Spectrophotometric determination of inorganic phosphate in the presence of thiol compounds

A spectrophotometric method for inorganic phosphate determination in the presence of thiol compounds is described. Thiol compounds, which interfere with the measurement of inorganic phosphate by a modification of the method of Gomori, are removed by carboxymethylation by iodoacetate prior to the formation and reduction of phosphomolybdate complex. A linear standard curve is obtained by this method, and the method is suitable for the assay of a phosphate-releasing enzyme when the measurement must be performed in the presence of thiol compounds.     

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

A radioimmunoassay for human pro-luteinizing hormone-releasing factor [pro-LRF(14-69)OH]

A radioimmunoassay (RIA) for human pro-LRF(14-69)OH was developed with an antiserum, generated in a rabbit, to [Tyr67]pro-LRF(47-67)NH2 conjugated to BSA. This antiserum bound 28-32% of [125I]pro-LRF(14-69)OH at a final dilution of 1:2500 and the binding was inhibited by pro-LRF(14-69)OH in a dose-dependent manner. The sensitivity of the RIA was 31.2-62.5 pg and the dose that inhibited 50% of the binding to the tracer was 280-320 pg. Intra- and inter-assay coefficients of variation at 50% inhibition were 8 and 12%, respectively. Neither LRF nor pro-LRF(14-37)OH was recognized by the antiserum. The dilution curve generated with human hypothalamic extract was parallel to that of pro-LRF(14-69)OH. In addition the extract yielded a major immunoreactive peak emerging in elution volumes concordant with [125I]pro-LRF(14-69)OH on Sephadex G-50 chromatography.     

An establishment of ELISA for murine IL-6

Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6 was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml.     

Interleukin-4 downregulates interleukin-6 production by human alveolar macrophages at protein and mRNA levels.

Effects of interleukin-4 (IL-4) on IL-6 production by human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors was examined at the protein and gene levels. IL-6 production was quantitated by enzyme immunoassay (EIA) and bioassay using the IL-6 dependent murine hybridoma cell line MH60.BSF2. Results showed that when activated with LPS, AM released significantly more biologically active IL-6 than blood monocytes. Human rIL-4 significantly suppressed IL-6 production by AM and monocytes stimulated with LPS. Northern blot analysis revealed that IL-4 reduced the expression of IL-6 mRNA in LPS-stimulated AM and monocytes. The inhibitory effect was most pronounced when IL-4 was added with LPS or within the first 4 hr after LPS to AM or monocytes. The suppressive effect of IL-4 was completely neutralized by pretreatment with anti-IL-4 antibody. IL-4 also showed a suppressive effect on IL-6 production by macrophages generated in vitro by maturation of blood monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF). These observations suggest that IL-4 may play a critical role in in situ regulation of immune responses through suppression of IL-6 production.     

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Bromodeoxyuridine (BrdUrd) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and 3H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0.05% proteinase at 37 degrees C for 20 min and hydrolysis with 1N HCl at 37 degrees C for 20 min was sufficient to detect BrdUrd immunoreactivity in 3H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd immunohistochemistry in cell-kinetic studies.     

Microorganisms of the San Francisco Sour Dough Bread Process: I. Yeasts Responsible for the Leavening Action

Two hundred isolates from San Francisco sour dough French bread fermentations (40 from each of five different bakeries) were screened by fermentation tests and for their ability to grow in the presence of cycloheximide (Actidione). All of the isolates from four of the bakeries and 70% of those from the fifth were unable to utilize maltose but grew well on other sugars, even in the presence of cycloheximide. The remaining few isolates from the fifth bakery utilized maltose but not galactose and were inhibited by cycloheximide. No bakers' yeast types were found. Sixteen of the maltose-negative and five of the galactose-negative isolates were subjected to more rigorous taxonomic procedures. All of the maltose-negative isolates were identified as asporogenous strains of Saccharomyces exiguus (Torulopsis holmii) and the galactose-negative ones, as S. inusitatus. The predominance of S. exiguus, its vigor in the particular acidic environment of the sour dough, and the correlation of its numbers with the leavening function constitute strong evidence on the role of this organism in the sour dough system.     

Establishment of five human myeloma cell lines

Summary Five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.