Dopamine Metabolism in Hypoxanthine-Guanine Phosphoribosyltransferase-Deficient Variants of PC12 Cells

Lesch-Nyhan syndrome results from a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). It is manifest by behavioral abnormalities, including self-mutilation, and evidence of abnormal 3,4-dihydroxyphenylethylamine (dopamine) metabolism. To assess whether an HPRT deficiency in a dopaminergic cell can adversely affect dopamine metabolism in that cell, dopamine metabolism was examined in HPRT-deficient variants of PC12 pheochromocytoma cells and in cells that had regained HPRT activity by virtue of transformation with a recombinant retrovirus containing the human gene for HPRT. There was no correlation between HPRT activity and endogenous dopamine levels, dopamine uptake, dopamine release, or monoamine oxidase activity. Transformation with the HPRT retrovirus did not adversely affect dopamine metabolism.     

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system

Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.     

Regional Amino Acid Concentration in the Brains of Rats Exposed to High Pressures

Regional amino acid concentrations were measured in rat brain fixed by microwave irradiation at three levels of elevated atmospheric pressure corresponding to different phases of the high-pressure neurological syndrome [20 atmospheres absolute (ATA), no clinical signs; 60 ATA, tremor; 85 ATA, severe tremor and myoclonic jerks]. No changes in amino acid content occurred at 20 or 60 ATA. At 85 ATA glutamine content increased in hippocampus, striatum, cerebellum, and substantia nigra, and gamma-aminobutyric acid content increased in hippocampus. It is suggested that enhanced glutamate release in various subcortical structures contributes to the myoclonic activity observed at 85 ATA.     

Membrane and cytoplasmic resistivity properties of normal and sickle red blood cells

The cytoplasmic resistivities and membrane breakdown potentials of normal (AA), sickle-cell-trait (AS), and sickle (SS) red blood cells have been measured by the biophysical methodology of resistive pulse spectroscopy over a range of osmolalities. At isotonicity, the average membrane breakdown potentials are virtually identical for the three types of cells occurring at about 1150 V/cm. Average isotonic cytoplasmic resistivities are somewhat higher for the SS cells (166.7±7.49 ohm-cm) compared to the AA (147.6±1.98 ohm-cm) or AS cells (148.7±1.79 ohm-cm). As medium osmolality is varied, the differences in resistive properties become enlarged, especially at very low and very high osmolalities. At high osmolalities, both types of sickle cells show a large increase in internal resistivity compared to the normals; at low osmolality, the SS samples exhibit a distinctly different membrane breakdown characteristic, decreasing in this parameter, whereas the other two groups increase. Of the 15 SS samples tested, three displayed much higher cytoplasmic resistivities at isotonicity: 218.2±5.25 ohm-cm, compared to an average of 153.5±3.46 ohm-cm for the other 12. The relationship between these high resistivities and the subfraction of irreversibly sickled cells in the sample is discussed.     

Effect of neon ions on synchronized Chinese hamster cells

The variation in radiosensitivity across the cell cycle after exposure to neon ions and 60Co gamma-rays is reported for cultured hamster cells. The cells were first synchronized by mitotic selection, then resynchronized in the region of the G1/S boundary by treatment with 10(-3)M hydroxyurea. Although the use of hydroxyurea improves the synchrony, it does sensitize cells at the G1/S boundary to some degree. The cells were exposed at the plateau and the distal peak position of a neon ion beam modified by a 10 cm wide ridge filter. The results indicate that the variation (ratio of maximum to minimum survival after fixed doses of radiation that are approximately matched to produce similar cell killing) was approximately 80 to 100-fold for 60Co gamma-rays and neon ions at the plateau, and 25-fold for distal peak neon ions. While the r.b.e. of distal peak neon ions decreased rapidly with increasing dose for cells in late S-phase, the r.b.e. is independent of dose for cells at the G1/S boundary.     

The kinetics of chemotactic peptide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils

Formyl-met-leu-phe (fMLP) induces actin assembly in neutrophils; the resultant increase in F-actin content correlates with an increase in the rate of cellular locomotion at fMLP concentrations less than or equal to 10(-8) M (Howard, T.H., and W.H. Meyer, 1984, J. Cell Biol., 98:1265-1271). We studied the time course of change in F-actin content, F-actin distribution, and cell shape after fMLP stimulation. F-actin content was quantified by fluorescence activated cell sorter analysis of nitrobenzoxadiazole-phallacidin-stained cells (Howard, T.H., 1982, J. Cell Biol., 95(2, Pt. 2:327a). F-actin distribution and cell shape were determined by analysis of fluorescence photomicrographs of nitrobenzoxadiazole-phallacidin-stained cells. After fMLP stimulation at 25 degrees C, there is a rapid actin polymerization that is maximal (up to 2.0 times the control level) at 45 s; subsequently, the F-actin depolymerizes to an intermediate F-actin content 5-10 min after stimulation. The depolymerization of F-actin reflects a true decrease in F-actin content since the quantity of probe extractable from cells also decreases between 45 s and 10 min. The rate of actin polymerization (3.8 +/- 0.3-4.4 +/- 0.6% increase in F-actin/s) is the same for 10(-10) - 10(-6) M fMLP and the polymerization is inhibited by cytochalasin D. The initial rate of F-actin depolymerization (6.0 +/- 1.0-30 +/- 5% decrease in F-actin/min) is inversely proportional to fMLP dose. The F-actin content of stimulated cells at 45 s and 10 min is greater than control levels and varies directly with fMLP dose. F-actin distribution and cell shape also vary as a function of time after stimulation. 45 s after stimulation the cells are rounded and F-actin is diffusely distributed; 10 min after stimulation the cell is polarized and F-actin is focally distributed. These results indicate that actin polymerization and depolymerization follow fMLP stimulation in sequence, the rate of depolymerization and the maximum and steady state F-actin content but not the rate of polymerization are fMLP dose dependent, and concurrent with F-actin depolymerization, F-actin is redistributed and the cell changes shape.     

