Plasmodium falciparum: expression of the adenine phosphoribosyltransferase gene in mouse L cells

Genomic libraries of Plasmodium falciparum were constructed in the pBR322 plasmid. Using the DNA-mediated gene transfer technique, the genomic libraries were introduced into tissue-cultured mouse cells lacking the enzyme adenine phosphoribosyltransferase. Following selection for the adenine phosphoribosyltransferse phenotype, several colonies were isolated. All clones were shown to possess adenine phosphoribosyltransferase activity and pBR322 sequences. In addition, the Km value of adenine phosphoribosyltransferase (for adenine) from a transformant was found to be identical to that from P. falciparum. These results indicate that the adenine phosphoribosyltransferase gene of P. falciparum was successfully cloned and expressed in a mammalian system.     

Leishmania major: excreted factor, calcium ions, and the survival of amastigotes

Mouse macrophages infected with amastigotes of Leishmania major contain about 40% more intracellular exchangeable calcium than control macrophages. Similar elevation of intracellular exchangeable calcium was observed in macrophages engulfing red blood cells coated with purified excreted factor from L. major. The rate of cytolysis of red blood cells coated with excreted factor was significantly lower than that of uncoated controls. Excreted factor strongly binds calcium; thus, the possible role of a microenvironment rich in calcium bound to excreted factor within the phagolysosome in protecting the amastigotes may be considered.     

Resealing of the proximal and distal cut ends of transected axons: Electrophysiological and ultrastructural analysis

The fates of the proximal and distal segments of transected axons differ. Whereas the proximal segment usually recovers from injury and regenerates, the distal segment degenerates. In the present report we studied the kinetics of the recovery processes of both proximal and distal axonal segment following axotomy and its temporal relations to the alterations in the cytoarchitecture of the injured neuron. The experiments were performed on primary cultured metacerebral neurons (MCn) isolated from Aplysia. We transected axons while monitoring the changes in transmembrane potential and input resistance (Rn) by inserting intracellular microelectrodes into the soma and axon. Correlation between the electrophysiological status of the injured axon and its ultrastructure was provided by rapid fixation of the neuron at selected times postaxotomy. Axotomy leads to membrane depolarization from a mean of ?55.7 S.D. 12.8 mV to ?12.7 S.D. 3.3 mV and decreased Rn from tens of MΩ to 1–3 MΩ. The transected axons remained depolarized for a period of 10–260 s for as long as the axoplasm was in direct contact with the bathing solution. Rapid repolarization and partial recovery of Rn was associated with the formation of a membrane seal over the cut ends by the constriction and subsequent fusion of the axolema. Prior to the formation of a membraneous barrier, electron-dense deposits aggregate at the tip of the cut axon and appear to form an axoplasmic “plug.” Electrophysiological analysis revealed that this “plug” does not provide resistance for current flow and that the axoplasmic resistance is homogenously distributed. The kinetics of injury and recovery processes as well as the ultrastructural changes of the proximal and distal segments are cannot be attributed to differences in the immediated response of the segments to axotomy. © 1993 John Wiley & Sons, Inc.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Modified Radioimmunoassay for Murine Sarcoma-Leukemia Virus Group-Specific Antigen

Iodination of disrupted Moloney strain murine sarcoma-leukemia virus resulted in labeled group-specific (gs) protein which was subsequently purified on an isoelectrofocusing column. This iodinated purified gs antigen, prepared from a relatively small quantity of purified virus, was used in a radioimmunoassay. A radioimmunoassay inhibition method was developed so that antibody specific for mammalian C-type gs antigen could be measured in undiluted or low dilutions of test serum without altering the known reagents of the test. The gs antigen isolated from purified Moloney strain murine sarcoma-leukemia virus has an isoelectric point (pH 5.95) which is significantly lower than that reported for other murine leukemia viruses.     

Expression of the Major Internal Viral Polypeptide in Cells Transformed by Wild-Type and Temperature-Sensitive Murine Sarcoma Virus

