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Pulmonary biochemical response to slate dust in rats.

The biochemical changes in rat lungs due to intratracheal instillation of 50 mg of slate dust have been studied up to 150 days of dust exposure. The remarkable feature of lung changes was the turnover of collagen in experimental animals after 90 days, reaching substantially higher values at 150 days. A concurrent increase in hexosamine and sialic acid contents was also observed. The phospholipid content in the whole lung tissue, as well as in the mitochondria, was generally higher in the dust-treated rats, particularly at the later stages. The mitochondrial cytochrome c oxidase and glutamate dehydrogenase activities increased, whereas monoamine oxidase was marginally affected. Mitochondria from experimental animals appeared to be in a swollen state, particularly at 120 days of exposure. The above results suggest that slate dust exerts its toxic effects by causing alterations in the tissue make-up as well as in the mitochondrial functioning of the lung.

Animals↗

Biochemical features of pseudomembrane at the bone-cement interface of loosened total hip prostheses.

Biochemical features of pseudomembrane formed at the bone-cement interface of 13 failed total hip replacements were studied and histological examination of the pseudomembrane was conducted. The results of biochemical analyses revealed on average hydroxyproline, 86 micrograms/mg; water content, 57%; hexosamine, 12 micrograms/mg; glucosamine/galactosamine, 1.4%; and calcium, 2.1%. Approximately 78% of collagen was extracted with pepsin, whereas only 8 and 0.3% collagen were extracted with acetic acid and neutral salt, respectively. On further differential salting out of the extracted collagen, about 77% was type I collagen, 19% type III, and 2.6% type V. Unlike pseudoarthrosis of fracture, no type II collagen was identified. Overall biochemical features in the light of the histological appearance suggested that pseudomembrane resembles granulation tissue. Bone healing mechanisms at the bone-cement interface appear to differ from that of fracture healing.

Adult↗

Isolation of intact aortic valve scaffolds for heart-valve bioprostheses: extracellular matrix structure, prevention from calcification, and cell repopulation features.

Extracellular matrix (ECM) scaffolds isolated from valvulated conduits can be useful in developing durable bioprostheses by tissue engineering provided that anatomical shape, architecture, and mechanical properties are preserved. As evidenced by SEM, intact scaffolds were derived from porcine aortic valves by the combined use of Triton X-100 and cholate (TRI-COL) or N-cetylpyridinium (CPC) and subsequent nucleic acid removal by nuclease. Both treatments were effective in removing most cells and all the cytomembranes, with preservation of (1) endothelium basal membranes, (2) ECM texture, including the D-periodical interaction of small proteoglycans with normally D-banded collagen fibrils, and (3) mechanical properties of the treated valves. Ultrastructural features agreed with DNA, hexosamine, and uronic acid biochemical estimations. Calcification potential, assessed by a 6-week rat subdermal model, was significantly reduced by TRI-COL/nuclease treatment. This was not true for CPC only, despite better proteoglycan preservation, suggesting that nucleic acids also are involved in calcification onset. Human fibroblasts, used to repopulate TRI-COL samples, formed mono- or multilayers on surfaces, and groups of cells also were scattered within the valve leaflet framework. A biocompatible scaffolds of this kind holds promise for production of durable valve bioprostheses that will be able to undergo probable turnover and/or remodeling by repopulating recipient cells.

Animals↗

Pexiganan-incorporated collagen matrices for infected wound-healing processes in rat.

The use of peptide-based drugs is limited by their rapid degradability and toxicity at high concentration during their therapeutic application. These problems could be managed by the use of a peptide delivery agent for sustained release in the site of action. Collagen is one of the most proven biomaterials of good biocompatibility with an exceptional ligand encapsulating property. In this work, we have shown that pexiganan, an antimicrobial, 22-amino-acid peptide could be incorporated and delivered to the wound-healing site against bacterial strains Pseudomonas aeruginosa and Staphylococcus aureus. The release profiles of pexiganan collagen films with different collagen concentration were studied. The release of pexiganan from 2.5% w/w of collagen film showed a sustainable activity over 72 h with effective antimicrobial concentrations. Pexiganan-incorporated collagen (PIC)-treated groups were compared with open wound (OW)- and collagen film (CF)-treated rats. PIC-treated animals showed a diminishing level of bacterial growth as compared with OW- and CF-treated animals. The biochemical parameters such as hydroxyproline, protein, DNA, uronic acid, hexosamine, SOD, and catalase content in the granulation tissue of the healing wound revealed increased proliferation of cells involved in tissue reconstruction in PIC-treated groups when compared with OW- and CF-treated groups. Furthermore, spectroscopic studies suggested that collagen structure is not perturbed by pexiganan incorporation. This study provides rationale for application of collagen membrane for antimicrobial peptide delivery in infected wounds.

