Studies on the Formation of 6-Hydroxydopamine in Mouse Brain After Administration of 2,4,5-Trihydroxyphenylalanine (6-HydroxyDOPA)

2,4,5-Trihydroxyphenylalanine (6-OH-DOPA) destroys central and peripheral noradrenergic neurons, while sparing dopaminergic neurons. Previous studies indicate that 6-OH-DOPA toxicity is mediated by the formation of 6-hydroxydopamine. However, levels of 6-hydroxydopamine in brain following peripheral administration of 6-OH-DOPA have not been documented. In the current study, 6-OH-DOPA and 6-hydroxydopamine were measured in brain by HPLC with electrochemical detection after intraperitoneal injection of 6-OH-DOPA. When mice were injected with 100 mg 6-OH-DOPA/kg, 6-hydroxydopamine levels in the striatum were highest (1.9 microgram/g) at 15 min and fell slowly to 24% of the peak value at 4 h. Experiments with reserpine indicated that the relatively stability of 6-hydroxydopamine was largely dependent upon storage in synaptic vesicles. Reserpine (10 mg/kg) lowered striatal 6-hydroxydopamine levels to 21.6% of control (non-reserpine-treated) values at 1 h, and to 8.9% of control values at 4 h. Levels of 6-hydroxydopamine in the striatum at 1 h were increased 113% by pargyline (100 mg/kg), 145% by alpha-methyldopahydrazine (carbidopa; 25 mg/kg), and 261% by pargyline and carbidopa together. Levels of dopamine in the striatum were unchanged at 2.5 h after 200 mg 6-OH-DOPA/kg (with pargyline and 50 mg carbidopa/kg), whereas levels of norepinephrine in the frontal cortex fell by 77%. At the same time, 6-hydroxydopamine levels were 8.8-fold higher in the striatum (5.54 micrograms/g) than in the cortex (0.63 micrograms/g).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF

Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis. One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions. Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine. The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants.     

Serotonin in human lumbar cerebrospinal fluid: a reassessment

An inter-laboratory comparison study was carried out in order to ascertain mean levels of serotonin (5-HT) in human lumbar cerebrospinal fluid (CSF). Analyses were performed using high performance liquid chromatography (HPLC) coupled with either electrochemical (LC-EC) or fluorometric (LC-F) detection. With the detection limits obtained (7-8 pg/ml for LC-EC, 7-15 pg/ml for LC-F) 5-HT was not usually detected in human lumbar CSF. The findings indicate that the true mean concentration of CSF 5-HT is less than 10 pg/ml. This upper limit is substantially lower than all previous reports of 5-HT concentrations in normal human lumbar CSF. The extremely low concentrations of 5-HT present in CSF make it unlikely that CSF 5-HT will be of clinical utility in assessing central serotonergic function.     

MHC antigen expression by melanomas recovered from mice treated with allogeneic mouse fibroblasts genetically modified for interleukin-2 secretion and the expression of melanoma-associated antigens

