Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma

By fusing Con A-activated BALB/c mice spleen cells with AKR thymoma BW5147 cells, we prepared a hybridoma producing a monoclonal nonspecific suppressor factor (MNSF). This factor inhibits a generation of LPS-induced immunoglobulin-secreting cells. We used ELISA for the bioassay of MNSF activity. With this method, a stable E17 hybridoma clone was selected, and its product in culture medium was isolated and characterized. MNSF fractionated on Sephadex G-100 in saline buffer shows a form with multiple m.w., but fractionated in 0.4 M pyridine-acetic buffer, it is limited to two species of approximately 24Kd and 16Kd. The MNSF was purified by hydroxyapatite chromatography, with marked effectiveness. MNSF activity was found exclusively in the 0.35 M sodium phosphate elution, and the content was further fractionated on subsequent gel filtration in the high ionic strength buffer described above. The purified factor exhibited two forms, of 24Kd and 16Kd, and showed peaks of pI 5.3 and 5.7, respectively, on isoelectric focusing. The MNSF preparation described here is stable at 56 degrees C and unaffected by 2-mercaptoethanol, but is unstable at pH 2.0 and is sensitive to tryptic proteolysis. We injected the hybridoma cells into the peritoneal cavity of pristane-primed F1 (AKR/J X BALB/c) mice, and a large amount of pure MNSF was obtained from the ascites, the characteristics of which were similar to those in the culture supernatant. Thus, the MNSF obtained from the E17 hybridoma consists of functionally identical but physicochemically different discrete proteins. This simple method of purification can serve as a probe for further characterization of MNSF and its application in in vivo experiments.     

The conformation of the lysyl side chain of substrates at the active center of trypsin

In order to study the conformation of the side chain of lysine substrates bound to the active center of trypsin, two lysine analogs, cis- and trans-2,6-diamino-4-hexenoic acids (4,5-dehydrolysines) were synthesized and kinetic parameters for the hydrolysis of benzoyl methyl esters and phenylthiazolones of these analogs by this enzyme were compared with those of the corresponding lysine derivatives. The derivatives of cis-4,5-dehydrolysine were hydrolyzed much more slowly than those of lysine, owing largely to the small kcat values for the former. On the other hand, the derivatives of the trans-isomer were hydrolyzed at about the same rates as those of lysine and the values of both Km and kcat of the former are also similar to those of the latter. These results indicate that the conformation of the side chain of the lysine derivatives hydrolyzed by trypsin is such that the beta- and epsilon-carbons are in a trans-like conformation, as suggested by X-ray crystallographic studies of inhibitor-trypsin complex.     

Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.     

Determination of 9α,11β-prostaglandin F2 by stereospecific antibody in various rat tissues

In view of the recent finding that prostaglandin D₂ is stereospecifically converted to 9α,11β-prostaglandin F₂, an isomer of prostaglandin F₂α, a highly specific and sensitive radioimmunoassay for 9α,11β-prostaglandin F₂ was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9α-11β-prostaglandin F₂ were raised in rabbits immunized with the bovine serum albumin conjugate, and [³H]9α,11β-prostaglandin F₂ was enzymatically prepared from [³H]prostaglandin D₂. The assay detected 9α,11β-prostaglandin F₂ over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-section with prostaglandin F₂α, prostaglandin F₂β and 9β,11β-prostaglandin F₂. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9α,11β-prostaglandin F₂ was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 ± 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D₂ (ca. 180 ng/g wet weight) and prostaglandin F₂α (ca. 70 ng/g) were produced in the brain; however, 9α,11β-prostaglandin F₂ was detected neither there nor in the blood. This result demonstrates that the conversion to 9α,11β-prostaglandin F₂ is a minor pathway, if one at all, of prostaglandin D₂ metabolism in the rat brain.     

Hydropiperoside,a novel coumaryl glycoside from the root of Polygonum hydropiper

From the methanol extract of the root of Polygonum hydropiper, a novel coumaryl glycoside hydropiperoside was isolated together with anthraquinone, ellagic acid 3,3′-di-O-methyl ether, gallic acid, two quercetin glycosides and an unidentified aromatic δ-lactone possessing antifertility activity. The structure of hydropiperoside was established as β-d-(1,3,6-tri-p-coumaryl)-fructofuranosyl-α-d-glucopyranoside by combination of extensive ¹H NMR and ¹³C NMR spectra, and the FD/MS spectrum.     

Sesquiterpenes from Porella species

The Porella liverworts contain abundant sesquiterpenes. ent-Biocyclogermacrene, three ent-aromadendrenes, a unique hydrocarbon, α-pinguisene and two drimane type sesquiterpenes were obtained together with the intensly pungent component, tadeonal, from P. vernicosa and P. gracillima. P. macroloba contained the same sesquiterpenes except for the absence of ent-bicyclogermacrene and the ent-aromadendrenes. The fragrant odor of P. perrottetiana was composed of α-pinene and camphor.     

Actin cytoskeleton of resting bovine platelets

Actin filaments in resting discoid bovine platelets were examined by fluorescence and electron microscopy. Rhodamine-phalloidin staining patterns showed a characteristic wheel-like structure which consisted of a central small circle connected by several radial spokes to a large peripheral circle. This wheel-like structure was composed of actin filaments forming a characteristic arrowhead structure with heavy meromyosin from muscle. Actin filaments were densely arrayed in parallel with a marginal microtubule band and radiated out from the center to the periphery. Platelets treated with colchicine lost their marginal microtubule band but retained their wheel-like structure and normal discoid form. Cytochalasin B disrupted the wheel-like structure but not the marginal microtubule band or the normal discoid form. After simultaneous treatment with both cytochalasin B and colchicine, platelets lost their discoid shape. These results suggest that actin filaments and microtubules both play important roles in the maintenance of the discoid shape of resting bovine platelets.