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Biomedical subjects

R A Bank

Publications and source records attributed to R A Bank.

51 records · Page 3Linked to original sources
PubMed1997

Sensitive fluorimetric quantitation of pyridinium and pentosidine crosslinks in biological samples in a single high-performance liquid chromatographic run.

A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pentosidine in mature collagen of a wide variety of connective tissue hydrolysates by a simple two-step isocratic assay using a reversed-phase column. The crosslinks (including the internal standard pyridoxine) were optimally detected by their native fluorescence by switching wavelengths of the detector during the assay. The method resulted in highly sensitive and accurate measurements, without need for precleaning of the samples: crosslink levels in 200 microm thin slices of the various zones of articular cartilage were easily quantified. The detection limit was as low as 0.4 pmol for the pyridinolines and 0.05 pmol for pentosidine. The intra-assay and inter-assay coefficients of variation were as low as 2% (pyridinolines) and 5% (pentosidine); calibration curves for all compounds were linear over a concentration range larger than two orders of magnitude. With our chromatographic system, the diglycosylated form of hydroxylysylpyridinoline in unhydrolyzed urine was separated as well.

Adolescent↗
PubMed1997

Pre-column derivatisation method for the measurement of glycosylated hydroxylysines of collagenous proteins.

Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (GH and GGH) in combination with other amino acids has been based on ion-exchange chromatography followed by reaction with ninhydrin. Here, a rapid and sensitive high-performance liquid chromatographic method with fluorimetric detection has been developed and employed to determine the glycosylated hydroxylysine residues in alkaline collagen hydrolysates. After hydrolysis, amino acids were derivatised with 9-fluorenylmethyl chloroformate and separated on a Micropak ODS-80TM reversed-phase column (150x4.6 mm). With a multistep gradient system all amino acids were separated in less than 30 min, including the collagen-specific hydroxylysine, hydroxyproline and the glycosylated hydroxylysines. The method was used to evaluate the glycosylation levels of human articular cartilage derived from femoral head, femoral condyle, tibial plateau and ankle. GGH was highest in cartilage from femoral head and ankle; GH showed no differences between the different sources of cartilage.

Animals↗
PubMed1997

Synthesis of collagen by bovine chondrocytes cultured in alginate; posttranslational modifications and cell-matrix interaction.

The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically. Predominantly type II collagen was present in the alginate bead, as reflected by the unique extent of lysyl hydroxylation, glycosylation, and pyridinoline crosslink formation measured. Collagen crosslinks, predominantly hydroxylysylpyridinoline (> 93%), were observed after 7 to 11 days of culture and their formation was effectively blocked by beta-aminopropionitrile (BAPN). Unexpectedly, BAPN treatment resulted in a 100% increase of collagen levels, without influencing cell proliferation and proteoglycan levels. In control cultures 90% of the synthesized collagen was retained in the cell-associated matrix, while in BAPN-treated cultures half of the collagen was found in the interterritorial matrix compartment further removed from the cells. This suggests that impaired crosslinking of collagen interferes with pericellular collagen deposition, causing upregulation of collagen synthesis by impaired cell-matrix interactions. Integrins are likely to be involved in this feedback inhibition by extracellular collagen since the cyclic RGD-containing peptide CGRGDSPC downregulated collagen synthesis by 37%.

Alginates↗
PubMed1997

A simplified measurement of degraded collagen in tissues: application in healthy, fibrillated and osteoarthritic cartilage.

