Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.     

Automated measurement of serum ferroxidase activity

A method is described for automated measurement of serum ceruloplasmin ferroxidase activity. In this method, Fe2+ ions are used as the substrate. In addition, a new calibration system without ceruloplasmin is also presented. Optimum assay reaction conditions were determined. Maximal catalytic activity was obtained at 0.45 mol/L acetate buffer, pH 5.8. The reagents and calibrator are stable for at least 6 months. Significant correlations between serum ferroxidase and p-phenylenediamine oxidase activities (r = 0.96; P <0.0001) and copper concentration (r = 0.93; P <0.0001) were found. The range for serum ceruloplasmin ferroxidase activity in healthy persons was 198-1107 U/L, and in patients with bronchial asthma it was 601-1912 U/L.     

Cellular immunity to the G1 domain of cartilage proteoglycan aggrecan is enhanced in patients with rheumatoid arthritis but only after removal of keratan sulfate

OBJECTIVE: To determine whether patients with rheumatoid arthritis (RA) express cellular immunity to the purified G1 globular domain of cartilage proteoglycan (PG) aggrecan and whether it is influenced by the removal of keratan sulfate (KS) chains from the molecule. METHODS: The G1 globular domain of PG was purified from mature bovine articular cartilage, digested with keratanase, and used in proliferation assays with peripheral blood lymphocytes (PBL) isolated from 43 patients with RA, 11 patients with nonarticular rheumatism (NAR), including soft tissue rheumatism and mechanical back pain, and 13 healthy age- and sex-matched control subjects. RESULTS: Removal of KS chains from the G1 globular domain resulted in significantly increased prevalence and values of cellular immune responses to G1 in RA patients compared with the control and NAR groups. In the majority of RA patients, KS chains on G1 significantly inhibited its immune recognition by PBL. There was no significant effect of KS removal on the immunity to G1 in patients with NAR and in the healthy control group. CONCLUSION: These results reveal that immune reactivity to the G1 globular domain of the cartilage PG aggrecan is enhanced in patients with RA but only when KS chains are removed. Thus, KS chains inhibit immune responses to this domain of aggrecan. Since immunity to the G1 globular domain of aggrecan induces an erosive polyarthritis in BALB/c mice after removal of KS chains, immunity to the G1 globular domain, cleaved by proteases to remove KS chains, may play a role in the pathogenesis of RA.     

13C-NMR spectroscopy of keratan sulphates--assignments for five sialylated pentasaccharides derived from the non-reducing chain termini of bovine articular cartilage keratan sulphate by keratanase II digestion

The effects of dexfenfluramine on the release of brain dopamine and serotonin into striatal dialysates were measured in freely moving rats. Samples collected every 20 min were assayed for dopamine and serotonin by high-performance liquid chromatography in a single run. The administration of a lower anorectic dose of dexfenfluramine (0.5 or 1.0 mg/kg intraperitoneally) significantly increased dialysate serotonin concentrations without affecting those of dopamine. A higher dexfenfluramine dose (2.5 mg/kg intraperitoneally) increased both serotonin and dopamine. The increase in dopamine could be blocked by administering the mixed-acting serotonin antagonist methiothepin (20 microM), and was reproduced by applying serotonin (3-10 microM) directly to striatal neurons. Tetrodotoxin (1 microM) added to the striatal perfusates decreased the basal release of dopamine and serotonin; it also blocked the effect of dexfenfluramine (2.5 mg/kg intraperitoneally) on dopamine release and decreased the increment in serotonin release (by approximately 70%). Amphetamine (1 mg/kg subcutaneously) or phentermine (2 mg/kg intraperitoneally) increased dialysate dopamine concentrations without affecting those of serotonin, and tetrodotoxin (1 microM) failed to block the response to amphetamine. These findings suggest that (1) lower anorectic doses of dexfenfluramine release serotonin but not dopamine, and (2) higher doses of dexfenfluramine also increase dopamine release by an indirect mechanism mediated via serotonin.     