Animals↗

Novel acetylation-aided migrating rearrangement of uridine-diphosphate-N-acetylglucosamine in electrospray ionization multistage tandem mass spectrometry.

Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) is the final product of hexosamine biosynthetic pathway (HSP) and the donor substrate for the modification of nucleocytoplasmic proteins at serine and threonine residues with N-acetylglucosamine (GlcNAc) catalyzed by O-GlcNAc transferase (OGT). Many analogs of UDP-GlcNAc were designed to interfere with the process of protein O-glycosylation by blocking OGT. A novel rearrangement reaction was observed in which phosphate-N-acetylglucosamine moiety migrated to 3' terminus of ribose in ESI-MS(n) of UDP-GlcNAc. Results from tandem mass spectrometry, control experiments and calculation showed that the phosphate-N-acetylglucosamine migration might undergo a pentacoordinate phosphoric intermediate. Furthermore, the acetylation of glucosamine in UDP-GlcNAc was essential in the migration process.

Acetylation↗

Fatty acid and long-chain base composition of gangliosides isolated from adult human brain.

A series of major and minor ganglioside species were isolated from a single human adult brain and analyzed for their lipophilic composition. Hematosides, GM3 and GD3, each showing double bands on thin-layer chromatograms, were separated into the upper band and lower band fractions to be analyzed for the heterogeneity of their ceramide moieties. The upper band fractions of GM3 and GD3 were found to contain relatively high amounts of longer-chain fatty acids (C20-C26), whereas the lower band fractions contained high amounts of shorter-chain fatty acids (C16-C18). Compared to other gangliosides which contain hexosamine, the two hematosides contained a smaller proportion of stearic acid. The proportion of 4-eicosasphingenine was found to increase with increasing sialic acid content in gangliosides. These findings suggest that, in the biosynthesis of various gangliosides, N-acetylhexosamine is preferentially transferred to hematosides rich in stearic acid, and sialic acid residues are preferentially transferred to 4-eicosasphingenine-containing species.

Brain Chemistry↗

Systemic administration of glycosaminoglycan polysulphate (arteparon) provides partial protection of articular cartilage from damage produced by meniscectomy in the canine.

Bilateral medial meniscectomy was undertaken in 14 mature beagles. Another two underwent arthrotomy (sham-operated controls). One week after surgery, six of the meniscectomised animals were administered glycosaminoglycan polysulphate (Arteparon) (2 mg/kg) subcutaneously three times a week for 3 weeks, then twice weekly until killed 23 weeks later. Two months before death all animals were given Na2(35)SO4 (1.0 mCi/kg) intravenously. At autopsy, articular cartilage (AC) from the medial and lateral compartments, as well as from the femoral trochlear groove and femoral head, was sampled. Proteoglycans (PGs) were isolated by 4.0 M Guanidine hydrochloride (GuHCl) extraction of AC and purified by ultracentrifugation. The PG monomers were assayed for hexuronic acid, protein, and hexosamines (galactosamine/glucosamine), and their ability to aggregate. The results indicated that Arteparon provided some protective effect to AC in the meniscectomised compartment as demonstrated histologically by reduced surface fibrillation, diminished chondrocyte cloning, and maintenance of alcianophilia. The levels of PGs and hexuronate-protein ratios in medial AC of drug-treated meniscectomised animals were found to be comparable to sham controls, whereas these parameters in the nondrug-treated meniscectomized group were depressed.

Animals↗

Tensile properties of human knee joint cartilage. II. Correlations between weight bearing and tissue pathology and the kinetics of swelling.