Intact triple helical collagen molecules are highly resistant to proteolytic enzymes, whereas degraded (unwound) collagen is easily digested. This fact was exploited to develop a simplified method for the quantification of the amount of degraded collagen in the collagen network of connective tissues. Essentially, the method involves extraction of proteoglycans with 4 M guanidinium chloride, selective digestion of degraded collagen by alpha-chymotrypsin, hydrolysis in 6 M HCl of the released fragments as well as the residual tissue, and then measurement of the amount of hydroxyproline in both pools. Since the digestion of degraded collagen by alpha-chymotrypsin and measurement of hydroxyproline is not restricted to a specific collagen type, this technique can be applied to a wide variety of connective tissues. The method was validated with articular cartilage. Levels of in situ degraded collagen were about four-fold higher in degenerated (fibrillated) cartilage than in its healthy counterpart derived from the same donor. More detailed investigations revealed that the collagen damage in degenerated cartilage is more extensive at the cartilage surface than in the region adjacent to bone. This was not the case in healthy cartilage; identical low values were obtained at the surface and close to the bone. An impaired collagen network has been hypothesized to be the reason for the swelling of cartilage in osteoarthritis (OA). The present paper presents the first experimental evidence to support this hypothesis: more damage to the collagen network (i.e., more degraded collagen molecules within fibrils) is linearly related to more extensive swelling of the OA tissue in hypotonic saline.

Adult↗
PubMed1996

Amino acid analysis by reverse-phase high-performance liquid chromatography: improved derivatization and detection conditions with 9-fluorenylmethyl chloroformate.

An improved method for the quantitative derivatization of amino acids with fluorenylmethyl chloroformate (FMOC-Cl) is described. Amino acids are derivatized in borate buffer at pH 11.4 for 40 min at ambient temperature. All amino acids resulted in stable derivatives. In particular, improved derivatization was obtained with the troublesome amino acids His and Tyr: exclusively monosubstituted His and disubstituted Tyr were formed, eluting as free peaks in the chromatogram. These derivatives show a higher fluorescence response than their disubstituted and monosubstituted counterparts, respectively, resulting from other protocols. Under the new conditions, considerable less of the hydrolysis product of FMOC-Cl is seen in the chromatograms. Baseline noise was substantially reduced at a higher emission wavelength (630 nm instead of 313 or 340 nm). With simple precautions, extensive adsorption of the disubstituted derivatives (Lys, Hyl, and Tyr) on plastic or glass surfaces could be prevented. Calibration curves were linear over a 10 to 300 molar ratio of FMOC-Cl to total amino acid. The detection limits are in the femtomole range and the derivatives are stable for more than 48 h, thus permitting automated analysis of multiple samples.

Amino Acids↗
PubMed1992

Variation in gene copy number and polymorphism of the human salivary amylase isoenzyme system in Caucasians.

The polymorphic patterns of human salivary amylase of a large number of individuals of Caucasian origin were determined by using isoelectric focusing and polyacrylamide gel electrophoresis. Nine different salivary amylase protein variants were found; three of them are recorded for the first time and their heredity is shown. Some of the variants are encoded by haplotypes expressing three allozymes. Most variants display low frequencies. Analysis of the relative intensities of variant-specific isozyme bands, combined with segregation analysis, show that extensive quantitative variation is present in the population. The numbers of salivary amylase genes in some families showing quantitative variation at the protein level have been estimated by the polymerase chain reaction. We present evidence that quantitative variations in amylase protein patterns do not always reflect variations in gene copy number but that other mechanisms are also involved.

Amylases↗
PubMed1992

Protease inhibitor (Pi) locus, fertility and twinning.

In a sample of 160 Dutch twin pairs and their parents, we found that mothers of dizygotic twins had frequencies of the S and Z alleles at the protease inhibitor (Pi) locus that were 3 times higher than a control sample. Mothers of identical twins also had a higher frequency of S than controls. The S allele may thus both increase ovulation rate and enhance the success of multiple pregnancies. There was also an increased frequency of the S allele in fathers of dizygotic twins; however, this may be a secondary effect of assortative mating for family size (indicating by the number of siblings of the parents), for which a correlation of 0.2 was observed. Parents of dizygotic twins came from larger families than parents of monozygotic twins, but no effect of Pi type on family size was seen.

Alleles↗
PubMed1992

Changes in saliva of epileptic patients.