Study of serum 3,5,3''-triiodothyronine sulfate concentration in patients with systemic non-thyroidal illness

The thyroid doses received by the juvenile population of Belarus following the Chernobyl accident ranged up to about 10 Gy. The thyroid cancer risk estimate recommended in NCRP Report No. 80 was used to predict the number of thyroid cancer cases among children during 1990-1992 in selected Belarussian regions and cities. The results obtained using this risk estimate show an excess of thyroid cancer cases being registered vs. the predicted cases. Thyroid cancer incidence rate among boys under investigation is higher than among girls in the postaccident period. The excess of the observed over the expected incidence in the general juvenile population is caused by the high thyroid cancer incidence rate among boys. These results, which can be considered part of the first stage of a thorough thyroid cancer risk estimation after the Chernobyl accident, demonstrate the critical need to complete these studies in depth.     

The structure of the keratan sulphate chains attached to fibromodulin from human articular cartilage

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase-2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial-to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4-MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen-induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen-stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.     

The structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage

Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion-exchange chromatography. Their structures have been determined by high-field 1H-NMR spectroscopy and high-pH anion-exchange chromatography. Both alpha(2-6)- and alpha(2-3)-linked N-acetylneuraminic acid have been found in the capping oligosaccharides, and, fucose which is alpha(1-3)-linked to N-acetylglucosamine has been found as a branch in both repeat region and capping oligosaccharides. These data demonstrate that there are fundamental differences between the structures present in the N-linked keratan sulphate chains attached to fibromodulin from articular cartilage and those from tracheal cartilage, which lack both alpha(2-6)-linked N-acetylneuraminic acid and alpha(1-3)-linked fucose. It has been confirmed that the keratan sulphate chains are short, being only eight or nine disaccharides in length. Very significant differences in the levels of galactose sulphation have been identified at the non-reducing end of the chain. The galactose residue adjacent to the non-reducing cap is sulphated in only 1-3% of chains, compared with a sulphation level of over 40% closer to the reducing end. This highlights the difference between the chain termini and the repeat region in terms of structure and points to the potential for functional importance. The repeat region and capping fragments of the N-linked keratan sulphates from bovine and equine articular cartilage fibromodulin have been found to have the following general structure: NeuAc-(alpha 2-3/6)Gal[6SO3-](beta 1-4)GlcNAc6SO3-(beta 1-3)Gal[6SO3-] (beta 1-4)?[Fuc(alpha 1-3)]0-1GlcNAc6SO3-(beta 1-3)Gal-[6SO3-](beta 1-4)? 6-7GlcNAc6SO3-.     

Prediction of serum IgG1 concentration in Holstein calves using serum gamma glutamyltransferase activity

We examined the relationship between serum gamma glutamyltransferase (GGT) activity and serum gamma immunoglobulin G (IgG1) concentration in Holstein calves. Blood samples were collected from calves aged 1 to 3 days. A follow-up sample was obtained from each calf 2, 7, or 15 days after the initial sampling. Serum GGT activity and IgG1 concentration were measured. Regression models were used to predict IgG1 concentration as a function of age and serum GGT activity measured 2, 7, or 15 days later. Serum GGT activity and calf age at the time of the second sample were directly related to serum IgG1 concentration in the initial sample in calves aged 3 to 17 days (r = .54) and in calves aged 3 to 10 days (r = .63). Models were used to estimate the serum GGT activity equivalent to a serum IgG1 concentration of 1,000 mg/dL. One-day-old calves should have serum GGT activities > 200 IU/L. Four-day-old calves should have serum GGT activities > 100 IU/L. One-week-old calves should have serum GGT activities > 75 IU/L. Calves with serum GGT activities < 50 IU/L should be classified as having failure of passive transfer.     

Evaluation of serum apolipoprotein C-III concentration by enzyme-linked immunosorbent assay and its higher concentration in cows during midlactation than during the nonlactating stage

OBJECTIVE: To determine serum apolipoprotein C-III (apoC-III) concentration in cows in various stages of lactation by use of an ELISA. SAMPLE POPULATION: Sera obtained from 29 Holstein cows during early lactation, 65 cows during midlactation, 42 cows during late lactation, and 23 cows during the nonlactating stage. PROCEDURE: A 7.3-kd bovine apoC-III antiserum raised in rabbits was purified by affinity chromatography, and an ELISA was developed. RESULTS: In the immunoblot analysis, the antiserum reacted with the 7.3-kd apoC-III and moreover with another 8.2-kd apoC-III isoform. The 2 isoforms of apoC-III were also indistinguishable in the developed ELISA, the 2 proteins being measured as total apoC-III. In the ELISA for serum apoC-III concentration, addition of 2-mercaptoethanol to the coating buffer (50 mM sodium carbonate buffer, pH 9.6) was required. Mean+/-SEM bovine serum apoC-III concentration (microg/ml of serum) was 71.6+/-12.1 for the early lactating stage, 115+/-14.0 for the midlactating stage, 104+/-18.8 for the late lactating stage, and 55.3+/-8.4 for the nonlactating stage. Concentration of apoC-III was significantly (P < 0.05) higher in cows during midlactation than in cows during the nonlactating stage and was correlated negatively with serum triglyceride concentration (r = -0.479; P < 0.01) and positively with total cholesterol (r = 0.421; P< 0.05) and phospholipids (r= 0.415; P< 0.05) concentrations. CONCLUSIONS: Changes of apoC-III concentration in various stages of lactation suggest that this apolipoprotein is involved in a function related to lactation.     

