This paper gives a survey of recent methodological studies of capillary electrophoresis at the Department of Biochemistry, Uppsala University. Several methods for on-and off-tube concentration of solutes have been developed. These methods also permit desalting of the sample, which is necessary for the creation of narrow starting zones in zone electrophoresis and to reduce the risk of precipitation and narrowing of the separation window in isoelectric focusing (IEF). A unique method designed particularly for desalting of samples for IEF is also discussed. To obtain high resolution the adsorption of solutes to the capillary wall must be eliminated. A pH stable, hydrophilic polymer coating has, therefore, been developed. Although polymer solutions give lower resolution than do gels they are often used as molecular-sieving media because they are replaceable and thus permit repetitive automated analyses in the same capillary. We have shown that low-melting methoxylated agarose gels are also replaceable and give the same high resolution as do polyacrylamide and dextran gels. Following an electrophoresis the methoxylated agarose gels can be displaced in the stationary capillary past the detection window. This scanning technique has several advantages. A capillary with strong electroendosmosis permits, in principle, separation of both acidic and basic proteins in one run. However, depending on whether the capillary wall is negatively or positively charged either the basic or the acidic proteins most often migrate with strong tailing caused by the electrostatic interaction with the capillary wall. The distortion of the zones is much less if the electroendosmotic flow is replaced by a hydrodynamic flow and the electrophoresis is performed in a coated electroendosmosis-free capillary to suppress adsorption of both basic and acidic proteins. By a unique technique we can prepare chromatographic beds with diameters as small as 5-25μm. These beds, which are more homogeneous than conventional beds built up of preformed beads, can be used with advantage for electrochromatography and for different modes of capillary chromatography of fractions obtained from micropreparative HPCE experiments. There is a general trend and desire to decrease the analysis time without sacrificing resolution. Therefore, we have developed low-conductivity buffers with satisfactory buffering capacity which permit field strengths as high as 2, 000V/cm with attendant very short run times. Many enantioselective agents gave strong UV-absorption. Therefore, the experimental conditions must be chosen so that they do not pass the detection window. Otherwise, the noisy background is very disturbing. When a zone migrates from a straightinto a curved section of a capillary the molecules at the outer lane will lag behind those at the inner lane. The attendant loss in resolution, which all commercial apparatus exhibits, can be decreased considerably by coiling the capillary into the figure-of-eight or a serpentine path. A straight capillary is, however, preferable. Methods for the determination of pH and electrical conductivity in small volumes are discussed. A technique for micropreparative HPCE and HPLC is presented. An HPCE apparatus designed according to new principles is described (straight capillary; one electrode vessel is closed to avoid hydrodynamic flow in the capillary; electroendosmotic pump for washing of the capillary; thermostated slit for the UV beam to obtain a straight base line; only a small part of the capillary is not cooled actively; thermal application of the sample; the sample rests as a small droplet on a carousel; the droplet is covered by a cap which can be lifted by a magnet; effective insulation for field strengths up to at least 2, 000V/cm).

View full abstract

We would like to focus on two topics which were recently made in our laboratoty on structure/function relationship of alpha-fetoprotein (AFP) by recombinant DNA technology: identification of estrogen binding site of rat AFP, and evaluation of glycosylation of two possible N-glycosylation sites in rat AFP. In order to identify estrogen binding site of rat AFP, a series of rat-human chimeric AFPs was designed, since rat AFP binds to estrogen while human AFP does not. Out of 7 rat-human chimeric AFPs, only those which carry a rat segment 426-467 in human back ground sequence conserved the estrogen binding activity, indicating the segment 426-467, where 15 amino acid substitutions were observed, contained the residues responsible for estrogen binding. A variety of human mutant AFPs in which some human-specific amino acid residues were substituted with those of rat AFP was obtained by site-directed mutagenesis employing polymerase chain reaction. Among 17 human mutant AFPs carrying different combinations of substitutions, those carrying 436 A→V, 437A→S, 438 T→I, 450 L→R, 451 L→S, 457 A→L, 461 I→Y conserved sufficient binding activities, strongly suggesting that these amino acid residues were responsible for the estrogen binding activity in rat AFP. The rat AFP resolves on polyacrylamide gel electrophoreses (PAGE) into two discrete bands, “Slow”and“Fast”, and this electrophoretic pattern was shown by us to depend on the heterogeneity of N-glycosylation. For evaluation of glycosylation at two possible N-glycosylation sites in rat AFP, we designed three mutant rat AFPs, which had either substitution of 93Asn→Gln, 229Asn→Gln, or 93Asn→Gln+229Asn→Gln, as well as recombinant rat AFP without mutation. SDS-PAGE before and after glycopeptidase F treatment of the mutant AFPs indicated that the“Fast”had one carbohydrate moiety at 229Asn and the“Slow”had two carbohydrate moieties at 93Asn and 229Asn. It was therefore indicated that 229Asn was fully glycosylated while 93Asn was partially.

