Radioimmunoassay for non-enzymatically glycated serum proteins

We have developed a radioimmunoassay (RIA) for nonenzymatically glycated serum proteins. The polyclonal antibodies prepared against reduced glycated human albumin were specific for the glucitollysine residues of serum proteins. Serum proteins from diabetic patients (n = 25) contained 5.3 +/- 2.8 nmoles of glucitollysine/mg protein, compared to 2.0 +/- 0.2 in controls (n = 20). The intra- and inter-assay variables were 3.2-6.2% and 4.4-8.6%, respectively. Results from this assay procedure correlated well with those from the boronate affinity chromatography procedure (r = 0.94; P less than 0.001). The data suggested that diabetic serum proteins contained at least 2.5 times as much immunochemically detectable glucitollysine residures as normal serum proteins after reduction of the proteins with sodium borohydride.     

Carboxyl-terminal deletion analysis of the Streptococcus mutans glucosyltransferase-I enzyme

Sequential deletion of the carboxyl-terminal amino acids (including the six direct repeating units) of the glucosyltransferase-I (GTF-I) enzyme of Streptococcus mutans revealed differential effects on sucrase and GTF activities. Removal of all but one repeating unit resulted in a truncated enzyme with significant sucrase activity but no detectable GTF activity. These results are compatible with the presence of two functional domains in the enzyme.     

Anomalous secondary vascular tissue inHelminthostachys zeylanica (Ophioglossaceae)

The vascular anatomy ofHelminthostachys zeylanica was examined with special reference to anomalous secondary tissue. Primary xylem development gradually takes place centrifugally. In branched rhizomes with destroyed apices, the vascular cylinder apical to the insertion of branch traces is generally composed of primary xylem, accessory xylem, inner parenchyma of radially arranged cells, outer parenchyma of irregularly arranged cells, and partly crushed phloem, listed in order going outwards. The accessory xylem as well as the inner parenchyma ofHelminthostachys zeylanica is probably secondarily produced, partly to contribute to the branch traces, in a position corresponding to that of secondary vascular tissue developed from a normal cambium inBotrychium sensu lato. It is suggested that although a cambium is lacking inHelminthostachys zeylanica, the secondary vascular tissues are comparable between the genera. The phylogenetic implication of this tissue is discussed.     

The polymerase chain reaction for Mycoplasma pulmonis

In vitro DNA amplification by polymerase chain reaction was examined to detect Mycoplasma pulmonis. A pair of synthetic oligonucleotide primers was constructed, and used to amplify a unique sequence of M. pulmonis DNA. Amplified products were detected by agarose gel electrophoresis and verified by blot hybridization with a synthetic oligonucleotide probe. This system detected cellular DNA of M. pulmonis but not M. arthritidis or M. neurolyticum, and thus appears to be useful for M. pulmonis diagnosis.     

Heterocyclic amine-DNA adducts analyzed by 32P-postlabeling method

DNA adducts formed by 12 heterocyclic amines were analyzed by 32P-postlabeling method. Several DNA adducts were detected in rat liver by administration of each heterocyclic amine. Total adduct levels ranged from 0.5 for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to more than 250 for 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) per 10(7) nucleotides 24 hr after intragastric administration of these compounds. The N-hydroxy derivative of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was reactive toward DNA in vitro to form adducts. Addition of acetic anhydride to N-OH-MeIQx greatly enhanced its reactivity to DNA. 32P-Postlabeling analysis revealed that the MeIQx-DNA adducts formed in vivo and in vitro were identical. Thus, MeIQx would be metabolized in vivo to N-hydroxy form and further esterified to produce more reactive species, such as N-acetoxy form, which modify DNA to form adducts.     

The heterogeneity of rat kidney lysosomes revealed by immunoelectron microscopic staining for cathepsins B and H

The heterogeneity of lysosomes was studied by analyzing the immunostaining behavior of cathepsins B and H in rat kidney proximal tubule cells. Rat kidneys were fixed by perfusion and embedded in Lowicryl K4M. A protein A-gold technique was applied to serial sections and a double labeling technique to conventional sections. By analyzing the immunostaining behavior of cathepsins B and H in the same lysosomes which were cut into separate sections, four types of lysosomes were found: Type 1 positive for both proteinases; type 2 strongly positive for cathepsin B, but weakly or negative for cathepsin H; type 3 strongly positive for cathepsin H, but weakly or negative for cathepsin B; and type 4 negative for both proteinases. The double labeling by two different sizes of the protein A-gold probes showed these four types of lysosomes. The results indicate that there exists the lysosomal heterogeneity of the proteinase content in the kidney proximal tubule cells.     