The nonequilibrium or kinetic swelling behavior of normal, fibrillated, and osteoarthritic (OA) (removed from total knee joint replacements) human knee joint cartilage has been measured using our isometric tensile apparatus (ITA). We found that large local variations exist in the manner with which human knee joint cartilage swells, including anisotropic effects, inhomogeneities, and dependence on local biochemical composition and pathological condition. The ITA provides three convenient biomechanical parameters--peak stress (sigma p), stress relaxation (sigma R), and diffusion coefficient (D)--to quantify the kinetics of swelling. We used these parameters to quantify and differentiate the kinetic swelling behavior of normal, fibrillated, and osteoarthritic cartilage, as well as the swelling behavior of cartilage from high and low weight-bearing areas. Also, these kinetic swelling parameters correlated very well, though by varying degrees, with such biochemical measures as collagen/proteoglycan ratio, hexosamine content/wet weight, and hydroxyproline content/dry weight, providing important insight into the mechanisms and processes involved during the course of swelling. Hence, the kinetic swelling behavior of cartilage should be used to provide important information not obtainable from equilibrium swelling studies.

Body Water↗

Calcitonin and fracture healing. An experimental study on rats.

The effects of systemically administered calcitonin (CT) on fracture healing were analyzed in an experimental study on rats. The healing of a fracture was followed from 3 days up to 9 weeks postoperatively. Half of the rats in each age group were given daily CT 10 MRC-U/kg body wt s.c. Mechanical properties of the healing tibial fractures (tension strength) as well as various connective tissue components of the callus tissue were analyzed. No difference in the radiological or microscopical appearance of the fractures was detectable between the animals receiving CT and the controls. In the biochemical analysis matrix production as assessed from the concentrations of nitrogen, hexosamines, and hydroxyproline within the callus followed the usual lines of undistributed fracture union without any difference between the groups with and without CT. No differences could be detected in the mineralization of the callus between the specimens from animals receiving CT and those without. The tensile strength values of the fractures increased almost linearly up to 9 weeks. At 1 week the tensile strength values for fractures union in the animals without CT were approximately 50% higher, but later on no differences could be detected between the groups. These results indicate that although in the early phases of long-term CT therapy collagen synthesis may be impaired, there will be no effect on the net content of collagen or calcifying tissue in the callus or on the mechanical strength of healing fractures.

Animals↗

Long-term explant culture of rabbit flexor tendon: effects of recombinant human insulin-like growth factor-I and serum on matrix metabolism.

The effects of human recombinant insulin-like growth factor-I (rhIGF-I, 50 ng/ml) on matrix metabolism in the deep flexor tendon from the tendon sheath region of the rabbit were studied in explants cultured for 3 weeks. Tendon segments cultured in medium supplemented with fetal calf serum (FCS) exhibited proliferation of the superficial cell layers. Synthesis of proteoglycan and non-collagen protein (NCP) increased threefold during the first week and remained elevated during the next 2 weeks of culture in medium supplemented with rhIGF-I or FCS, but not in medium without supplements (bovine serum albumin, BSA). The estimated halflife (t1/2) for elimination of newly labeled proteoglycans from the tendon explants ranged from 5.1 to 8.5 days and from 4.9 to 6.8 days for NCP in supplemented medium. Presence of rhIGF-I or FCS did not affect degradation of matrix as compared with BSA. The total hexosamine content per tendon segment was stable during the culture period, but the non-collagen protein content decreased by 25%. Collagen synthesis decreased to 10% of the initial level after 3 weeks in supplemented medium, but to 3% in unsupplemented medium. There was no measurable turnover of collagen in explants cultured in either medium, and the collagen content remained unchanged. Our results suggest that rhIGF-I, as well as FCS, stimulates matrix synthesis but does not influence matrix turnover in rabbit flexor tendon explants in long-term culture as compared with medium without supplements. We conclude that rhIGF-I may be used as a defined growth-promoting factor in serum-free media and may be of importance in tendon healing.

Animals↗

Immunologic and chemical studies on mucopolysaccharide derived from murine lymphosarcoma.

Mucopolysaccharide was prepared from media in which the Gardner's lymphosarcoma cells were cultured. Multiple intraperitoneal injections of the preparation into syngeneic mice resulted in enhancement of the tumors which were inoculated prior to the injections. Sera of these hosts contained antibodies reacting with antigens of the outer layer of the tumor cells. Chemical analyses of the preparation showed that the mucopolysaccharide is composed of hexosamine, uronic, acid, hexose, and proteins. The mucopolysaccharide was shown to possess unique migration rate on electrophoresis and thin layer chromatography.

Animals↗

Preliminary results of nuclear magnetic resonance studies of healthy human saliva.