View full abstract

Human AFP molecule has one asparagine-linked sugar chain. Recently, the difference in these sugar chain exists between several diseases, has been revealed. In order to study changes in these sugar chains, Con A, LCA and PHA-E4 lectins have been used. By Con A lectin-binding analysis of AFP, it was possible to distinguish HCC from YST and metastatic liver cancer, but impossible to differentiate HCC from liver cirrhosis. By LCA and PHA-E4 lectin-binding analyses of AFP, HCC was distinguished from liver cirrhosis in many cases. The increase of LCA or PHA-E4 bound AFP fraction strongly suggested HCC development in very near future in the patients with liver cirrhosis.

View full abstract

Alpha-fetoprotein (AFP) and albumin gene expression are specific for hepatocytes. Since liver contains not only hepatocytes but also sinusoidal lining cells such endothelial cell, Kupffer cell, Ito cell and pit cell, the liver specific gene expression may be regulated by paracrine, autocrine and endocrine mechanism. In the present study, we analyzed the factors involved in the regulation of AFP and albumin gene expression using cultured human hepatoma cell lines. Epidermal growth factor and phorbol ester synergistically suppressed the AFP enhancer activity, resulting in the down-regulation of both AFP and albumin gene expression. Hepatocyte growth factor and transforming growth factor β₁ suppressed the AFP gene expression through the inhibition of its promoter activity. Δ¹²-Prostaglandin J₂, an active form of prostaglandin D₂, repressed both AFP and albumin promoter activity. AFP and albumin mRNA levels were reversibly suppressed by the increased colloid osmotic pressure. Butyric acid, a natural fermentation product of colonic bacterial flora, inhibited the proliferation of HuH-7 human hepatoma cells in conjunction with the reciprocal regulation of AFP and albumin gene expression. These results indicate that AFP gene expression as well as albumin gene expression is physiologically regulated by varing factors in a paracrine fashion or an endocrine fashion through the arterial and the portal blood flow.

View full abstract

Leukocytes including neutrophils, monocytes/macrophages and lymphocytes are essential cellular components 3 involving in body defense and immunological reactions. In the normal inflammatory response, the peripheral leukocytes migrate across the blood vessels to the site of infection in response to chemotactic factors released in the site. The importance of cell to cell adhesion between the leukocytes and vascular endothelial cell in the migration of the leukocytes were substantiated by the finding of a recently defined heritable disease called leukocyte adhesion deficiency (LAD). LAD is a disorder characterized by recurrent bacterial infections, impaired pus formation and wound healing, persistent periodontitis, and an abnormally high peripheral leukocyte count. The profound pathogenesis of the LAD has been defined as impairments in adhesion-dependent leukocyte functions such as chemotaxis due to a lack of leukocyte cell-surface expression of adhesion molecules of LFA-1, Mac-1 and p 150/95, the β₂-integrin. Very recently, a new disorder having almost the same clinical features and leukocyte dysfunctions as the LAD but normal expression of the β₂-integrin was identified. The disease is characterized by the lack of cell-surface sugar components, the sialyl Lewis antigens which are the ligands of another kind of adhesion molecules of selectins expressed on the vascular endothelial cells. The presence of these two diseases indicates that the leukocyte locomotion requires cell to cell adhesion through both the β₂-integrin and selectins.

View full abstract

We analyzed midbands (abnormal bands) appearing in the serum lipoprotein fractionation by a polyacrylamide disc gel electrophoresis (Lp-DISC) and immunoblotting. Lp (a) and remnant lipoproteins were to be considered as lipoproteins which possibly appeared as the midbands on disc gel electrophoresis. Even though serum LDL was glycosylated in vitro, they appeared at the cathodal side of LDL. However, those bands migrated to the anodal side of LDL after additional incubation more than 72h at 37°C. It is conceivable that this migration of lipoprotein was due to oxidative reaction of LDL with reactive oxygen by inhibition effects following addition of EDTA and SOD. We therefore think that modified-LDL, glycated-LDL and oxidized-LDL may also appear as the midbands. Attention may need to be paid to the Lp-DISC midband including Lp (a), remnant lipoprotein and modified-LDL as a risk factor for arteriosclerosis from the viewpoint of its association in the formation of foam cells.

View full abstract

We amplified the tymidine kinase region of feline herpesvirus-1 (FHV-1) genomes of several strains by the polymerase chain reaction (PCR) using the extract of DNA with instaGene-DNA purification matrix and analyzed the PCR products by using restriction endonucleases. The detection limit of purified FHV-1 DNA using PCR was 0.5pg. The lysate of infected cells (100cells/μl) by instaGene was diluted with sterile distilled water, and this diluted lysate was analysed using PCR. DNA was detected to 100 times dilution. From these results, it may be suggested that the detected DNA is obtained from only one infected cell. DNA detection limit indicated by viral titer from the viral fluid using instaGene was 10TCID₅₀/0.1ml. Restriction patterns of PCR product cleaved with EcoR V, Mbo II, Pvu II, Mva I and Hap II revealed same genomic variation among tested FHV-1 strains.

View full abstract