Phenotypic expression of the murine intraspecies and interspecies antigenic determinants of the major type C viral structural 30,000-dalton polypeptide, p30, was measured by radioimmunoassay inhibition in cell lines from different species. Uninfected normal rat kidney (NRK) cells did not contain detectable levels of murine intraspecies and interspecies p30 antigen, whereas rat cells transformed by and producing murine sarcoma virus (MSV)-Moloney leukemia virus (M-MSV-MuLV) contained high levels of both murine intraspecies and interspecies p30 antigen. Significant amounts of murine intraspecies and interspecies p30 antigen were detected in wild-type MSV-transformed nonproducer NRK cells. The control of p30 antigen expression was examined in temperature-sensitive MSV-transformed nonproducer cells [NRK(MSV-1b)] which are cold sensitive for maintenance of the transformed phenotype. Both murine intraspecies and interspecies p30 antigens were detected in NRK(MSV-1b) cells when grown at the permissive (39 C) or nonpermissive (33 C) temperature, suggesting that p30 antigen expression is not correlated with maintenance of the transformed phenotype. The results demonstrate that previously undetectable p30 antigens are expressed in MSV-transformed nonproducer NRK cells, and suggest that the expression of p30 antigen may be a useful marker for viral gene expression in mammalian cells.     

Predation of Arternia cysts by water-tiger larvae of the genus Anacaena (Coleoptera,Hydrophilidae)

Artemia cysts are preyed upon by the larvae of Anacaena (Coleoptera, Hydrophilidae) in the wet supralittoral of the hypersaline Solar Lake near Elat. The larvae are specialized feeders of the cysts, as shown by laboratory experiments.     

Low-molecular-weight (61K) mu chain in P3HR-1 cells and various derived somatic hybrids

The expression of a truncated 61K mu chain in the Burkitt lymphoma lien P3HR-1 and a derived ouabain and TG-resistant subline, PUT, and in various somatic cell hybrids with PUT as one of their parents is described. Both PUT and P3HR-1 contain intracellular mu and kappa chains, but express no membrane immunoglobulin. Immunoprecipitation of 14C-labeled amino acid or [3H]glucosamine-labeled P3HR-1 extracts with anti-mu serum brought down the same 61K mu chain. Anti-light-chain sera did not precipitate the truncated mu chain. P3HR-1 is a clonal derivative of the Burkitt lymphoma (BL) line Jijoye. The parental Jijoye line is membrane-IgM positive and contains two normal-sized mu chains. Both are precipitable by anti-mu and anti-kappa sera. In addition, anti-mu also precipitated a 61K mu chain. A 61K mu chain was also identified in the following somatic hybrids: PICATPO, an autohybrid of two different P3HR-1 sublines, PUTRAL and PUT/ARH-77, derived from the fusion of PUT with the membrane-IgG-positive BL line Rael and the lymphoblastoid cell line (LCL) ARH-77, respectively, and the HP-1 (PUT/HL-60) hybrid, derived from the fusion of PUT with the granulocytic leukemia line, HL-60. The 61K mu chain could not be detected in some other BL/BL hybrid combinations, namely RAMPUT (PUT/Ramos) and NAMPUT (PUT/Namalva). The anti-light-chain serum (lambda or kappa) had no detectable effect on the truncated 61K mu chain in any of the cases tested, suggesting a lack of assembly between the 61K mu chain and the light chain.     

A microsomal fraction of cryptococcus neoformans induces lymphocyte blastogenesis in infected guinea pigs

Differential centrifugation of a homogenate from a mechanically disrupted, acapsular isolate of Cryptococcus neoformans resulted in a 105 000 X g supernatant (105 K) and a microsomalfraction (MS), both of which were capable of eliciting specific delayed cutaneous hypersensitivity and in vitro blastogenesis in infected guinea pigs. Polyacrylamide gel electrophoresis revealed two major proteins in the MS and seven proteins in the 105 K fractions. Electron microscopy of the MSshowed both membranes and ribosomes. In vitro lymphocyte blastogenesis elicited by 1 to 10 g/ml of antigens was maximal after 4 days of incubation; the reacting populations were peripheral blood leukocytes (PBL) and peritoneal exudate cells (PEC). Spleen cells of infected animals were unresponsive to in vitro antigenic stimulation. A simplified schedule of priming animals was infection with a single dose of virulent cryptococci. Under these conditions 3 of 6 animals' PBL responded with stimulation ratios of 6.55, 21.1, 35.42 to the MS and 1.41, 9.33, 17.39 to the 105 K antigens at l ug/ml. Four of six animals' PEC response were positive with stimulation ratios of 2.62, 2.72, 4.02 and 7.20 towards MS, and 2.62, 5.13, 5.71, 10.01 to the 105 Kantigens at 1 g/ml. When small capsule and large capsule isolates were used for infection, the small capsule form was not isolated from the brain, in contrast to its isolation from 2 of 3 animals receiving large capsule forms. Two of three animals in each group responded with blastogenic indices more vigorous in the PBL, and the most potent antigen was MS. There was no obvious difference in lymphocyte reactivity between the two groups.Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.