Identification of the 190 kD microtubule-associated protein in cultured fibroblasts and its association with interphase and mitotic microtubules

We previously investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of the adrenal medulla and adrenal cortex and found that they contain a new kind of MAP with a molecular weight of 190,000 (190 kD MAP) as a major species (Kotani, S., H. Murofushi, S. Maekawa, C. Sato, and H. Sakai. Eur. J. Biochem. 156, 23-29, 1986). We now have used an affinity purified anti-(190 kD MAP) antibody and show by indirect immunofluorescent microscopy the association of this MAP with microtubules in situ in TIG-3 cells (human embryonic lung fibroblasts). The 190 kD MAP was present along the interphase and mitotic microtubules, and there was no marked difference between the staining pattern with anti-tubulin and that with anti-(190 kD MAP) antibodies, evidence that the localization of 190 kD MAP is not restricted to the subset of microtubules. We also isolated MAPs from TIG-3 cells and identified their 190 kD MAP as a major heat-stable component. Several other unidentified polypeptides were recovered in the MAP fraction specifically.     

Correlation between bilayer destabilization and activity enhancement by diacylglycerols in reconstituted Ca-ATPase vesicles

Using the reconstituted Ca-ATPase vesicles as a model system, we demonstrated that the presence of 1,2-dioleoyl-sn-glycerol (diolein) in the membrane introduces a pronounced enhancement in the Ca-transport function of Ca-ATPase, while the 1,2-dipalmitoyl-sn-glycerol (dipalmitin) does not. We also found by both 31P NMR and freeze-fraction electron microscopy that diolein destabilized lipid bilayers to a greater extent than did dipalmitin. We conclude that the tendency of diacylglycerols to destabilize the phospholipid bilayer is related to their capacity to enhance the activity of the membrane calcium pump.     

Determination of trace levels of fatty acid metal salts by high-performance liquid chromatography with fluorescence prelabeling

A simple method is described for picomole determinations of fatty acid metal salts. Fatty acid salts are directly labeled with 4-bromomethyl-7-methoxycoumarin in the presence of excess ethylenediaminetetraacetic acid tripotassium salt without any solvent extractions. The fluorescence derivatives of fatty acids are separated by reverse-phase high-performance liquid chromatography followed by fluorometric detection. The response of each fatty acid (C8-C18) calcium salt is linear from 1 to 50 micrograms/ml of samples. The detection limit is about 7 pmol. Good recoveries are obtained for the calcium salts of myrystic acid and soap (C8-C18, C18:1,2). The new method is successfully applied to the study on biodegradation of fatty acids in river water.     

Islet cell and 64K autoantibodies are associated with plasma IgG in newly diagnosed insulin-dependent diabetic children

There is a high prevalence of islet cell antibodies (ICA) and autoantibodies detected against an islet cell protein of Mr 64,000 at the time of clinical diagnosis of insulin-dependent diabetes (IDDM). In view of the biphasic immune response after antigen presentation, the purpose of this study was to determine the presence of ICA and antibodies against the 64,000 islet antigen after separation of IgM from IgG to prevent interference between the two antibody classes. Plasma samples from 10 newly diagnosed IDDM children and 10 healthy controls were precipitated with polyethylene glycol (PEG), and the crude Ig was subjected to Sephacryl S-300 chromatography to separate IgM and IgG. ICA determined by indirect immunofluorescence on frozen sections of human pancreas showed reduced background immunofluorescence intensity in the purified fractions compared with crude plasma. The number of ICA-positive samples among the IDDM patients increased from 7/10 in plasma to 9/10 in the IgG fraction. There was an increase in the ICA titer in 6/9 of the positive samples. All purified IgM samples were ICA negative. Immunoprecipitation experiments by using Nonidet P-40 detergent lysates of [35S]methionine-labeled neonatal rat islets demonstrated that the 64,000 autoantibodies were in the IgG fraction. We found 7/10 IDDM samples to be positive, whereas all controls were negative. The background in the autoradiographic analysis was markedly reduced in the IgG fractions compared with immunoprecipitates with crude or PEG-purified plasma and the IgM fraction. ICA titers did not correlate to the ability of the IgG fraction to precipitate the 64,000 autoantigen. It is concluded that both the ICA and 64,000 autoantibodies are primarily of the IgG class at the time of clinical onset of IDDM, and that purification of IgG from human IDDM plasma facilitates the detection of the rat islet cell 64,000 antigen.     

Glycerol models of the ciliated epithelium of bronchial mucosa and their use in the diagnosis of chronic nonspecific lung diseases

The method of extraction of ciliated epithelium from biopsy samples of human bronchial mucosa with glycerol is suggested. Permeabilized cilia of glycerol-extracted cells can be easily reactivated by exogenous ATP. This method was used for the study of ciliary dyskinesia in patients with chronic lung diseases. It was shown that in patients with Kartagener's syndrome neither freshly-isolated, nor glycerol-extracted ATP-treated cilia were motile. On the other hand, in some patients with bronchial asthma ATP reactivated glycerol-extracted cilia, while cilia of freshly-isolated cells remained immotile. The study shows that glycerol permeabilization and reactivation by ATP can be used for the analysis of cilial contractile apparatus in patients with chronic lung disease.     

Enzymic synthesis ofL-Lysine fromDL-α-Amino-ε-caprolactam by new microbial strains

The production of L-lysine fromDL-α-amino-ε-caprolactam (DL-ACL) by new strains producingL-α-amino-ε-caprolactamase and aminocaprolactam racemase is described. Optimal conditions for hydrolysis ofL-ACL byCryptococcus sp. and for racemization of ACL by cells of a strain isolated in nature and identified asPseudomonas sp. were determined. Synthesis ofL-α-amino-ε-caprolactamase is induced byDL-ACL orL-lysine with the same effectivity. A positive effect of phosphates (potassium salts) on reduction of the induction lag was detected, the synthesis of this enzyme was found to be repressed by glucose and some possibilities of the reversion of this repressive effect were demonstrated. Under conditions optimal for the production of both enzymes a quantitative theoretical conversion of 10 % aqueousDL-ACL toL-lysine by a mixture of native cells in a mass ratio of 1: 2 (producer of ACL-hydrolase to producer of ACL-racemase) occurred in 8 h at 40 °C and pH 8.0     

Detection of Staphylococcal Enterotoxin A,B, and C in Milk By an Elisa Procedure

Enterotoxigenic reference strains of Staphylococcus aureus were cultivated in sterile whole and skim milk for 18 h at 37°G. Staphylococcal enterotoxin A, B, and C were detected directly in the milk by an enzyme linked immunosorbent assay (ELISA), sensitive down to 1 ng/ml. Enterotoxins in the range of 1 ng–20 µg/ml milk were detected without any concentration or extraction. Skim and whole milk were almost identical as medium for enterotoxin production.     

Glycosylation of catalase inhibitor necessary for activity

A protein isolated from maize scutella which inhibits catalase in vitro has been shown to contain 12% carbohydrate in the form of galactose. This corresponds to four galactose molecules per inhibitor subunit. Removal of the carbohydrate with β-galactosidase or blockage with a galactose-specific lectin abolished activity of the inhibitor.