High-pressure polymorphism of titanium dioxide.

X-ray diffraction studies made in situ under conditions of high pressure and high temperature revealed the direct transition of rutile to the alpha lead dioxide form in titanium dioxide. Compressibility studies of this alpha lead dioxide form at room temperature showed anomalous behavior in that its molar volume converges close to, but not equal to, that of the rutile form. Under this circumstance an unexpectedly large error appears in the calculations of the equilibrium pressure for the two forms at 298 degrees K.

Enthalpy of transformation of a high-pressure polymorph of titanium dioxide to the rutile modification.

The enthalpy of transformation of a high-pressure form of titanium dioxide, which has the alpha lead dioxide structure, to the rutile modification was measured by the method of "transposed temperature-drop calorimetry." For the reaction, titanium dioxide (alpha lead dioxide form) transforming to rutile, the change in the heat content is -0.76 +/- 0.17 kilocalorie per mole. From this value and the volume change (+ 2.8 percent) associated with the transformation, we estimate the equilibrium pressure at 294 degrees K to be 60 +/- 20 kilobars.

Shock-wave compression and x-ray studies of titanium dioxide.

The Hugoniot of the rutile phase of titanium dioxide has been determined to 1.25 megabars, and data show the existence of a phase change at about 0.33 megabar. The volume decrease associated with this transformation appears to be quite large (approximately 21 percent). Rutile, when recovered from shockloading in excess of the transformation pressure, is found to be irreversibly transformed to the orthorhombic lead dioxide structure (a distortion of the fluorite structure) with parameters a, 4.529; b, 5.464; and c, 4.905 angstroms and a calculated density of 4.374 grams per cubic centimeter. The new phase reverts to rutile at temperatures above 450 degrees C. It is suggested that the new phase may be another diagnostic indicator of meteorite impact on the earth's surface.

The role of the carbohydrate chains of Gal beta-1,4-GlcNAc alpha 2,6-sialyltransferase for enzyme activity.

Gal beta-1,4-GlcNAc alpha 2,6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2,6 sialyltransferase, EC 2.4.99.1) is a glycoprotein containing carbohydrate chains of the complex type (Jamieson, J.C. (1989) Life Sci. 43, 691-697). The carbohydrate chains may be important for controlling the expression of sialyltransferase catalytic activity during transit of the enzyme from the rough endoplasmic reticulum to the Golgi complex where it is active as a membrane bound enzyme anchored to the luminal face. To study the role of the carbohydrate chains of sialyltransferase for enzyme activity, conditions were established in which the native enzyme was deglycosylated with N-Glycanase and endo F. It was found that Glycanase removed the carbohydrate chains from native sialyltransferase, but methanol or ethanol had to be present for rapid and complete deglycosylation. Presence of methanol or ethanol were not essential for removal of carbohydrate chains with endo F. There was a correlation between the loss of catalytic activity of sialyltransferase with increased deglycosylation. After deglycosylation with Glycanase for 18 h catalytic activity was largely eliminated and there was a reduction in molecular mass of about 5 kDa compared to the untreated enzyme when examined by immunoblot analysis; this reduction was identical to that found when the denatured enzyme was deglycosylated with Glycanase. At shorter times of incubation partially deglycosylated forms of the enzyme were detected. Complete deglycosylation of native or denatured sialyltransferase with endo F could not be achieved. However, incubation with endo F for 24 h resulted in a loss of catalytic activity of about 60%. Immunoblot analysis showed the presence of three forms of the enzyme corresponding in molecular mass to the native and deglycosylated enzyme and a third form corresponding to a partially deglycosylated enzyme. Sialyltransferase was also subjected to sequential treatment with exoglycosidases. Removal of NeuAc and Gal had little effect on catalytic activity, but subsequent removal of GlcNAc resulted in a significant loss in catalytic activity suggesting that the presence of the trimannose core with GlcNAc attached is important for the expression of catalytic activity. The presence of organic solvents during deglycosylation with Glycanase may be a useful method that can be applied to other glycoproteins.

Studies on the effect of the hepatocyte-stimulating factor on galactose-beta 1----4-N-acetylglucosamine alpha 2----6-sialyltransferase in cultured hepatocytes.

