N-glycosylation of recombinant human interferon-gamma produced in different animal expression systems

Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.     

The diagnostic and treatment characteristics of focal liver lesions using computed tomography

Under analysis is an experience with examining and treatment of 167 patients with focal lesions of the liver using computed tomography. Percutaneous transhepatic puncture and draining the hepatic abscesses and cysts under control of computed tomography is an independent method of treatment used in 53 patients. The technique of performing the puncture and drainage is described. Specific features of surgical treatment of focal lesions of the liver are described. Decompression of bile ducts in the postoperative period is shown to be necessary. A conclusion is made about high efficiency of computed tomography in diagnosis and treatment of focal lesions of the liver.     

Central motor conduction in motor neuron disease

Central motor conduction time (CMCT) to abductor digiti minimi (ADM) and tibialis anterior (TA) was measured in 21 patients of motor neuron disease (MND). In the upper limb, the motor pathways were inexcitable in 13 and central motor conduction time (CMCT-ADM) was prolonged in 7 sides. In the lower limbs the motor pathways were inexcitable in 10 and CMCT-TA was prolonged in 14 sides. The CMCT abnormalities did not follow a constant pattern but were randomly distributed and were asymmetric in the upper limbs in 7 and lower limbs in 3 patients. Asymmetric and randomly focal abnormalities in central motor conduction in our patients are consistent with asymmetric and focal neuronopathy in MND.     

Carbohydrate molecular weight profiling, sequence, linkage, and branching data: ES-MS and CID

This section summarizes several strategies for a more complete understanding of carbohydrate structure with a focus on glycolipids and glycoprotein glycans. The techniques include periodate oxidation to impart greater molecular specificity to isomeric glycans, methylation to improve sensitivity and the information content within CID spectra, electrospray for "soft" and efficient ionization, and CID to obtain structural detail. The lipophilicity of the products following derivatization contributes to product cleanup by solvent extraction and enhances sensitivity during ES. When combined with CID information, this yields sequence, linkage, and branching information. Oxidation and reduction preceding methylation augments CID analysis with an altered structure that can be profiled at the same sensitivity. Within the context of established motifs, these contrasting profiles corroborate glycan structure and specifically identify isobaric elements transparent in the initial profile. An earlier report indicating ring-opening fragments were essentially absent in low-energy collisions of methylated and natriated oligosaccharides contrasts our observations. However, as this report used a methylated oligomer containing an internal N-acetylhexose as an illustration, the conclusion is plausible (cf., Figure 9). The poor ionization efficiency of FAB and the high matrix background limit the dynamic range in the CID spectrum and, thereby, the ability to unambiguously identify weaker peaks. It would be expected that high-energy CID affords a broader range of fragment types, including ring-opening fragments. In terms of a structural methodology, this is ambivalent since the increase in fragmentation pathways also applies to small molecule eliminations which are usually less informative. In ES-CID-MS, the carbohydrate chemist has a powerful new tool in hand for structural elucidations that can be conducted at the low-picomole level. Parallel developments can be expected to continue for other ionization methods, in particular matrix-assisted desorption/ionization on linear and reflectron time of flight mass spectrometers, and improvement in the performance and sensitivity of high-resolution mass analyzers through the use of focal plane detectors and more sophisticated hardware and software for Fourier transform ion cyclotron resonance mass measurements. These have, as yet, only begun to be applied to carbohydrate structural analysis but should add still more versatility to experimental design in the future.     

Low- and high-density lipoproteins as mitogenic factors for vascular smooth muscle cells: individual, additive and synergistic effects

The mitogenic activities of low (LDL)- and high (HDL)-density lipoproteins have been examined in cultures of human vascular smooth muscle cells (VSMC). LDL and HDL3 dose-dependently (EC50 values approximately 50 micrograms/ml) stimulated DNA and protein synthesis ([3H]-thymidine and [3H]-leucine incorporation, respectively) in the absence of exogenously added mitogens. The synthetic responses of VSMC to combinations of LDL and HDL3 were additive, indicating that each lipoprotein mediates discrete effects. LDL or HDL3 promoted VSMC proliferation under strict mitogen-free conditions, but this growth response was not sustained. VSMC exposed to combinations of lipoproteins (either LDL or HDL3) and growth factors (either PDGF-BB, EGF, bFGF or IGF) exhibited synergistic DNA synthesis responses. In the combined presence of PDGF-BB and either LDL or HDL3, VSMC proliferation was sustained. Anionized lipoprotein preparations (oxidized, acetylated, carbamylated or malonimylated) also stimulated DNA and protein synthesis. Since the antioxidant beta-hydroxylated toluene did not block the effect of native LDL on DNA synthesis, and fucoidin, a specific competitor for the 'scavenger' receptor, did not inhibit oxidized LDL-induced DNA synthesis, activation of mitogenic signals by lipoproteins does not depend on lipid peroxidation. Rather, the apparent intrinsic mitogenic potential of lipoproteins may depend upon their direct activation of replication-coupled signal transduction systems.     

Activity of threonine deaminase and biosynthesis of avermectins in the culture of Streptomyces avermitilis

The influence of ammonium, threonine, isoleucine and valine on the activity of threonine deaminase and the biosynthesis of avermectins in the culture of two mutants of Streptomyces avermitilis, i.e. a sensitive one and a resistant one with respect to alpha-amino-beta-oxyvaleric acid, a threonine antimetabolite, was studied. It was shown that the synthesis of threonine deaminase was induced by threonine and valine in the mycelium of both the mutants. The level of threonine deaminase was higher in the mycelium of the antimetabolite resistant mutant. The antibiotic activity of the resistant mutant was lower while the relative content of the group B avermectins in the pool of the synthesized avermectins was higher than that in the culture of the sensitive mutant.