Relaxation times T1 and T2 of water protons for stimulated and unstimulated parotid and submandibular saliva from healthy patients were measured in the conventional way using the Bruker PC 20 Minispec. Some measurements of dispersion were also carried out. An interpretation of the average values for T1 and T2 observed in the 20 MHz range is proposed for these types of saliva in relation to biochemical composition (total proteins, amylase activity, sialic acids and hexosamins) and viscosity. For parotid saliva T2(-1)-T1(-1) correlates significantly with amylase activity. In the submandibular saliva T2(-1)-T1(-1) correlates significantly with both mucins and alpha-amylase activity.

Amylases↗

Hepatic D-galactosamine toxicity studied with localized in vivo 31P magnetic resonance spectroscopy in intact rats.

Spatially resolved 31P magnetic resonance spectroscopy (MRS) at 4.7 T was applied to noninvasively assess liver phosphorus metabolites in a biochemically well-characterized model of hepatotoxicity induced by injection of a sublethal dose of D-galactosamine (galN). A newly developed hybrid method based on spectral localization with B0 and B1 gradients was employed to obtain multivoxel spectra in intact anesthesized rats. Spatially localized in vivo spectra were recorded 0 to 26 h after galN injection of female rats. In response to galN exposure, diphosphodiester peaks ascribed to UDP-hexosamines became detectable by 4 h and persisted up to 26 h. A metabolite coresonating with inorganic phosphate increased rapidly in intensity by 2 h after galN and returned to baseline by 18 h; this resonance was shown not to be Pi and was assigned to galN-1-phosphate by subsequent high resolution MRS experiments on extracts prepared from these livers. These results confirmed in vivo the metabolic perturbations described previously for this model of hepatotoxicity following biochemical studies based on classical extraction methods. Unlike the in vitro studies, however, these noninvasive experiments provided additional information on the time course of metabolic alterations on the same animal.

Adenosine Triphosphate↗

Wound healing properties of Hylocereus undatus on diabetic rats.

Aqueous extracts of leaves, rind, fruit pulp and flowers of Hylocereus undatus were studied for their wound healing properties. Wound healing effects were studied on incision (skin breaking strength), excision (percent wound contraction) and the nature of wound granulation tissues, which were removed on day 7 and the collagen, hexosamine, total proteins and DNA contents were determined, in addition to the rates of wound contraction and the period of epithelialization. In streptozotocin diabetic rats, where healing is delayed, topical applications of H. undatus produced increases in hydroxyproline, tensile strength, total proteins, DNA collagen content and better epithelization thereby facilitating healing. H. undatus had no hypoglycemic activity.

Animals↗

Glycosides derived from Hemidesmus indicus R. Br. root inhibit adherence of Salmonella typhimurium to host cells: receptor mimicry.

For centuries, indigenous plants have been used against enteritis but their molecular targets and mode of action remain obscure. The present study was carried out to elucidate the protective and therapeutic role, if any, of glycosides from Hemidesmus indicus against S. typhimurium-induced pathogenesis. Studies were carried out in a human intestinal cell line (Int 407) and a murine macrophage cell line (P388D1) in order to evaluate its potency in local as well as systemic infections. The inhibitory role of the glycosides present in Hemidesmus indicus root extract (GHI) were tested by pre-coating the cells (both Int 407 and P388D1) with GHI prior to infection, and by neutralizing the wild-type bacteria with GHI before cell infection. In both cases, GHI protected the host cells from the cytotoxic effects of the wild S. typhimurium. This suggests that the biologically significant sugars (hexose, hexosamine, fucose and sialic acid etc) present in GHI might be mimicking host cell receptor saccharides and thereby blocking the bacterial ligands from binding to the host cells. Int 407 cells infected with wild-type bacteria had a diffused adherence pattern after 4 h incubation, but this typical character was not observed in cells infected with GHI-treated bacteria and the cells were normal in appearance at 4 h. After 18 h cells infected with wild-type bacteria were hypertrophoid with a disintegrated membrane and wrapped in a bacterial coat, whereas cells infected with treated bacteria had comparatively less morphological changes and few defective shrunken rods adhered locally. This suggests that the glycosides can change the adherence pattern of S. typhimurium from diffused to local. Treated bacteria had less adherence and invasion capability in Int 407 as well as P388D1 cells. The results show the decreased ability of adherence of GHI-treated S. typhimurium was due to a loss of surface hydrophobicity. A nonspecific binding between S. typhimurium and the glycosides was confirmed using ELISA. In summary, the glycosides of H. indicus root inhibited S. typhimurium induced pathogenesis nonspecifically, by reducing bacterial surface hydrophobicity and perhaps also by mimicking host cell receptors, thereby blocking its attachment to host cell and further pathological effects.

