Heterogeneity of big-big hPRL in hyperprolactinemia

Sera from a patient with macroprolactinoma (case 1) and from a hyperprolactinemic woman with regular menstruation (case 2) were analyzed for prolactin activity by gel filtration using Sephadex G-100, Sephadex G-200 and TSK G3000SW columns. The chromatographic profile by Sephadex G-100 showed that the percentage of immunoreactive big-big hPRL was 10.7% in case 1 and 64.1% in case 2. On Sephadex G-200 and TSK G3000SW columns, the molecular weight of big-big hPRL was estimated to be more than 500,000 daltons (big-big1 hPRL) in case 1 and approximately 250,000-300,000 daltons (big-big2 hPRL) in case 2. Big-big1 hPRL in case 1 was converted to big and little hPRLs when the serum was treated with 2-mercaptoethanol (2-ME), but part of the big-big2 hPRL in case 2 was converted to a larger molecule. Radioactive big-big hPRL generated by mixing labeled hPRL with the serum from case 1 was eluted with the void volume on Sephadex G-100 column and was not converted to the other molecular forms after 2-ME treatment. There were two radioactive big-big hPRL on TSK G3000SW column and these estimated molecular weights were more than 300,000 daltons. The data demonstrated the existence of at least two forms of big-big hPRL in the serum and indicated that radioactive big-big hPRL may be different from these hPRLs in the serum.     

Plasmid dependence of Pseudomonas sp. strain NK87 enzymes that degrade 6-aminohexanoate-cyclic dimer.

A bacterial strain, Pseudomonas sp. strain NK87, that can use 6-aminohexanoate-cyclic dimer as the sole source of carbon and nitrogen was newly isolated from wastewater of a factory which produces nylon-6. Two responsible enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (P-EI) and 6-aminohexanoate-dimer hydrolase (P-EII), were found in the NK87 strain, as is the case with Flavobacterium sp. strain KI72, another 6-aminohexanoate-cyclic-dimer-metabolizing bacterium (H. Okada, S. Negoro, H. Kimura, and S. Nakamura, Nature [London] 306:203-206, 1983). The P-EI enzyme is immunologically identical to the 6-aminohexanoate-cyclic-dimer hydrolase of KI72 (F-EI). However, antiserum against the 6-aminohexanoate-dimer hydrolase purified from KI72 (F-EII) did not react with cell extracts of NK87, indicating that the F-EII and P-EII enzymes are immunologically different. Restriction endonuclease analyses show that the NK87 strain harbors at least six plasmids ranging in size from 20 to 80 kilobase pairs (kbp). The P-EI and P-EII genes were cloned in Escherichia coli. Both the P-EI and F-EI probes strongly hybridized with a 23-kbp plasmid in Southern hybridization analyses. The P-EII probe hybridized specifically with an 80-kbp plasmid, but the F-EII probe hybridized with none of the plasmids harbored in NK87. These results indicate that the P-EI gene and P-EII gene are encoded on the 23-kbp and 80-kbp plasmids, respectively.     

Changes of DNA methylation level during pre-and postnatal periods in mice

DNA methylation in an adult mammalian body shows tissue-specificity. But when and how the specificity is established in the process of development has not yet been elucidated. Here we have investigated age-dependent changes in the amount of 5-methyldeoxycytidine (5mdC) that DNA of various mouse tissues contains during the late-fetal and postnatal periods, using high-performance liquid chromatography. The tissue-specificity in the 5mdC level was observed in the late-fetal stage, and the level continued to change during the subsequent periods. The most pronounced alterations were observed in brain and liver, where similar biphasic changes were seen, but at different ages. At maturation, the 5mdC levels were high in thymus, spleen and brain, intermediate in lung, and low in liver and sperm. The data demonstrate the importance of the peri- and postnatal periods in establishment of tissue-specificity in 5mdC content.     

Multiple expression of keratins, vimentin, and S-100 protein in pleomorphic salivary adenomas.

Immunohistochemical staining for S-100 protein and the intermediate filaments keratin and vimentin, was made in 41 salivary adenomas. In pleomorphic adenomas, great heterogeneity in the staining, as well as multiple and co-expressions of these proteins were found in the outer tumor cells of tubulo-ductal structures and modified myoepithelial cells, but not in the luminal tumor cells. All the outer tumor cells stained for S-100 protein, 97% for K8.12 keratin and 85% for vimentin. Of these cells, 29% showed multiple expression of K8.12 keratin, vimentin, and S-100 protein, and 17% showed co-expression of K8.12 and S-100 protein. Modified and neoplastic myoepithelial cells showed similar expressions of these proteins to those of outer tumor cells; myoepithelioma cells displayed the most complicated pattern, being positive for KL1, PKK1, and K8.12 keratins, vimentin and S-100 protein. In luminal tumor cells there was a heterogeneous expression of KL1 and PKK1 in 82%, and of KL1, PKK1, and K8.12 in only 14.7%. Based on the immunohistochemical findings obtained with different monoclonal antibodies in pleomorphic salivary adenomas, outer tumor cells may be derived from ductal basal cells and luminal tumor cells from intercalated duct cells.     

