreased until adolescence when values were comparable to those of infancy (5.29 versus 3.35). CONCLUSIONS: We recommend calculating urinary calcium oxalate saturation for diagnostic purposes as well as for therapy control. Normal age and sex related values must be considered.     

Cell surface proteoglycans are necessary for A27L protein-mediated cell fusion: identification of the N-terminal region of A27L protein as the glycosaminoglycan-binding domain

We previously showed that vaccinia virus infection of BSC40 cells was blocked by soluble heparin, suggesting that cell surface heparan sulfate mediates vaccinia virus binding (C.-S. Chung, J.-C. Hsiao, Y. -S. Chang, and W. Chang, J. Virol. 72:1577-1585, 1998). In this study, we extended our previous work and demonstrated that soluble A27L protein bound to heparan sulfate on cells and interfered with vaccinia virus infection at a postbinding step. In addition, we investigated the structure of A27L protein that provides for its binding to heparan sulfate on cells. A mutant of A27L protein, named D-A27L, devoid of a cluster of 12 amino acids rich in basic residues, was constructed. In contrast to the soluble A27L protein, purified D-A27L protein was inactive in all of our assays, including binding to heparin in vitro, binding to heparan sulfate on cells, and the ability to block virus infection. These data demonstrated that the N-terminal region acts as a glycosaminoglycan (GAG)-binding domain critical for A27L protein binding to cells. Previously A27L protein was thought to be involved in fusion of virus-infected cells induced by acid treatment. When we investigated whether cell surface GAGs also participate in A27L-dependent fusion, our results indicated that soluble A27L protein blocked cell fusion, whereas D-A27L protein did not. Taken together, the results therefore demonstrated that A27L-mediated cell fusion is triggered by its interaction with cell surface GAGs through the N-terminal domain.     

Uropontin in urinary calcium stone formation

Normal urine is frequently supersaturated with respect to calcium oxalate. Thus, urinary inhibitors of crystallization appear to have an important role in preventing urinary stone formation. Uropontin was isolated by monoclonal antibody immunoaffinity chromatography and has the same N-terminal sequence as osteopontin derived from bone. This urinary form of osteopontin is a potent inhibitor of calcium oxalate monohydrate crystal growth at concentrations (approximately 0.1 microM) that normally prevail in human urine. Interaction with calcium oxalate monohydrate in vivo was shown by analysis of EDTA extracts of calcium stones. Uropontin is an abundant component of calcium oxalate monohydrate stones and present in only trace quantities in calcium oxalate dihydrate and hydroxyapatite stones. However, the precise role of uropontin in the pathogenesis of urinary stone formation is not known and is the subject of ongoing investigations.     

Localization of glycosaminoglycans (GAGs) in pleomorphic adenoma (PA) of salivary glands: an immunohistochemical and histochemical evaluation

The tumor matrix of salivary pleomorphic adenoma (PA) is characteristically rich in glycosaminoglycans (GAGs), which contribute to its complex histoarchitecture. This study evaluated the microscopic localization of various GAGs in 17 PAs, using a panel of anti-GAG monoclonal antibodies and biotinylated hyaluronic acid (HA)-binding protein. Both epithelial and mesenchymal-like tissues were confirmed to contain GAGs. Luminal epithelial cells mostly lacked GAGs, whereas GAGs were seen both in the cytoplasm and cell membrane of non-luminal epithelial cells. In addition, small intercellular accumulations of GAGs were often present in solid epithelial areas, implying the epithelial origin of GAGs. GAGs did not appear to be a main component of the hyaline matrix. The myxoid region was consistently stained for both chondroitin 6-sulfate (CS-6) and HA but variably for chondroitin 4-sulfate (CS-4), dermatan sulfate (DS) and keratan sulfate (KS); heparan sulfate (HS) was not detected. The chondroid region showed increased staining for CS-6 but reduced staining for HA when compared with the myxoid region. In addition, CS-4, DS and KS were seen both in chondroid cells and the territorial matrix, whereas HS was present only in the cells. It is suggested that GAGs in PA are mainly produced by non-luminal cells and influence the proliferation, differentiation, secretory activity and shape of tumor cells, thus contributing to the morphological diversity of this tumor.     