Uronic acid-containing glycosaminoglycans and keratan sulfate are present in the tectorial membrane of the inner ear: functional implications

The use of growth hormone (GH) as an anabolic agent is limited by its tendency to cause hyperglycemia and by its inability to reverse nitrogen wasting in some catabolic conditions. In a previous study comparing the anabolic actions of GH and IGF-I (insulin-like growth factor I), we observed that intravenous infusions of IGF-I (12 micrograms/kg ideal body wt [IBW]/h) attenuated nitrogen wasting to a degree comparable to GH given subcutaneously at a standard dose of 0.05 mg/kg IBW per d. IGF-I, however, had a tendency to cause hypoglycemia. In the present study, we treated seven calorically restricted (20 kcal/kg IBW per d) normal volunteers with a combination of GH and IGF-I (using the same doses as in the previous study) and compared its effects on anabolism and carbohydrate metabolism to treatment with IGF-I alone. The GH/IGF-I combination caused significantly greater nitrogen retention (262 +/- 43 mmol/d, mean +/- SD) compared to IGF-I alone (108 +/- 29 mmol/d; P < 0.001). GH/IGF-I treatment resulted in substantial urinary potassium conservation (34 +/- 3 mmol/d, mean +/- SE; P < 0.001), suggesting that most protein accretion occurred in muscle and connective tissue. GH attenuated the hypoglycemia induced by IGF-I as indicated by fewer hypoglycemic episodes and higher capillary blood glucose concentrations on GH/IGF-I (4.3 +/- 1.0 mmol/liter, mean +/- SD) compared to IGF-I alone (3.8 +/- 0.8 mmol/liter; P < 0.001). IGF-I caused a marked decline in C-peptide (1,165 +/- 341 pmol/liter; mean +/- SD) compared to the GH/IGF-I combination (2,280 +/- 612 pmol/liter; P < 0.001), suggesting maintenance of normal carbohydrate metabolism with the latter regimen. GH/IGF-I produced higher serum IGF-I concentrations (1,854 +/- 708 micrograms/liter; mean +/- SD) compared to IGF-I only treatment (1,092 +/- 503 micrograms/liter; P < 0.001). This observation was associated with increased concentrations of IGF binding protein 3 and acid-labile subunit on GH/IGF-I treatment and decreased concentrations on IGF-I alone. These results suggest that the combination of GH and IGF-I treatment is substantially more anabolic than either IGF-I or GH alone. GH/IGF-I treatment also attenuates the hypoglycemia caused by IGF-I alone. GH/IGF-I treatment could have important applications in diseases associated with catabolism.     

Molecular cloning and sequence analysis of the aggrecan interglobular domain from porcine, equine, bovine and ovine cartilage: comparison of proteinase-susceptible regions and sites of keratan sulfate substitution

Oligonucleotide primers which were designed based on identical peptide sequences flanking the interglobular domain (IGD) of human, bovine and rat aggrecan were used in RT-PCR reactions containing human, porcine, equine, bovine and ovine cartilage RNA. Novel cDNAs encoding the IGD of the latter four species were obtained and sequenced. The deduced amino acid sequences for these cDNAs were aligned and compared with those described for six other species. Amino acid sequences surrounding the major proteolytic cleavage sites in the IGD are highly conserved, with some species-specific substitutions. Similarly, known sites of keratan sulfate attachment in the IGD are highly conserved in all species. The results provide essential amino acid sequence data for species commonly used in model systems of cartilage degeneration.     

