MPTP, MPP+ and mitochondrial function

1-Methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inhibited NAD(H)-linked mitochondrial oxidation at the level of Complex I of the electron transport system. MPTP and MPP+ inhibited aerobic glycolysis in mouse striatal slices, as measured by increased lactate production; MPTP-induced effects were prevented by inhibition of monoamine oxidase B activity. Several neurotoxic analogs of MPTP also form pyridinium metabolites via MAO; these MPP+ analogs were all inhibitors of NAD(H)-linked oxidation by isolated mitochondria. 2'-Methyl-MPTP, a more potent neurotoxin in mice than MPTP, was also more potent than MPTP in inducing lactate accumulation in mouse brain striatal slices. Overall, the studies support the hypothesis that compromise of mitochondrial oxidative capacity is an important factor in the mechanisms underlying the toxicity of MPTP and similar compounds.     

In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes

The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes.     

Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes

T-cell receptor (Ti) gene restriction fragment patterns (RFPs) were determined by Southern blots of genomic DNA obtained from T-lymphocyte colonies isolated from a single normal individual. 4 wild-type colonies and 11 in vivo derived 6-thioguanine-resistant mutant colonies with previously characterized hprt gene structural alterations were studied. Among the hprt mutants, 10 of the 11 showed unique Ti RFPs indicating their origins in different in vivo progenitors. Unique Ti RFPs were also seen among the wild-type T-cell colonies. One, however, shared its Ti RFP with a mutant. These results suggest that mutation in vivo of the hprt gene in human T-lymphocytes occurred after thymic maturation and that the 11 recovered hprt mutants probably resulted from 11 independent mutational events.     

Lupus prone (SWR x NZB)F1 mice produce potentially nephritogenic autoantibodies inherited from the normal SWR parent

The incidence of nephritis in autoimmune NZB mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed 65 monoclonal anti-DNA autoantibodies derived from SNF1 mice and compared them with those obtained from the NZB parent. The majority of the SNF1-derived anti-DNA antibodies were IgG and cationic in charge. By contrast, 77% of the NZB-derived antibodies were IgM. Moreover, all three NZB-derived IgG anti-DNA antibodies were anionic. The cationic property of the SNF1-derived IgG autoantibodies was not restricted to any particular antigenic specificity pattern or IgG subclass, nor was there a preference for the allotype of either parent. However, we identified a set of highly cationic (pI at 8.2 to 8.8 pH) IgG2b anti-DNA antibodies from SNF1 hybrids that had the SWR allotype. Isoelectric focusing of intact antibodies and isolated heavy and light chains showed that the highly cationic charge of these antibodies was determined by the variable regions of their heavy chains. Because IgG anti-DNA antibodies with cationic charge are especially pathogenic, those antibodies bearing the allotype of the normal SWR parent may account for the high incidence of severe nephritis in the F1 hybrids. The results indicate that pathogenic autoantibodies, which are encoded by genes of the nonautoimmune SWR parent, are expressed in the SNF1 mice due to some cellular and genetic regulatory influence of the NZB parent.     

Thymic epithelial genotype influences the production of recombinant leukemogenic retroviruses in mice

By using T1 oligonucleotide fingerprinting and mapping techniques, we analyzed the genomic structure of retroviruses produced by thymocytes and splenocytes of reciprocal bone marrow-and thymus-grafted chimeras. We found that the genetic factor(s) derived from NZB mice that suppresses the development of thymic leukemia in (AKR X NZB)F1 mice also prevents the formation of recombinant leukemogenic viruses and the expression of preleukemic changes in the (AKR X NZB)F1 thymocytes. The NZB mouse gene or genes appeared to exert this suppressive effect by acting on the thymic reticuloepithelial cells and not on the thymic lymphocytes of (AKR X NZB)F1 hybrids. Prospective studies with thymic epithelial grafts from young mice showed that the AKR thymic epithelium could mediate the formation and expression of leukemogenic recombinant viruses and preleukemic changes in thymocytes that lead to the development of thymic leukemia, whereas the (AKR X NZB)F1 thymic epithelium was deficient in this regard. Our results also confirmed a previous observation that during in vivo generation of recombinant leukemogenic viruses, the acquisition of polytropic virus-related sequences in the 3' portion of the p15E gene and the U3 region and in the 5' part of the gp70 gene can occur independently.     

CHROMOSOME MICROMANIPULATION : III. Spindle Fiber Tension and the Reorientation of Mal-Oriented Chromosomes

Kinetochore reorientation is the critical process ensuring normal chromosome distribution. Reorientation has been studied in living grasshopper spermatocytes, in which bivalents with both chromosomes oriented to the same pole (unipolar orientation) occur but are unstable: sooner or later one chromosome reorients, the stable, bipolar orientation results, and normal anaphase segregation to opposite poles follows. One possible source of stability in bipolar orientations is the normal spindle forces toward opposite poles, which slightly stretch the bivalent. This tension is lacking in unipolar orientations because all the chromosomal spindle fibers and spindle forces are directed toward one pole. The possible role of tension has been tested directly by micromanipulation of bivalents in unipolar orientation to artificially create the missing tension. Without exception, such bivalents never reorient before the tension is released; a total time "under tension" of over 5 hr has been accumulated in experiments on eight bivalents in eight cells. In control experiments these same bivalents reoriented from a unipolar orientation within 16 min, on the average, in the absence of tension. Controlled reorientation and chromosome segregation can be explained from the results of these and related experiments.     

Compartmentation of glutamic acid metabolism in brain slices

— (1) Compartmentation of glutamate metabolism in brain cortex previously observed only in vivo, has now been demonstrated in vitro.This was shown by using [U-¹⁴C]aspartate and [U-¹⁴C]glutamate as tracer substrates. (2) Preparation and maintenance of the slices at 0° resulted in reversible inhibition of glutamine synthesis. Preincubation at 37° for 10 min or preparation of the slices at room temperature partially overcame this inhibition. (3) Transfer to fresh medium after preincubation had an added stimulatory effect on glutamine synthesis. (4) Incubation in high K⁺ medium (27 mm ) altered the relative specific activity of glutamine. (5) The data are in keeping with the postulate of the existence of at least two different pools of citric acid cycle intermediates in the cerebral cortex.     

CHROMOSOME VELOCITY DURING MITOSIS AS A FUNCTION OF CHROMOSOME SIZE AND POSITION

Chromosome velocity has been studied in living Melanoplus differentialis spermatocytes by phase contrast cinemicrography. Melanoplus chromosomes (and bivalents) differ in length by as much as 1:3.5. As expected, no size-dependent velocity differences were detected in anaphase, and this is also shown to be true for the less predictable movements during prometaphase congression. The size of the X chromosome can change during observation following x-irradiation, but this is equally without influence on velocity. However, an effect of position on velocity is found in both prometaphase and in anaphase: the chromosomes furthest from the central interpolar axis move 25 per cent faster than more central chromosomes. A simple mechanical model relating frictional resistance and mitotic forces to chromosome velocity is discussed in detail. Calculations from the model suggest that a significant difference in the force acting on a large, as compared with a small chromosome is necessary to account for the observed similarity in velocity. Therefore, it is concluded that the mitotic forces are so organized or regulated that velocity is, within limits, independent of load. The implications of velocity-load independence in relation to the molecular origin of mitotic forces are discussed.