Comparative acute toxicity and primary irritancy of the ethylidene and vinyl isomers of norbornene

Perlecan and aggrecan are proteoglycans that receive primarily heparan sulfate and chondroitin sulfate side chains, respectively. Their large multidomained core proteins have little or no homology to each other and their glycosaminoglycan (GAG) attachment sites are restricted to certain domains only. We examined the involvement of the non-GAG bearing domains in designating the GAG type added to the GAG attachment domain by preparing cDNA constructs that expressed perlecan/aggrecan chimeras as recombinant products in COS-7 cells and then determining the size and GAG composition of the recombinant products. The results showed that domain I of perlecan receives primarily (73-81%) heparan sulfate when coupled with domain II and III of perlecan, but when coupled with the G3 domain of aggrecan, it receives primarily (59-63%) chondroitin sulfate. Furthermore, the chondroitin sulfate attachment region of aggrecan received GAG side chains more readily when coupled to the G3 domain of aggrecan than when coupled to domains II and III of perlecan. The GAG side chains on all these recombinant products were small and similar in size. These findings indicate that the utilization of attachment sites for heparan and chondroitin sulfate or the sulfation of these GAGs can be influenced, in part, by non-GAG bearing domains.     

Zeta potential measurement and particle size analysis for a better understanding of urinary inhibitors of calcium oxalate crystallization

To better understand urinary inhibitors of calcium oxalate crystallization, both zeta potential measurement and particle size analysis were chosen to illustrate: (1) the potential therapeutic efficacy of G872, a semi-synthetic sulfated polysaccharide, in stone prevention; and (2) the relative contribution of various urinary fractions ?e.g., ultrafiltered urine (UFU), Tamm-Horsfall protein (THP), urinary polyanions precipitated with cetylpyridinium chloride (CPC), urinary macromolecular substances with different concentration ratios (UMS10,50,90 and UMS'10,50,90) and THP-free urine (THPFU)? to total urinary inhibitory activity. The results showed: (1) addition of G872 significantly enhances urinary inhibitory activity and negative zeta potential values; (2) re-addition of the CPC to UFU completely restores urinary inhibitory activity; and (3) artificial urines prepared by mixing UMS'10,50,90 from THPFU with UFU differed in inhibitory activity from that prepared by mixing UMS10,50,90 from a pooled normal urine with UFU. Based on these experimental results, the following speculations can be made: (1) normal human urines are considered to be a protective colloidal system; (2) urinary inhibitory activity originates mainly from CPC and/or UMS; (3) normal THP is a protective material to maintain urinary inhibitory activity; and (4) mutual interaction between urinary inhibitors may change the total urinary inhibitory activity.     

Overexpression of transforming growth factor-beta1 in scirrhous carcinoma of the stomach correlates with decreased survival

BACKGROUND: Several reports in the 1970s suggested that etidronate disodium might be clinically useful to prevent calcium stones, but the use of etidronate in the urolithiasis field was discontinued due to adverse effects of this drug on skeletal turnover and mineralization. Because the drug might affect not only crystallization, but also crystal-tubular interactions, we investigated the minimum dose of etidronate necessary to effectively prevent stone recurrence without adverse side effects. METHODS: We examined the effect of etidronate on the crystallization of calcium oxalate, calcium phosphate and magnesium ammonium phosphate using synthetic urine and measured by an aggregometer. We also studied its effect on the adhesion of calcium oxalate monohydrate crystals to Madin-Darby canine kidney (MDCK) cells in vitro. RESULTS: Etidronate affected the crystallization+ of not only calcium phosphate and calcium oxalate, but also magnesium ammonium phosphate in synthetic urine. The inhibitory activities on these crystallizations were detected at extremely low drug concentrations. Etidronate also had a strong inhibitory activity against the adhesion of calcium oxalate crystals to MDCK cells. CONCLUSION: Although further studies are necessary regarding the effects of etidronate on crystallization and crystal adhesion both in vivo and in vitro, and the appropriate schedule of dosing to prevent side effects, it is possible that etidronate may be useful in the treatment of urinary stones.     