Unstimulated whole saliva samples of 27 indoor epileptic patients were studied on their protein composition using biochemical and immunochemical methods. A number of salivary proteins appeared at least partially to be hydrolyzed. In a number of saliva samples the concentration of carbohydrate-containing isoenzymes of amylase was reduced. In addition, the concentration of the 20 kD glycoprotein EP-GP was reduced by 60%. Sialic acid, the terminal sugar of the glycoproteins and mucins, was released for about 50% and in three salivas even nearly completely. Moreover, sialic acid- and fucose-containing epitopes could hardly be detected by monoclonal antibodies to human salivary mucins. As a consequence of this hydrolytic breakdown the saliva mediated aggregation of two S. sanguis strains had been reduced. In contrast, the aggregation of S. oralis had been maintained.

Adolescent↗
PubMed1991

High-performance liquid chromatography: purification and chromatographic behaviour of molecular variants of pepsinogen A from human urine.

By combining conventional DEAE chromatography with high-performance liquid chromatography on Sephacryl S-200 HR and Mono-Q columns, we have been able to isolate and fractionate human pepsinogen A (PGA) isozymogens from large amounts of urine. This method of fractionation is simple and allows one to obtain pepsinogen in a native non-denatured conformation. The isozymogens are homogeneous by electrophoretic and chromatographic criteria; this was confirmed by N-terminal amino acid sequencing. Purified PGA-3 and PGA-5 can be converted into an additional, more anionic, isoform on incubation at 37 degrees C. This isoform exists not only in vitro but also in vivo. The net negative charge of the PGA isozymogens is in the order PGA-5 less than deamidated PGA-5 less than PGA-3 less than deamidated PGA-3. Surprisingly, the elution order on the Mono-Q column was PGA-5/PGA-3/deamidated PGA-5/deamidated PGA-3. We have performed molecular modelling on PGA to investigate this phenomenon in terms of surface charge (not net charge) of the proteins. The model provides evidence that (1) only a fraction of the protein surface interacts with the support and (2) regions of localized charge at the protein surface may allow portions of the external surface to dominate chromatographic behaviour, resulting in a steering of the proteins with respect to the oppositely charged matrix. Pepsinogens may serve as model proteins for elucidating some of the variables that determine the chromatographic behaviour of proteins on ion-exchange columns.

Chromatography, High Pressure Liquid↗
PubMed1991

Electrophoretic characterization of posttranslational modifications of human parotid salivary alpha-amylase.

Human salivary alpha-amylase displays multiple bands upon native polyacrylamide gel electrophoresis. In fresh saliva, due to posttranslational modifications, a pattern of 5-6 isozymes is observed. The isozymes are designated 1-6, in the order of increasing anodal mobility. As a result of the development of a rapid and sensitive electrophoresis system, with markedly higher resolution than previously reported, we concluded that a previously proposed model (Karn et al., Biochem. Genet. 1973, 10, 341-350) is inadequate to explain the origin of the various bands. We propose an alternative model that fits in with our new and previously made observations. According to this model, band 2 is the primary gene product and band 1 is its glycosylated counterpart--with only one neutral oligosaccharide present on each molecule. Band 3 originates from band 1 by the transialidase-catalyzed incorporation of sialic acid into the biantennary chain. Bands 4 and 6 originate from bands 2 and 4, respectively, by deamidation; band 5 is the deamidation product of amylase with an acidic oligosaccharide (band 3). Only a minor part of band 3 consists of the deamidation product of band 1. Peptide Asn-Gly-Ser (residues 427-429) is the most probable candidate for glycosylation; literature data suggests that deamidation occurs in the stretch Glu-Asn-Gly-Lys-Asp (residues 364-368) and Asn-Gly-Asn-Cys (residues 474-477). Both glycosylation and deamidation might play a role in the clearance of amylase from the systemic circulation. The electrophoresis system described is a powerful tool to determine amylase isozyme distributions in health and disease, especially for the screening of alterations seen in ectopically produced amylase.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of three non-mucinous human salivary proteins with affinity for hydroxyapatite.