A chondroitin sulfate/keratan sulfate proteoglycan, PG-1000, forms complexes which are concentrated in the reticular laminae of electric organ basement membranes

Previously, we identified PG-1000 as part of a disulfide-linked complex of two large proteoglycans (PG-1000 and the beta component) and three smaller proteins purified from the extracellular matrix of elasmobranch electric organ (Iwata and Carlson, 1991, J. Biol. Chem. 266: 323-333). PG-1000 is a chondroitin sulfate/keratan sulfate proteoglycan with a molecular mass of about 1.2 x 16(6) daltons. When visualized in the electron microscope, PG-1000 has the typical "bottle-brush" appearance expected for a proteoglycan with an average total length of about 345 nm and about 20 chains of approximately 110 nm (Carlson and Wight, 1987, J. Cell Biol. 105: 3075-3086). Using immunocytochemical methods, we now demonstrate that PG-1000 is a component of the interstitial extracellular matrix of the electric organ. PG-1000 immunoreactivity is found throughout the interstitial matrix, but it is highly concentrated in that region of the matrix immediately adjacent to the basal lamina, the reticular lamina. The reticular and basal laminae together form the basement membrane. PG-1000 immunoreactivity is especially apparent on basal laminae that surround nerve fibers and nerve terminals. When the disulfide-linked PG-1000 complexes are purified and examined in the electron microscope following rotary shadowing, they appear as bottle-brush structures which are often attached at a central region and radiate like spokes of a wheel. These aggregates contain two to six proteoglycan monomers. We hypothesize that the PG-1000 complexes are disulfide-stabilized parts of an extended network of linked proteoglycans in the reticular lamina.     

Osteoadherin, a cell-binding keratan sulfate proteoglycan in bone, belongs to the family of leucine-rich repeat proteins of the extracellular matrix

Osteoadherin is a recently described bone proteoglycan containing keratan sulfate. It promotes integrin (alphav beta3)-mediated cell binding (Wendel, M., Sommarin, Y., and Heinegard, D. (1998) J. Cell Biol. 141, 839-847). The primary structure of bovine osteoadherin has now been determined by nucleotide sequencing of a cDNA clone from a primary bovine osteoblast expression library. The entire translated primary sequence corresponds to a 49,116-Da protein with a calculated isoelectric point for the mature protein of 5.2. The dominating feature is a central region consisting of 11 B-type, leucine-rich repeats ranging in length from 20 to 30 residues. The full, primary sequence contains four putative sites for tyrosine sulfation, three of which are at the N-terminal end of the molecule. There are six potential sites for N-linked glycosylation present. Osteoadherin shows highest sequence identity, 42%, to bovine keratocan and 37-38% identity to bovine fibromodulin, lumican, and human PRELP. Unique to osteoadherin is the presence of a large and very acidic C-terminal domain. The distribution of cysteine residues resembles that of other leucine-rich repeat proteins except for two centrally located cysteines. Northern blot analysis of RNA samples from various bovine tissues showed a 4.5-kilobase pair message for osteoadherin to be expressed in bone only. Osteoadherin mRNA was detected by in situ hybridization in mature osteoblasts located superficially on trabecular bone.     

火焰原子吸收分光光度法测定矿石样品中银的不确定度评定

文中对火焰原子吸收分光光度法测定矿石样品中银的不确定度进行了评定，测量结果的不确定度由称样质量、标准工作溶液、工作曲线拟合、试液定容体积等引入的不确定度分量组成。在对各个不确定度分量进行量化的基础上，通过合成得到测量结果的标准不确定度，并确定影响银含量测量不确定度的主要分量是工作曲线拟合引入的不确定度。     

Evaluation of an immunoseparation method for quantitative measurement of remnant-like particle-cholesterol in serum and plasma

The structure of a nonspecific lipid transfer protein from barley (ns-LTPbarley) in complex with palmitate has been determined by NMR spectroscopy. The structure has been compared to the structure of ns-LTPbarley in the absence of palmitate, to the structure of ns-LTPbarley in complex with palmitoyl coenzyme A, to the structure of ns-LTPmaize in its free form, and to the maize protein complexed with palmitate. Binding of palmitate only affects the structure of ns-LTPbarley moderately in contrast to the binding of palmitoyl coenzyme A, which leads to a considerable expansion of the protein. The modes of binding palmitate to the maize and barley protein are different. Although in neither case there are major conformational changes in the protein, the orientation of the palmitate in the two proteins is exactly opposite.