Formate production from methanol by formaldehyde dismutase coupled with a methanol oxidation system

Summary Formaldehyde dismutase was greatly stabilized by immobilization in a urethane prepolymer (PU-6). The immobilized enzyme exhibited stochiometrical dismutation of formaldehyde to methanol and formate in several repeated reactions. Conversion of methanol to formate occurred in a reaction with an immobilized enzyme system consisting of alcohol oxidase, catalase and formaldehyde dismutase, and with an intact cell-mixture of Hansenula polymorpha and Pseudomonas putida. Furthermore, the stability of the cell-mixture during repeated reactions was greatly improved by the immobilization, the 600 mM methanol added periodically being converted to formate in a 75% yield in 12 h. The immobilized cellsystem was also effective for the conversion of several aliphatic alcohols, C₁ to C₄, to the corresponding acids.     

Comparative Study of Glial Marker Proteins in the Hypoplastic Cerebellum of Jaundiced Gunn Rats

The behavior of marker proteins of glial cells [alpha-enolase, beta-S100 protein, and glial fibrillary acidic protein (GFAP)] was investigated quantitatively by using enzyme immunoassay systems during the development of cerebellar hypoplasia in jaundiced Gunn rats. A neuronal marker protein, gamma-enolase, was also measured as a reference. At postnatal day 8 corresponding to the early stage of cerebellar damage, the amount of beta-S100 on a protein basis was significantly higher in jaundiced homozygotes (jj) than in control nonjaundiced heterozygotes (j+), whereas no differences in alpha- and gamma-enolases and GFAP were observed between the two groups of rats. At days 15 and 30, which correspond, respectively, to the advanced and late stages of cerebellar damage, the three glial proteins, especially GFAP, were higher and the neuronal protein was lower in the jj rat cerebellum than in the control. These results are consistent with the reported histological observations that neuronal cells are vulnerable and damaged by bilirubin, whereas glial cells seem to be less sensitive. On the other hand, the amounts of beta-S100 and alpha-enolase per cerebellum were significantly lower in jj rats at days 15 and 30, as in the case of gamma-enolase, whereas that of GFAP remained at the same level as the control at day 15 and showed a slight but significant decrease at day 30. The possibility is suggested that beta-S100 and GFAP may be available as biochemical indicators of glial cells, especially in the early and advanced stages of cerebellar damage, respectively, but that alpha-enolase is less available.     

Cellular and Vacuolar Fusion of Protoplasts Electrofused Using Platinum Microelectrodes

Protoplasts isolated from cultured cells of Coptis japonicaand Euphorbia millii were electrically fused using platinummicroelectrodes. The process involved two stages, cellular andvacuolar fusion, which are characterized respectively by transientwrinkling of the membrane and the formation of a dark-red precipitate. (Received June 12, 1987; Accepted October 13, 1987)     

Effects of Physical Parameters upon Protoplast Electrofusion

The effects of three physical parameters upon protoplast electrofusionwere studied using protoplasts from cultured cells of Coptisjaponica and Euphorbia millii. The osmotic potential of themedium did not appreciably affect the AC-field-induced protoplast-pairformation, but significantly influenced the fusion process ofthe paired protoplasts in response to DC pulses. The optimumosmotic potential was 0.55 to 0.60 Osm/kg H₂O in our system.The density of the medium markedly influenced both pair formationand fusion process. The optimum density was 1.13 to 1.14 g/cm₃,and at this density the yield of the fused protoplasts increasedto more than twice that of the control (1.10 g/cm₃). Hydrophiliccoating of the bottom surface of the chamber with Gellan gumor polyacrylamide gel was also effective for both pair formationand the fusion process, while coating with hydrophobic siliconewas entirely inhibitory. Possible interpretations of the effectsof these physical parameters upon protoplast electrofusion arepresented. ¹Permanent address: Biochemical Research Laboratories, KanegafuchiChemical Industry Co., Ltd., Takasago, Hyogo 676, Japan. (Received December 21, 1987; Accepted March 18, 1988)