Rapid purification of human DNase I using mouse monoclonal anti-DNase I antibodies and characterization of the antibodies

Five anti-human deoxyribonuclease I (DNase I) monoclonal antibodies were obtained from BALB/c mice immunized with DNase I purified from human urine. Four of them inhibited DNase I enzyme activity, as did a rabbit polyclonal antibody; these 4 did not have immunostaining ability. The remaining one had immunostaining ability but no inhibitory activity. A Sepharose 4B column conjugated with 1 of the 4 antibodies that had inhibitory activity effectively adsorbed and eluted the DNase I enzyme; this did not occur with the rabbit polyclonal antibody. We showed that adding an immunoaffinity chromatography step made the purification of human DNase I easier and faster than the conventional procedure.     

Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in treatment of corneal alkali burns in mice

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 microg/microl) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semi-quantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-kappaB activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-alpha in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-alpha, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-alpha-/- mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-alpha/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

Two distinct antigenic types of the polysaccharide chains of Helicobacter pylori lipopolysaccharides characterized by reactivity with sera from humans with natural infection

We have purified lipopolysaccharides (LPS) from 10 Helicobacter pylori clinical isolates which were selected on the basis of chemotype and antigenic variation. Data from immunoblotting of the purified LPS with sera from humans with H. pylori infection and from absorption of the sera with LPS indicated the presence of two distinct epitopes, termed the highly antigenic and the weakly antigenic epitopes, on the polysaccharide chains. Among 68 H. pylori clinical isolates, all smooth strains possessed either epitope; the epitopes were each carried by about 50% of the smooth strains. Thus, H. pylori strains can be classified into three types on the basis of their antigenicity in humans: those with smooth LPS carrying the highly antigenic epitope, those with smooth LPS carrying the weakly antigenic epitope, and those with rough LPS. Sera from humans with H. pylori infection could be grouped into three categories: those containing immunoglobulin G (IgG) antibodies against the highly antigenic epitope, those containing IgG against the weakly antigenic epitope, and those containing both specific IgGs; these groups made up about 50%, less than 10%, and about 40%, respectively, of all infected sera tested. In other words, IgG against the highly antigenic epitope were detected in more than 90% of H. pylori-infected individuals with high titers. IgG against the weakly antigenic epitope were detected in about 50% of the sera tested; however, the antibody titers were low. The two human epitopes existed independently from the mimic structures of Lewis antigens, which are known to be an important epitope of H. pylori LPS. No significant relationship between the reactivities toward purified LPS of human sera and a panel of anti-Lewis antigen antibodies was found. Moreover, the reactivities of the anti-Lewis antigen antibodies, but not human sera, were sensitive to particular alpha-L-fucosidases. The human epitopes appeared to be located on O-polysaccharide chains containing endo-beta-galactosidase-sensitive galactose residues as the backbone. Data from chemical analyses indicated that all LPS commonly contained galactose, glucosamine, glucose, and fucose (except one rough strain) as probable polysaccharide components, together with typical components of inner core and lipid A. We were not able to distinguish between the differences of antigenicity in humans by on the basis of the chemical composition of the LPS.     

Liberation of serotonin from rabbit blood platelets by bacterial cell walls and related compounds.

A study was made on the activity of various bacterial cell walls and peptidoglycans to liberate serotonin from rabbit blood platelets. All of the test cell walls or peptidoglycans prepared from 27 strains of 21 bacterial species were shown to cause a marked release of serotonin, regardless of differences in types of peptidoglycan and non-peptidoglycan moieties and in some biological properties. The assay made with the water-soluble "digests" of Staphylococcus epidermidis cell wall peptidoglycans, which were prepared by use of appropriate enzymes, revealed that a polymer of peptidoglycan subunits (a disaccharide-stempeptide) was definitely active in the release of serotonin, but a structural unit monomer was inactive. Among a variety of synthetic muramylpeptides and their 6-O-acyl derivatives, only 6-O-(3-hydroxy-2-docosylhexacosanoyl)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-lysyl-D-alanine was found to hold a strong serotonin-liberating activity.     

Nine-bp repeat polymorphism in exon 1 of thehMSH3 gene

Summary We have identified a polymorphic 9-bp repeat sequence in exon 1 of thehMSH3 gene using polymerase chain reaction (PCR). Five alleles were observed in unrelated Japanese individuals with heterozygosity of 0.57.     

Dipole-tracing of 'awareness' attenuating the cortical components of somatosensory evoked potentials

Using the dipole-tracing method, the source generators of N18, P22 and P40 of the somatosensory evoked potential (SEP) were estimated as the equivalent dipole. After voluntary action of the thumb flexion, no changes were observed in N18 or P40, but the amplitude of P22 was suppressed. The after-effects of intention accompanied by a voluntary action or the subject's awareness that electrical stimulation will be given after the voluntary action were treated as 'awareness'. By subtracting the pure SEP from SEP during 'awareness', it was found that the equivalent dipole of 'awareness' of P22 was located at the same region of pure P22, but the vector was of opposite orientation. 'Awareness' attenuated the perceptive potential of SEP like P22 generated in the cortex.