Rat hepatic Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase is released into the blood at elevated levels following an inflammatory challenge: this is a typical response of the group of plasma proteins known as acute-phase reactants. In the present study, primary cultures of liver parenchymal cells are used to demonstrate that the same hepatic cell type that produces plasma proteins such as fibrinogen also produces and releases sialyltransferase. Hepatic production of sialyltransferase is stimulated by a major regulator of hepatic acute-phase reactant production, the hepatocyte-stimulating factor (HSF), while another monokine, interleukin-1, does not affect hepatocyte sialyltransferase production. The maximum increase in sialyltransferase occurs 48 h after exposure to HSF which is considerably later than the fibrinogen response. The sialyltransferase that is stimulated by HSF is the Gal beta 1----4GlcNAc alpha 2----6 isozyme.

Increased levels of GALbeta1-4GLCNACalpha2-6 sialyltransferase pretransplant predict delayed graft function in kidney transplant recipients.

BACKGROUND: Galbeta1-4GlcNAcalpha2-6 sialyltransferase (ST6GalI) is an acute phase reactant whose release from cells can be induced by proinflammatory cytokines. Because patients with chronic renal failure have high circulating levels of proinflammatory cytokines, we hypothesized that patients on the renal transplant waiting list would have high circulating levels of ST6GalI, which might adversely affect post-transplant events. METHODS: Levels of ST6GalI were measured in the serum of 70 patients immediately before renal transplant; these were correlated with posttransplant events, such as delayed graft function and rejection. RESULTS: The mean serum level of ST6GalI was significantly higher in the patients (3162+/-97 U) than in 19 controls (2569 +/- 125 U; P<0.003). Patients who required dialysis posttransplant for treatment of delayed graft function (n=20) had significantly higher levels of ST6GalI pretransplant (3735+/-228 U) than patients (n=50) who did not require dialysis (2933+/-83 U; P<0.0001). In a multivariate analysis the ST6GalI level and cold ischemic time were found to be independent risk factors for the development of delayed graft function. CONCLUSIONS: ST6GalI levels are high in renal failure patients awaiting a renal transplant and may be a risk factor for the development of delayed graft function. The assessment and perhaps modulation of a potential transplant recipient's ST6GalI systemic level may be beneficial.

Differential effects of glycoprotein processing inhibition on experimental metastasis formation by T24-H-ras transformed fibroblasts.

Highly metastatic mouse 10T1/2 cell lines (Ciras 2, Ciras 3 and dGC2M5) which have been T24-H-ras transfected, are shown to have differential responses in metastatic properties when grown in the presence of the processing inhibitors, swainsonine, castanospermine and deoxymannojirimycin. Concanavalin A binding data indicated the inhibitors caused similar shifts in oligo-saccharide structures, resulting in more high mannose character for all cell lines. However, swainsonine inhibited the experimental metastasis of dGC2M5, but did not affect the metastatic properties of Ciras 2 and Ciras 3. Inversely, castanospermine reduced experimental metastasis of Ciras 2 and 3 and did not inhibit dGC2M5. These results show that closely related metastatic cell lines respond differently in their metastatic ability when changes occur in N-linked oligosaccharide content. This observation emphasizes the importance of oligosaccharide structure in the malignant phenotype and indicates that some caution should be used when generalizing about the effects of processing inhibitors on a complex process like metastasis.

Investigation of different combinations of derivatization, separation methods and electrospray ionization mass spectrometry for standard oligosaccharides and glycans from ovalbumin.

Derivatization procedures using 1-phenyl-3-methyl-5-pyrazolone (PMP) and 2-aminonaphthalene trisulfone (ANTS) were selected among a number of well known methods for labelling carbohydrates. PMP derivatives were selected owing to our laboratory's previous high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) experience with these, whereas the ANTS-labelled compounds were prepared for fluorophore-assisted carbohydrate electrophoresis (FACE) separation. ANTS-oligosaccharide standards were characterized to study their ionization patterns. Reversed-phase and normal-phase HPLC systems were coupled on-line with ESI-MS. Each necessitated its own mobile phase system which, in turn, imposed some important changes in the ionization conditions used and/or on the ionization patterns and spectra obtained. Following characterization of the intact glycoprotein ovalbumin with ESI-MS, its glycans were detached using the enzyme PNGase-F. The glycans were subjected to PMP and ANTS derivatization. It was very difficult to separate ANTS derivatives by reversed-phase HPLC owing to lack of retention, and normal-phase HPLC offered reasonable retention with limited separation. PMP compounds overall yielded better normal- and reversed-phase separations and improved sensitivity over the ANTS-labelled sugars, for which negative mode ESI had to be used. The combination of ESI of intact ovalbumin and ESI of PMP-glycans gave rise to the detection of over 20 different glycoforms, excluding the possible presence of structural isomers for each sugar composition detected.

Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody.

The murine B-lymphocyte hybridoma, CC9C10, was grown at steady state in serum-free continuous culture at dissolved oxygen (DO) concentrations of 10, 50, and 100% of air saturation. The secreted mAb, an IgG1, was purified and subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Both methods resulted in complete removal of N-linked oligosaccharide chains. Isolated N-glycan pools were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The FACE profiles and corresponding HPAEC-PAD chromatograms of N-linked oligosaccharides obtained by PNGase F digestion and hydrazinolysis provided complementary and corroborating information. The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialylated, and trace amounts of afucosyl, oligosaccharides. A definite shift towards decreased galactosylation of glycan chains was observed as DO concentration in continuous culture was reduced. The vast majority of N-linked glycosylation occurred on the heavy chain. There was no evidence for N-linked glycosylation of the light chain or for O-linked glycosylation of the mAb.

Concanavalin A binding to fibroblasts from Duchenne muscular dystrophy patients and age-matched controls.

An investigation of [125I]Con A binding to skin fibroblasts from Duchenne muscular dystrophy patients and age-matched controls was carried out. The age groups examined were 5-6 years, 11-12 years, and 15-17 years. Only small differences in binding abilities were observed between dystrophic cells and matched controls. When data was examined as micrograms Con A bound/micrograms protein, dystrophic fibroblasts bound slightly more lectin compared to controls with the 5-6 and 11-12 year age groups, whereas the 15-17 years age group bound slightly less Con A compared to normal controls. However, analysis of binding data as lectin bound/cell showed slightly reduced binding of Con A to dystrophic cells from all age groups when compared to matched controls. It was also found that the amount of Con A bound by both normal and dystrophic fibroblasts markedly increased with the age of the donor. Obviously several factors must be taken into account when analyzing lectin binding data obtained with human fibroblasts. Taken as a whole, our studies do not provide evidence for significant modification of cell surface Con A receptors on fibroblasts from Duchenne muscular dystrophy patients.

Studies on the effect of 1-deoxynojirimycin on the release of albumin, sialyltransferase and alpha 1 acid glycoprotein from liver slices from normal and inflamed rats.

Liver slices from control and 24hr inflamed rats were incubated for up to 20hr with 5mM 1-deoxynojirimycin (DN), an inhibitor of the processing glucosidases. The amounts of albumin and alpha 1-acid glycoprotein (AGP) and the activities of sialyltransferase were determined in liver and medium. The presence of DN significantly inhibited the release of AGP and sialyltransferase. The inhibitory effect of DN was most pronounced with slices from inflamed rats. Secretion of albumin was not inhibited. Incorporation studies with labelled leucine and mannose showed that the inhibitor did not significantly affect protein synthesis, but it did inhibit mannose incorporation into AGP and sialyltransferase. The results show that DN inhibits the secretion of acute phase AGP and sialyltransferase in liver slices and further suggests that sialyltransferase is a glycoprotein.

Studies on the effect of inflammation on the synthesis of glucosylated intermediates of glycoprotein biosynthesis in rat liver microsomes.

Glycoprotein biosynthesis is substantially increased in liver during experimental inflammation. This study describes the effect of inflammation on the incorporation of labelled Glc from UDP-Glc into glucosylated lipid-linked intermediates of glycoprotein biosynthesis in liver microsomes. Maximum incorporation of Glc into lipid sugar and lipid oligosaccharide fractions occurred using microsome fractions from rats suffering from inflammation for 48-72 hr. Incorporation of Glc into lipid-sugar fractions was increased about three-fold over controls and incorporation into lipid-oligosaccharide fractions was increased about four-fold compared to controls. Maximum incorporation of Glc into lipid-sugar and lipid-oligosaccharide occurred at pH 6.0 and pH 5.2, respectively.

Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors.