Animals↗

Electron ionization-tandem mass spectrometry of glycosphingolipids. Part II. The identification of a carbohydrate sequence corresponding to a novel repetitive blood group A heptaglycosylceramide.

In a previous paper, the presence in human kidney vein tissue of a novel blood group A heptaglycosylceramide based on the type-3 carbohydrate chain GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-4Glc beta 1-1 Ceramide, was suggested based on thin-layer immunostaining and electron ionization mass spectrometry. Ions corresponding to a structure containing two deoxyhexoses, two hexosamines and three hexoses were identified, but no information was obtained from mass spectrometry concerning the carbohydrate sequence. In the present paper, we report the identification of carbohydrate sequence ions corresponding to a type-3 chain A heptaglycosylceramide by electron ionization-tandem mass spectrometry of a permethylated-reduced glycosphingolipid mixture isolated from human kidney vein tissue. The use of a microchannel-plate-array detector increased the sensitivity for collision-induced dissociation spectra by a factor of at least ten over a conventional electron multiplier.

Blood Group Antigens↗

Identification of the lipid moiety and further characterization of the novel lipophosphoglycan-like glycoconjugates of Trichomonas vaginalis and Trichomonas foetus.

The lipid moiety of the lipophosphoglycan (LPG)-like glycoconjugates of Trichomonas vaginalis and Trichomonas foetus, parasites of the urogenital tract of human and cattle, respectively, has been isolated and characterized by a combination of enzymatic and chemical degradation, chromatography, and mass spectrometry. The carbohydrate composition of the glycan inositol lipid core is also reported. The glycan inositol core of trichomonad glycoconjugates is unique in having more than one GlcN and is significantly larger than any other glycan core reported so far. T. vaginalis glycoconjugate binds strongly to the lectin RCA-I, which suggest that the macromolecule possesses terminal beta 1,4-linked galactosyl residues. The binding of T. foetus glycoconjugate to the lectin UEA-I suggests the presence of terminal alpha 1,2-linked fucose. Acid hydrolysis of deaminated and reduced LPG products yields a [3H]anhydromannitol-containing product, indicating the presence of unacetylated glucosamine in the trichomonad LPGs. Reductive radiomethylation has been applied to label free amino groups in the hexosamine or other free amine-containing residues of the trichomonad glycoconjugates. Treatment of the LPGs with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis liberates a ceramide substituent. Treatment of LPGs with nitrous acid releases a phospholipid moiety containing myo-inositol and ceramide, implying that the LPGs are anchored in the membrane via an inositol-phosphate-ceramide. Structural characterization of the ceramide by gas-liquid chromatography (GC) and GC-mass spectrometry indicated the presence of the major long-chain base sphinganine (d 18: 0 dihydrosphingosine) and a C 16:0 N-acyl group. Lipophosphoglycans from both parasites contain ceramide as their only lipid moiety. These results suggest that T. vaginalis and T. foetus anchor their LPG-like glycoconjugates on the cell surface via inositol-phosphoceramide and also the glycan inositol core of the macromolecule appears to be unique in nature.

Animals↗

Elevated O-linked N-acetylglucosamine metabolism in pancreatic beta-cells.

High intracellular glucose concentrations increase flux though the hexosamine biosynthetic pathway, resulting in elevated UDP-N-acetylglucosamine (GlcNAc) concentrations. The nucleocytoplasmic enzyme O-linked N-acetylglucosaminyltransferase (OGT) uses UDP-GlcNAc as a donor to modify numerous critical substrates, including nuclear pore proteins and transcription factors. Here, we document (a) the overwhelming enrichment of pancreatic OGT transcripts in the beta-cells of the islets of Langerhans, (b) the physiologically significant increase in the level of O-GlcNAc residues present in beta-cells, and (c) the action of streptozotocin, a close analogue of GlcNAc, to selectively inhibit O-GlcNAcase, an enzyme involved in the removal of O-GlcNAc residues. Taken together, these findings suggest that pancreatic beta cells maintain a highly elevated O-GlcNAc metabolism and that the diabetes inducing drug streptozotocin inhibits O-GlcNAcase.

Acetylglucosamine↗