Peri-operative immunotherapy with OK-432

Pre- and postoperative intradermal administration of OK-432 enhanced the SU-PS skin reaction in patients with gastric cancer, but failed to prevent a fall in the NK activity induced by the operation.The change in NK activity was not associated with a change in the proportion of Leu 7-positive cells, but was related to Leu 11a-positive cells. Intradermal injection of OK-432 increased the proportion of Leu 7-positive cells in the patients in whom they accounted for less than 20% of lymphocyte population. The case was the same with Leu 11a-positive cells.Intravenous injection of OK-432 tended to increase suppressor-inducer T cells (CD4⁺2HA⁺ cells), B cells and Leu 7-positive cells. Particularly, the proportions of OK-M₁-positive cells and MHC class II antigen-positive cells increased in all patients. Immunotherapy with OK-432 given intravenously at a dose of 0.1 KE appeared to be safe because no side effects were essentially observed.     

The precursor of a metalloendopeptidase from human rheumatoid synovial fibroblasts. Purification and mechanisms of activation by endopeptidases and 4-aminophenylmercuric acetate.

Two active forms (Mr 45,000 and 28,000) of a metalloendopeptidase that digest proteoglycans and other extracellular matrix components of connective tissues have previously been purified from rheumatoid synovial cells and characterized [Okada, Nagase & Harris (1986) J. Biol. Chem. 261, 14245-14255]. To study the mechanisms of activation the precursor of this metalloendopeptidase has now been purified. The final products are homogeneous on SDS/polyacrylamide-gel electrophoresis and identified as a set of zymogens of Mr 57,000 and 59,000, in which the latter form is probably the product of post-translational glycosylation of the Mr 57,000 zymogen, as it binds to concanavalin A. The zymogen can be activated by trypsin, chymotrypsin, plasma kallikrein, plasmin and thermolysin, but not by thrombin. Although the activated metalloendopeptidase is further degraded by trypsin, plasma kallikrein and thermolysin during a prolonged incubation, it is relatively stable against plasmin and chymotrypsin. Activation with 4-aminophenylmercuric acetate is dependent on its concentration. It requires the reaction with the zymogen, possibly through thiol groups, and the continued presence of the agent. During this treatment the zymogen undergoes a sequential processing; first it becomes active without changing its apparent molecular mass, and then it is processed to low-Mr species of Mr 46,000, 45,000 (HMM) and 28,000 (LMM). The rate of conversion of the precursor into an initial intermediate of Mr 46,000 follows first-order kinetics (t1/2 2.0 h with 1.5 mM-4-amino-phenylmercuric acetate at 37 degrees C) and is independent of the initial concentration of the zymogen or the presence of up to a 676-fold molar excess of substrate, whereas the generation of HMM and LMM species is affected by these parameters. These results indicate that activation of the prometalloendopeptidase by an organomercurial compound is initiated by the molecular perturbation of the zymogen that results in conversion into the 46,000-Mr intermediate by an intramolecular action; the subsequent processing of this intermediate in HMM and LMM species is a bimolecular reaction. In vivo it is probable that the precursor of this metalloendopeptidase is activated either by direct limited proteolysis by tissue or plasma endopeptidases, or, alternatively, by factors that cause certain conformational changes in the zymogen molecule.     

Purification and Characterization of Thermostable Pullulanase from Bacillus stearothermophilus and Molecular Cloning and Expression of the Gene in Bacillus subtilis

A thermostable pullulanase (alpha-dextrin 6-glucanohydrolase [EC 3.2.1.41]) from a newly isolated Bacillus stearothermophilus strain (TRS128) was purified and characterized. The enzyme hydrolyzed (1-->6)-alpha-d-glucosidic linkages of pullulan to produce maltotriose, and the optimum temperature was 65 degrees C. About 90% of the enzyme activity was retained after treatment at 65 degrees C for 60 min. By using pTB522 as a vector plasmid, the pullulanase gene was cloned and expressed in Bacillus subtilis.     

C1q induces chemotaxis and K+ conductance activation coupled to increased cytosolic Ca2+ in mouse fibroblasts

Cultured mouse fibroblasts (L cells) respond to whole C with a slow hyperpolarization. Among the C components tested, C1q was found to be most effective. In contrast, the cell did not respond to C1, in which the collagen-like region of the C1q molecule is masked. The C1q-induced hyperpolarizing response was inhibited by collagen or C1q-specific antisera. Human diploid skin fibroblasts (Flow 1,000 cells) also exhibited similar membrane potential changes in response to whole C or C1q. After repeated applications of C1q, the cell membrane became unresponsive (desensitized). The treatment of L cells with pronase E inhibited the C1q-induced response, whereas the response to ATP, which is known to interact to its own receptor, was still preserved. The reversal potential of C responses was close to the K+ equilibrium potential. The hyperpolarizing response was inhibited by a blocker of Ca2+-activated K+ channels in fibroblasts (quinine), by deprivation of extracellular Ca2+ or by a Ca2+ channel blocker (nifedipine). By means of Ca2+-selective microelectrodes, the cytosolic free Ca2+ concentration was found to increase from 126 to 206 nM upon stimulation of L cells with C1q. Using an agarose-well method, L cells were observed to migrate predominantly toward C1q or whole C. It is concluded that the fibroblasts have the C1q receptor sensitive to pronase E and that activation of C1q receptors gives rise to Ca2+ influx, triggering an increase in the cytosolic free Ca2+ ions, which in turn induces a hyperpolarizing response as a result of the stimulation of Ca2+-activated K+ channels and initiates chemotaxis to C1q.