Recent approaches toward the immunotherapy of neoplastic disease involve the introduction of expression-competent genes for interleukin-2 (IL-2) into autologous malignant cells. Treatment of tumor-bearing experimental animals with the IL-2-secreting cells successfully induces partial and at times complete remissions. In most instances, however, although delayed, progressive tumor growth continues. Here, certain of the characteristic of B16 melanomas (H-2^b) persisting in C57BL/6 mice (H-2^b) treated with an IL-2-secreting, melanoma-antigen-positive cellular immunogen (RLBA-IL-2 cells) are described. Unlike the melanoma cells first injected, B16 cells recovered from mice treated with RLBA-IL-2 cells were deficient in the experssion of MHC class I, but not class II determinants. Deficient MHC class I expression correlated with the cells' resistance to cytotoxic T lymphocytes (CTL) from the spleens of mice immunized with RLBA-IL-2 cells. Melanomas persisting in mice treated with non-IL-2-secreting, melanoma-antigen-positive cell constructs (RLBA-ZipNeo cells) were also deficient in the expression of MHC class I determinants, and the melanoma cells were resistant to CTL from mice immunized with RLBA-ZipNeo cells. Thus, the expression of melanoma-associated antigens rather than IL-2-secretion correlated with deficient MHC class I expression by the persistent melanomas. This point was substantiated by the expression of MHC class I antigens by melanomas persisting in mice treated with IL-2-secreting, melanoma-antigen-negative LM cells (LM-IL-2); it was equivalent to that of melanomas in untreated mice. The involvement of MHC class I antigens in the immune resistance of persistent melanoma cells from mice treated with the melanoma-autigen-positive immunogens was indicated by the effect of interferon (IFN) orN-methyl-N-nitro-N-nitrosoguanidine (MNNG) on the susceptibility of the cells to anti-melanoma CTL. Treatment of the resistant melanomas with IFN or MNNG stimulated MHC class I antigen expression and restored the cells' sensitivity to CTL from mice immunized with IL-2-secreting or nonsecreting, melanoma-antigen-positive cellular immunogens. Prior treatment of the treated cells with antibodies to MHC class I determinants inhibited the cells' susceptibility to CTL from mice immunized with RLBA-IL-2 cells.     

Decline in sperm counts: an artefact of changed reference range of "normal"?

OBJECTIVE--To investigate a reported fall in sperm counts during 1940-90 in relation to the reduced lower reference value of "normal" during the same period by assuming the null hypothesis that no change had occurred in the probability distribution of the sperm concentration. DESIGN--Analysis by using various mathematical models of the probability distribution of sperm concentration together with experimental data which supported a model employing a logarithmic distribution. SUBJECTS--235 men presenting for stimulated in vitro fertilisation at Midland Fertility Services, Aldridge, in 1992 together with samples of 20 ejaculates from each of five men attending the same centre during 1992-3. RESULTS--The effect of the change in lower reference value for the "normal" sperm concentration (from 60 x 10(9) to 20 x 10(9)/l) depended on the probability distribution of the concentration in the population. If that distribution was normal or uniform, then very little of the reported decline was a consequence of the change in lower reference value. If it was heavily skewed, then most or all of the reported decline may have been a consequence of that change. The limited experimental data available indicate that the distribution was heavily skewed. CONCLUSIONS--Depending on the actual distribution of sperm concentration in the population, the reported decline in concentration may have been accounted for entirely or in part by the change in lower reference value. The original evidence does not support the hypothesis that the sperm count declined significantly between 1940 and 1990.     

Role of the imp operon of the Streptomyces coelicolor genetic element SLP1: two imp-encoded proteins interact to autoregulate imp expression and control plasmid maintenance.

The Streptomyces coelicolor genetic element SLP1 can exist either integrated into the host chromosome or as an autonomously replicating plasmid. The integrated form of SLP1 includes a locus (imp, for inhibition of plasmid maintenance) that can act both in cis and in trans to prevent propagation of SLP1 as an extrachromosomal replicon (S. R. Grant, S. C. Lee, K. Kendall, and S. N. Cohen, Mol. Gen. Genet. 217:324-331, 1989). We report here that a 1.8-kb Eco47III DNA fragment previously shown to encode the Imp+ phenotype contains two genes (impA and impC) that must be expressed in cis to each other and whose products interact functionally and probably physically to interfere with SLP1 plasmid maintenance and repress expression of the imp operon. Partial repression of the imp promoter (P(imp)), which is located immediately 5' of impA, by the 29.7-kDa ImpA protein is enhanced by the impC gene product. Gel shift analysis indicates that ImpA binds to a 16-bp sequence located within the DNA segment containing P(imp) and that ImpC interferes with this binding. Our data suggest that binding of ImpA to the P(imp) region mediates DNA looping in this region.