Exogenous heparin induces an increase in glycosaminoglycans of the chick embryo chorioallantoic membrane: its possible role in the regulation of angiogenic processes

In this study heparin (HE) was injected into the allantoic sac of chick embryo eggs at 5, 9, 14 days of incubation. 48 h after injection glycosaminoglycan (GAG) concentration was measured in the chorioallantoic membrane (CAM) in order to verify if HE-related CAM angiogenic activity previously demonstrated [Ribatti et al: Acta Anat 1987; 130:257-263] might be correlated with changes in GAG concentration. The results showed that HE inoculation induced an increase of 3H-glucosamine incorporation into total GAGs in comparison to control CAMs. Furthermore, HE altered the balance between the GAG classes, and in particular it produced a significant increase in the accumulation of hyaluronic acid and heparan sulfate between 7 and 11 days of incubation in comparison to control CAMs.     

Direct in vivo gene transfer to canine myocardium using a replication-deficient adenovirus vector

Inter-alpha-inhibitor (I alpha I) is a serine protease inhibitor present in human plasma. It has a molecular weight of about 220 kDa which encompasses 3 chains including two heavy chains and one light chain. The light chain, known as bikunin, is responsible for the antitryptic activity of I alpha I in the inhibition of various enzymes, such as trypsin and chymotrypsin. Under physiologic or certain pathologic circumstances, several macromolecules related to I alpha I appear in plasma and urine. However, the physiologic role of I alpha I remains unclear. As far as urolithiasis is concerned, two urinary macromolecules related to I alpha I have been isolated and shown to be potent inhibitors of calcium oxalate formation. One of these inhibitors, uronic-acid-rich protein (UAP), has been identified and well characterized. The sequence of the first 18 amino acid residues of UAP is identical with that of bikunin. Furthermore, the immunoreaction between UAP and I alpha I antibody using immunoblot analysis was positive. UAP isolated from the urine of stone formers exhibited less inhibitory activity towards calcium oxalate crystallization than that derived from the urine of healthy subjects. This suggests a structural abnormality of the inhibitor obtained from stone patients. The organic matrix extracted from kidney stones contained a protein antigenically related to I alpha I. We conclude that UAP is a member of I alpha I family taking part in inhibiting calcium oxalate crystallization, and modulating the formation of stones in the urinary tract.     

Clinical resistance to topoisomerase-targeted drugs

The objectives of this study were to determine whether dietary manganese deficiency alters total glycosaminoglycan (GAG) concentration and composition and glycosyltransferase activity in rat aortas. Sprague-Dawley rats were fed either a manganese-deficient or a manganese-sufficient diet. Arterial GAGs were isolated and quantified by measuring uronic acid content. The individual GAGs were separated and quantified with cellulose acetate electrophoresis. The activity of the enzyme galactosyltransferase I was measured using a 100,000 g particulate fraction and 4-methylumbelliferylxyloside (Xyl-MU) as an acceptor. There was a significant decrease (p < or = 0.05) in uronic acid content in the manganese-deficient (1.18 +/- 0.08 mg/g) rat aortas as compared with the manganese-sufficient (1.59 +/- 0.10 mg/g) ones. Chondroitin sulfate and heparan sulfate concentrations were decreased by 38% (p < or = 0.01) and 36% (p < or = 0.05), respectively, in the manganese-deficient rat aortas. The incorporation of UDP-galactose to acceptors by the manganese-deficient rat aorta preparations was increased by 28% as compared to the manganese-sufficient preparations. These results indicate that manganese is involved in arterial GAG metabolism by affecting the enzyme galactosyltransferase and that changes in GAG concentration and composition with manganese deficiency may ultimately affect arterial wall integrity and subsequently cardiovascular health. This is the first work to demonstrate that manganese nutrition is important in arterial GAG metabolism.     

Activation of the autophagy, c-FOS and ubiquitin expression, and nucleolar alterations in Schwann cells precede demyelination in tellurium-induced neuropathy

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.     