The isolation and chemical analysis of three human salivary proteins is reported. Using cation-exchange, FPLC anion-exchange chromatography and gel filtration, three human salivary proteins with affinity for hydroxyapatite were isolated, having molecular weights of 60 kD, 20 kD and 14 kD, respectively. The 60 kD protein was identified as albumin, and the 14 kD protein as a member of the cystatin family. Isoelectric focusing of the 14 kD protein revealed a single band, having an isoelectric point (pl) of 4.7. The third protein, not described yet, is appearing as a 20 kD doublet on SDS-PAGE. Isoelectric focusing resolved the 20 kD protein into four bands having pl's of 4.8, 5.0, 5.2 and 5.4, respectively. All bands were recognized by monoclonal antibodies to the 20 kD protein indicating that these four protein bands share the same epitope. The 20 kD protein is a glycoprotein with a carbohydrate content of 13% and a molar ratio of Fuc: Man: Gal: GlcNAc: NeuAc = 3.4:2.6:2.9:4.0:0.4. All three proteins bind strongly to a hydroxyapatite-based HPHT column at pH 6.0. With increasing pH, the binding diminished, especially of the 14 kD protein.

Albumins↗
PubMed1989

Isolation of high molecular weight mucins from human whole saliva by ultracentrifugation.

A high molecular weight mucin fraction was prepared from human whole saliva using relatively mild conditions. The method involves ultracentrifugation of human whole saliva in the presence of 7.2 M urea and 0.5 M sodium chloride. The resulting preparation consists of a highly purified salivary mucin fraction, as judged by several purity criteria: molecular weight analysis of the final preparation by gel electrophoresis and analytical gel filtration indicated an apparent molecular weight greater than 10(6). Analytical isopycnic density centrifugation demonstrated that the preparation consisted of a mucin fraction with a buoyant density of approximately 1.47 g/ml. The final preparation comprised 12.8% protein, 31.8% N-acetylglucosamine, 11.5% N-acetylgalactosamine, 9.5% fucose, 21.4% galactose, 0.8% mannose, 10.3% N-acetylneuraminic acid, 1.7% sulphate and 0.16% fatty acids.

Centrifugation, Isopycnic↗
PubMed1988

Identification of a Glu greater than Lys substitution in the activation segment of human pepsinogen A-3 and -5 isozymogens by peptide mapping using endoproteinase Lys-C.

The isozymogens PGA-3 and PGA-5 of human pepsinogen A were digested with endoproteinase Lys-C. The peptides were separated by reverse-phase HPLC. PGA-5 showed a peak strongly absorbing at 254 nm absent in PGA-3. Analysis of amino acid composition using the Pico-Tag methodology combined with DABITC-sequencing reveals the sequence Tyr-Phe-Pro-Gln-Trp-Lys (peptide 37-43 of the activation segment). This confirms a study at the DNA level by our group [16] suggesting a Glu greater than Lys mutation at position 43 in the activation segment of PGA-5. Furthermore, it is proposed that the number of genetic variants of PGA is higher than is actually seen by electrophoresis.

Amino Acid Sequence↗
PubMed1988

Renal handling of pepsinogens A and C in man.

1. Fractional excretions of pepsinogens A and C in the urine were investigated in 21 healthy subjects and in 38 patients with chronic renal insufficiency. In eight of the healthy subjects fractional excretions were measured again after oral administration of omeprazole for 9 days. 2. The mean fractional excretion of pepsinogen A was 27.6% (range 4.4-73.9%) in healthy subjects and remained unchanged after omeprazole administration. In patients with renal failure the mean fractional excretion of pepsinogen A was 37.9% (range 7.0-81.9%). The mean fractional excretion of pepsinogen C was 1.0% (range 0.04-6.8%) in healthy subjects and decreased after omeprazole. In patients with chronic renal diseases a sharp rise in fractional excretion of pepsinogen C was observed once glomerular filtration rate was less than 40 ml/min. 3. Fractional excretion of pepsinogen A was unexpectedly high for a negatively charged protein with a molecule mass of 40,000 daltons. This might be explained by the presence of the positively charged activation peptide. Furthermore, pepsinogen C seemed to be almost entirely reabsorbed from the glomerular filtrate and a tubular reabsorption maximum appeared to be present. Pepsinogen C may, therefore, be a new marker of tubular function. The cause of the remarkable difference in tubular handling of two quite similar low-molecular-mass proteins remains to be elucidated.

Adult↗