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.     

Genetic structure of the IncN plasmid N3

N3, a plasmid of incompatibility group N (IncN) was mapped by cleavage with restriction endonucleases. The restriction fragments were cloned into vector plasmids. All of the genes unique to IncN plasmids such as specific replication machinery, a restriction-modification system, and repair functions were located on a large portion which had no cleavage sites for many of the site-specific six base-identifying restriction endonucleases tested.     

Mitochondrial degeneration after organic phosphate poisoning in prosimian primates

Summary The degenerative reaction of mitochondria to tricresylphosphate (TCP) poisoning in spinal ganglion cells of Slow Loris (Nycticebus coucang coucang) were studied with the electron microscope. In neurones of animals treated with TCP, mitochondria display various stages of alterations which confirm mitochondrial involvement in TCP poisoning. The role of degenerated mitochondria in the formation of neuronal lipofuscin is discussed. It is suggested that the lipofuscin granule is a metabolic product inherently related to mitochondrial degeneration, irrespective of the primary cause: ageing or intoxication.Fellow of the Deutscher Akademischer Austauschdienst on study leave from the Medical Faculty, University of SingaporeA grant from the Deutsche Forschungsgemeinschaft (Gl. 28/20) is gratefully acknowledgedThe skilfull technical assistance of Mr. Tajuddin b.M. Ali, Mr. P. Gopal, Mr. R. Dungan and Mrs. C. Weinrichter is gratefully acknowledged     

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.     

Anthelmintic-induced destrobilation and its influence on calculated drug efficacy in Hymenolepis diminuta infections in rats

Anthelmintic efficacy studies typically involve direct counts of worms remaining in the host shortly after drug treatment. Few such studies, however, have considered the phenomenon of tapeworm destrobilation when determining effective dosages. The present study reports on the frequency of drug-induced destrobilation and the subsequent regeneration of Hymenolepis diminuta in rats following treatment with niclosamide or praziquantel and its implications with respect to the apparent efficacy of these anthelmintics. Drug efficacies very similar to those reported in the literature were determined upon examination of infected animals 24 hr posttreatment. Small regenerating worms were, however, observed in the small intestine of rats 8 days after treatment, indicating that destrobilated worms were present, but overlooked, during the initial examination. Within several days posttreatment, destrobilated worms can regenerate to a size that is readily apparent in the gut contents, allowing the effective dosage to be determined with much greater confidence. Due to the demonstrated ability of these destrobilated worms to regenerate to the gravid state, it is imperative that a fully effective anthelmintic dosage be determined and administered.     

The presence of an adenosine-5'-triphosphatase dependent on 6S tubulin and calcium ions in rat brain microtubules.

An ATPase activity was found in rat brain microtubules prepared by successive cycles of polymerization and depolymerization. On phosphocellulose column chromatography, the ATPase activity was recovered in the fraction eluted with 0.6 M KCl and containing the microtubule associated proteins. The ATPase activity was markedly stimulated by the addition of purified brain 6S tubulin, and the stimulation was dependent on the presence of Ca2+ ions. Approximately 50 pmol of purified 6S tubulin was required for the maximal stimulation in the presence of 8 microgram of microtubule associated proteins. The specific activity was 8 to 13 nmol of ATP hydrolyzed per min per mg of protein at 37 degrees C, and the Km value for ATP was 3 X 10(-5) M in the presence of added tubulin.     

Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.