Bulinus tropicus, a natural intermediate host for Schistosoma margrebowiei in Lochinvar National Park, Zambia

Snails, provisionally identified as Bulinus tropicus, on the basis of chromosome number, egg protein profile, AcP and HBDH enzymes of the digestive gland, and radular morphology, from Lochinvar National Park, Zambia have been demonstrated to transmit Schistosoma margrebowiei naturally. The identification of the unpaired male schistosomes was confirmed by PGM and AcP analyses. The observations confirm earlier epidemiological predictions, and add another species of mollusc to the two, B. forskalii and B. scalaris, known to be natural intermediate hosts of S. margrebowiei.     

Prophylactic immunization against experimental leishmaniasis. IV. Subcutaneous immunization prevents the induction of protective immunity against fatal Leishmania major infection

Durable immunity against fatal L. major infection in genetically susceptible mice can be induced by immunization with 150,000-rad irradiated or heat-killed promastigotes administered i.v. or to a lesser extent i.p. Conversely, subcutaneous (s.c.) and intramuscular (i.m.) injections are not only totally ineffective but generally increase susceptibility to and enhance the progression of the disease, leading to earlier mortality. This detrimental effect is particularly evident with lower infecting challenge doses. Disease exacerbation is apparent in mice given 4 X s.c. injections of as few as 2 X 10(4) irradiated promastigotes, but it appears most potent after doses of 2 X 10(7). When mice given 4 X s.c. injections were subsequently immunized i.v. with 2 X 10(7) irradiated promastigotes, they failed to develop any evidence of protection against infection with 2 X 10(5) promastigotes, whereas mice given i.v. immunization alone were strongly protected. Thus, s.c. injections are capable of blocking the prophylactic effect of i.v. immunization with irradiated parasites. This inhibitory effect can be achieved with a single s.c. injection, although rather less potently than with four, and is even effective against four repeated weekly i.v. immunizations. Once induced, the effect persists undiminished after 100 days. A weaker effect is also inducible by s.c. injection given after i.v. immunization. The blocking effect of s.c. injection is not dependent on continuing viability of the promastigotes, as it can be induced equally readily with heat-killed, formalin-fixed, or sonicated parasites. The phenomenon extends to mouse strains genetically resistant as well as susceptible to L. major infection and, in congenic mice of BALB background, is independent of the major histocompatibility (H-2) gene complex.     

Prophylactic immunization against experimental leishmaniasis. V. Mechanism of the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major infection

The responsiveness of BALB/c mice to protective i.v. immunization with 150,000-rad irradiated or heat-killed Leishmania major promastigotes can be totally suppressed by prior subcutaneous (s.c.) injection of the same "vaccine." Induction of this effect is leishmania specific for although prevention of protection against L. major infection can be obtained with either homologous or Leishmania donovani promastigotes, it does not follow s.c. administration of an immunogenic Trypanosoma cruzi epimastigote preparation. Multiple s.c. injections of irradiated L. major promastigotes do not inhibit the subsequent antibody response of any major isotype to i.v. immunization, but rather induce some priming. The same s.c. injections induced delayed-type hypersensitivity (DTH) reactivity that could be transferred locally or systemically, although it was weaker than in mice with cured infections. Parallel cell-mediated immunity (CMI) responses were also reflected in vitro in specific lymphocyte transformation assays. Despite this evidence of a DTH/helper type of T cell response, transfer of 5 X 10(7) viable T cell-enriched spleen cells from 4 X s.c. immunized donors to normal recipients completely abrogated the protective response to i.v. immunization. Conversely, T cell-depleted (anti-Thy-1.2 + C treated) cells were without effect. The inhibitory T cells were defined by monoclonal antibody pretreatment as possessing an Lyt-1+2-,L3T4+ phenotype. T cells from s.c. immunized donors were also shown, by mixed transfer experiments, to counteract completely the protective effect of T cells from i.v. immunized donors in 550-rad irradiated recipients. They were as potent as suppressor T cells from donors with progressive disease both in this capacity and in abrogating the prophylactic effect of sublethal irradiation itself. The similarities and differences between suppressor and immune effector T cells induced by s.c. or i.v. immunization and those arising in response to leishmanial infection itself are discussed.