The murine B-lymphocyte hybridoma cell line, CC9C10, was grown in serum-free continuous culture at steady-state dissolved oxygen (DO) concentrations of 10%, 50%, and 100% of air saturation in both LH Series 210 (LH) and New Brunswick Scientific (NBS) CelliGen bioreactors. All culture parameters were monitored and controlled and were nominally identical at steady state in the two bioreactors. The secreted monoclonal antibody (mAb), an immunoglobulin G(1), was purified and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGase F). Asparagine-linked (N-linked) oligosaccharide pools released from mAb samples cultured in each bioreactor at each of the three DO setpoints were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The predominant N-linked structures were core-fucosylated asialo biantennary chains with varying galactosylation. There were also minor amounts of monosialyl oligosaccharides and trace amounts of afucosyl oligosaccharides. The level of DO affects the glycosylation of this mAb. A definite reduction in the level of galactosylation of N-glycan chains was observed at lower DO in both bioreactors, as evidenced by prominent increases in the relative amounts of agalactosyl chains and decreases in the relative amounts of digalactosyl chains-with the relative amounts of monogalactosyl chains being comparatively constant. However, the quantitative results are not precise matches between the two bioreactors. The effect of DO on galactosylation is less pronounced in the NBS bioreactor than in the LH bioreactor, particularly the shift between the relative amounts of agalactosyl and digalactosyl chains in 10% and 50% DO. There are also perceptibly higher levels of sialylation of the mAb glycans in the NBS bioreactor than in the LH bioreactor at all three DO setpoints. The results indicate that the DO effect is not bioreactor specific and that nominally identical steady-state conditions in different chemostat bioreactors may still lead to some incongruities in glycosylation, possibly due to the particular architectures of the bioreactors and the design of their respective monitoring and control systems. The observed differences in N-linked glycosylation of the mAb secreted by the hybridoma grown in the LH and NBS bioreactors may be explained by the differences in oxygen supply and control strategies between the two bioreactors.

Release of sialyltransferases from rat liver Golgi membranes by a cathepsin D-like proteinase: comparison of the release of Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase, Gal beta 1-3(4)GlcNAc alpha 2-3 sialyltransferase and lactosylceramide alpha 2-3 sialyltransferase (SAT-1).

The activities of Gal beta 1-3(4)GlcNAc alpha 2-3 sialyltransferase and SAT-1 were measured in rat liver Golgi in inflammation; both enzymes decreased by about 50%. This compares with increases of about 3-fold for the Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase. All three sialyltransferases were released from disrupted Golgi membranes by incubation at reduced pH which activates an endogenous cathepsin D which is believed to be the lysosomal enzyme. Pepstatin A was found to block the release of all three sialyltransferases providing support for the role of cathepsin D as the proteinase that clips the catalytic portions of the enzymes from their membrane anchor and stem regions.

90-kilobar diamond-anvil high-pressure cell for use on an automatic diffractometer.

A gasketed diamond-anvil high-pressure cell is described which can be used on a four-circle automatic diffractometer to collect x-ray intensity data from single-crystal samples subjected to truly hydrostatic pressures of over 90 kilobars. The force generating system exerts only forces normal to the diamond faces to obtain maximum reliability. A unique design allows exceptionally large open areas for maximum x-ray access and is particularly well suited for highly absorbing materials, as the x rays are not transmitted through the sample. Studies on ruby show that high-pressure crystal structure determinations may be done rapidly, reliably, and routinely with this system.

Studies on the site of addition of sialic acid and glucosamine to rat alpha 1-acid glycoprotein.

Ultrasonic extracts of rough and smooth endoplasmic reticulum fraction and Golgi fractions from rat liver were examined by immunoelectrophoresis using antiserum to alpha 1-acid glycoprotein. Rough endoplasmic reticulum fractions contained only sialic acid free alpha 1-acid glycoprotein, whereas smooth endoplasmic reticulum and Golgi fractions also contained sialic acid containing alpha 1-acid glycoprotein. Determination of the sialic acid contents of immune precipitates isolated from the extracts suggested that the Golgi complex was the main site of addition of sialic acid to alpha 1-acid glycoprotein. Immunological studies on puromycin extracts of polyribosomes showed that polypeptide chains of alpha 1-acid glycoprotein and albumin were assemble mainly on membrane-bound polyribosomes. Evidence is presented from incorporation studies with labelled leucine and glucosamine that initial glycosylation of alpha 1-acid glycoprotein occurs mainly or entirely after release of nascent polypeptide from the ribosomal site.

Studies on incorporation of mannose into rat alpha 1-acid glycoprotein: evidence for precursor forms in rough membranes.

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and alpha 1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and alpha 1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of alpha 1-acid glycoprotein by isolation of a high mannose containing precursor species of alpha 1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of alpha 1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of alpha 1-acid glycoprotein with extensions of the polypeptide chain may also exist.