Life circumstances, lifestyles and oral health among older Canadians

Nucleation, growth and aggregation are thought to be the most important crystallization processes in stone formation. Since crystallization properties change with urinary dilution, centrifugation and filtration, crystallization should always be studied in freshly voided and not pretreated urine. Recently we developed an automated method where calcium oxalate crystallization is induced in native urine by an exogenous oxalate load and nucleation and growth are monitored by an ion-selective calcium electrode. The method has now been supplemented with the spectrophotometric measurement of crystal aggregation. Repeated experiments in the same urine with different oxalate loads enable the determination of the critical oxalate additionable to induce crystallization (metastable limit) and the calculation of an oxalate load-independent growth rate constant. Preliminary results obtained in the native urine of healthy controls showed an extremely high limit of metastability and a complete absence of crystal aggregation. These findings may explain why, despite frequent urinary calcium oxalate supersaturation, healthy people do not form stones.     

Serum antibodies to heparan sulfate glycosaminoglycans in Guillain-Barré syndrome and other demyelinating polyneuropathies

We tested for serum antibodies to glycosaminoglycans (GAGs), including heparan sulfate, in patients with Guillain-Barré syndrome (GBS) and other disorders. We used ELISA methods that optimize immunoglobulin binding to carbohydrate antigens to measure serum antibodies to heparan sulfate GAGs in GBS, and control neuromuscular and immune disorders. We found serum IgM or IgG antibodies to heparan sulfate GAGs in 34% of patients with GBS. Serum IgM binding to heparan sulfate GAGs was also found in some chronic demyelinating polyneuropathies, with the highest frequency (33%) in patients with IgM anti-MAG M-proteins. Antibodies to heparan sulfate GAGs were rare (1%) in control serums from patients with other disorders. This result is the first demonstration of high titer serum antibodies to a specific antigen in a substantial group of, and with some specificity for, patients with the classically described GBS syndrome of acute-onset, motor-sensory polyneuropathy with demyelinating features.     

Morphological and biochemical features affecting the antithrombotic properties of the aorta in adult rabbits and rabbit pups

We hypothesised that there are important physiologic differences in arterial wall structure and function with respect to antithrombotic activity in the very young (pre-puberty) compared to adults. Electron microscopy, gel electrophoresis, and activity assays were used to examine differences in aorta structure and function comparing prepubertal rabbits (pups) to adult rabbits. Differences in endothelial function, extracellular matrix structure, proteoglycan (PG) distribution and glycosaminoglycan (GAG) content and function were shown. In both intima and media, total PG, chondroitin sulfate (CS) PG and heparan sulfate (HS) PG content were significantly increased in pups compared to adult rabbits. These findings corresponded to increased concentrations by mass analyses of CS GAG and DS GAG in aortas from pups. There was also a significant increase in antithrombin activity in pups due to HS GAG. In conclusion, differences in both structure and antithrombin activity of aortas from pups compared to adult rabbits suggest that young arteries may have greater antithrombotic potential that is, at least in part, related to increased HS GAG.     

Role of agglomeration in the early stages of papillar stone formation

Possible effects of crystal agglomeration on the early stages of calcium oxalate papillar stone formation are evaluated. The collecting ducts are filled with liquid that flows laminarly as established through hydrodynamical and physicochemical considerations. Under such conditions, agglomeration due to laminar shear forces proceeds. Agglomeration of calcium oxalate monohydrate crystals present in urine at a concentration typical for clinically observed crystalluria cannot result in the formation of a particle sufficiently large enough to be retained in the Bellini's duct and become a papillar stone nidus (nucleus). Formation of such an aggregate during the passage time of urine through the duct requires an unrealistically high concentration of crystals in urine, one that exceeds the normal content of urinary oxalate by several orders of magnitude. Aggregates obstructing the Bellini's duct as assumed in the free particle theory cannot represent a major factor in stone formation. This conclusion is corroborated by experimental results and other observations.     

Uric acid-related nephrolithiasis

Abnormalities in uric acid metabolism are associated with uric acid and calcium oxalate urolithiasis. Clinical stone formation depends on multiple identifiable risk factors that affect uric acid and calcium oxalate solubility. The understanding of urinary pH is critical to direct appropriate treatment of uric acid-related nephrolithiasis. Understanding uric acid metabolism and the pathophysiology of uric acid and calcium oxalate stone formation leads to a rational treatment approach to uric acid and hyperuricosuric calcium oxalate stone disease.