Efficient production of IGG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide,pokeweed mitogen,and interleukin 4

Summary Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed forin vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulations of human IgG production was a culture medium containing 20 μg/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.     

Putative morphogen, DIF, of Dictyostelium discoideum induces apoptosis in rat pancreatic AR42J cells

We have recently shown that differentiation-inducing factor-1 (DIF-1) of Dictyostelium discoideum is capable of raising intracellular calcium concentration ([Ca²⁺]_i) and suppressing cell proliferation of rat pancreatic AR42J cells in a dose-dependent manner, and that DIF-1 at a concentration of 40 μmol/L is toxic to the cells. In this study, we have further characterized the cytotoxic effect of DIF-1 on AR42J cells and have analyzed the effect of DIF-1 on [Ca²⁺]_i. In the presence of 40 μmol/L DIF-1, cells began to bleb after approximately 6 h, and most had died within 48 h. Biochemical analysis revealed that DNA fragmentation was accompanied by cell death. Monitoring the changes in [Ca²⁺]_i induced by DIF-1, it was found that cells were able to adapt to stimulation with DIF-1 so that they did not respond to subsequent stimulation by DIF-1. These results indicate that DIF-1 induced apoptosis in AR42J cells probably via a cell signaling system.     

Enzyme- and immuno-histochemical localization of kallikrein

Summary Localization of kallikrein in the human kidney was investigated by two markers: kallikrein-like activity and kallikrein antigenicity. Kallikrein-like activity was demonstrated enzyme-histochemically by using a synthetic substrate for kallikrein, pro-phe-arg-naphthyl-ester. Kallikrein antigenicity was demonstrated by the unlabelled antibody peroxidase-antiperoxidase method using an antiserum against human urinary kallikrein. The kallikrein-like activity was localized in the proximal tubular cells without any corresponding kallikrein antigenicity. Neither kallikrein-like activity nor kallikrein antigenicity was noticed in any other tubular cell. These results are contrary to those in the ductal cells of the human parotid gland where the kallikrein-like activity and the kallikrein antigenicity were identical in their locations. The peroxidase-antiperoxidase method revealed, for the first time, kallikrein antigenicity both in the interstitium and in the basement membrane region of Bowman's capsule and of all the tubules, possibly representing circulating glandular kallikreins deposited in the renal tissue. Thus, the present findings are consistent with the hypothesis that the urinary (renal) kallikreins are derived from circulating glandular kallikreins.     

Separation of RRBC-RFC and non-rosette forming cells (non-RFC) of guinea pig T-cell populations and their functional difference. 1. Response of RRBC-RFC and non-RFC to either Con-A or LPS.

Guinea pig lymph node cells suspension (LNC-O) was filtered through a glass wool column and the effluent (LNC-G) was further filtered through a nylon column. In this effluent (LNC-NE) about 30 per cent of the lymphocytes was identified as non-rosette forming cells (non-RFC). The non-RFC fraction was separated from LNC-NE fraction by Ficol-Conray specific gravity centrifugation or effluent cells reacted previously to rabbit red blood cells (RRBC). The upper layer after centrifugation, designated non-RFC fraction, was separated. In this fraction 96% of the cells were lymphocytes and about 95% of them were non-RFC, which lacked receptors for rabbit red blood cells (RRBC) or EAC and detectable surface immunoglobulin by conventional techniques. Though the response of the lymphocytes in the non-RFC fraction to mitogenic (Con-A, LPS) or antigenic stimulation was lower in comparison with that in RFC-rich fraction, the response of non-RFC to ConA exceeded the response to LPS. These facts suggest that at least a portion of the non-RFC may be cells from the T-cell line.     

Microtubular structures in a stable staphylococcal L-form.

Microtubular structures, which were demonstrated as straight, dense-walled cylinders attached to cell membranes, were found in a stable staphylococcal L-form grown in the absence of antibiotic.     

New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.     

Stereoselective hydrolysis of dl-menthyl succinate by gel-entrapped Rhodotorula minuta var. texensis cells in organic solvent

Summary dl-Menthyl succinate was successfully hydrolyzed stereoselectively by Rhodotorula minuta var. texensis cells entrapped within photo-crosslinked or polyurethane resin gels in water-saturated n-heptane. The hydrolyzed product was found to be pure l-menthol. The catalytic activity of the immobilized cells, especially those entrapped in urethane polymers, was far more stable than that of the free cells. The half-life of the polyurethaneentrapped cells was estimated to be 55–63 days in the organic solvent.Dedicated to the 65th birthday od Professor Dr. G. Manecke     

The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica charantia)

The complete amino acid sequence of ribonuclease (RNase MC) from the seeds of bitter gourd (Momordica charantia) has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with trypsin, lysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The protein contains 191 amino acid residues and has a calculated molecular mass of 21,259 Da. Comparison of this sequence with sequences of the fungal RNases, RNase T2, and RNase Rh, revealed that there are highly conserved residues at positions 32-38 (TXHGLWP) and 81-92 (FWXHEWXKHGTC). Furthermore, the sequence of RNase MC was found to be homologous to those of Nicotiana alata S-glycoproteins involved in self-incompatibility sharing 41% identical residues.