d-Aspartate in Human Brain

The presence of the biologically uncommon D-aspartic acid (D-aspartate) in human brain white matter has been previously reported. The earlier study has now been expanded to include D/L-aspartate ratios from 67 normal brains. The data show that the D-aspartate content increases rapidly from 1 year to approximately 35 years of age, levels off in middle age, and then appears to decrease somewhat. The D-aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L-aspartate ratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D-aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.     

Binding of pyrimidin-2-one ribonucleoside by cytidine deaminase as the transition-state analogue 3,4-dihydrouridine and the contribution of the 4-hydroxyl group to its binding affinity

Cytidine deaminase, purified to homogeneity from constitutive mutants of Escherichia coli, was found to bind the competitive inhibitors pyrimidin-2-one ribonucleoside (apparent Ki = 3.6 x 10(-7) M) and 5-fluoropyrimidin-2-one ribonucleoside (apparent Ki = 3.5 x 10(-8) M). Enzyme binding resulted in a change of the lambda max of pyrimidin-2-one ribonucleoside from 303 nm for the free species to 239 nm for the bound species. The value for the bound species was identical with that of an oxygen adduct formed by combination of hydroxide ion with 1,3-dimethyl-2-oxopyrimidinium (239 nm), but lower than that of a sulfur adduct formed by combination of the thiolate anion of N-acetylcysteamine with 1,3-dimethyl-2-oxopyrimidinium (259 nm). The results suggest that pyrimidin-2-one ribonucleoside is bound by cytidine deaminase as an oxygen adduct, probably the covalent hydrate 3,4-dihydrouridine, rather than intact or as an adduct involving a thiol group of the enzyme. In dilute solution at 25 degrees C, the equilibrium constant for formation of a single diastereomer of 3,4-dihydrouridine from pyrimidin-2-one ribonucleoside was estimated as approximately 4.7 x 10(-6), from equilibria of dissociation of water, protonation of 1-methylpyrimidin-2-one, and combination of the 1,3-dimethylpyrimidinium cation with the hydroxide ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

Mouse tumors are heterogeneous in their susceptibility to syngeneic lymphokine-activated killer cells and delineate functional subsets in such effectors

We have analyzed whether lymphokine-activated killer (LAK) cells, generated from C57BL/6J (B6) spleen cells at different times after recombinant interleukin-2 (rIL-2) culture, could be heterogeneous in their ability to lyse a variety of tumor targets. When tested 3 days after exposure to 250 U/ml rIL-2 (day-3 LAK cells) a significant lysis was detected with the natural-killer(NK)-sensitive YAC lymphoma, the NK-resistant P815 mastocytoma, three different syngeneic melanomas and a syngeneic fibrosarcoma (group 1 targets), whereas no lysis was observed with a reticulum cell sarcoma, two different lymphomas or concanavalin A blasts, all of B6 origin (group 2 targets). LAK cells cultured for 5 days, however, lysed group 2 targets and showed a parallel increase of cytotoxic activity against group 1 targets. At day 7, LAK activity declined on all targets examined. In cold-target inhibition studies, the lysis of group 1 tumor targets by day-3 or day-5 LAK cells could be inhibited only by group 1 and not by group 2 unlabelled tumor cells. All group 1 tumors could effectively compete each other. Conversely, the lysis of group 2 tumor targets by day-5 LAK cells was inhibited by both group 1 and group 2 targets. These data indicate the presence of separate LAK effectors that appear to arise with different time kinetics and have different recognition structures. In vitro antibody depletion at the effector level showed that day-3 LAK cells with cytotoxic activity against group 1 tumors were ASGM1+. Day-5 LAK cells included both ASGM1+ and Lyt2+ effectors and both populations, although to a different extent, contributed to the lysis of all targets. Our results indicate that LAK cells are functionally heterogeneous. This heterogeneity is defined by their susceptible target cells and cannot be ascribed to different (Lyt2+ versus ASGM1+) lineages.     

Adoptive immunotherapy of a mouse colon carcinoma with recombinant interleukin-2 alone or combined with lymphokine-activated killer cells or tumor-immune lymphocytes

Summary We have used a BALB/c colonic adenocarcinoma (C-26) to evaluate the therapeutic potential of recombinant interleukin-2 (rIL-2) at high and low dosages in combination with or without lymphokine-activated killers (LAK) or tumor-specific, immune lymphocytes in either an adjuvant spontaneous or an artificial metastasis system. Most (80%) of the mice that underwent s.c. C-26 tumor excision were shown to die of spontaneous metastasis with lung involvement by 1–4 months after excision. Postsurgical systemic treatment with low-dose rIL-2 (3 × 10⁴ U/day, i.p.) increased the survival rate to 31% as compared to 21% (not significant) in excised controls while administration of high-dose rIL-2 (8 × 10⁴ U/day) led to 53% survival (P <0.01). Both LAK cells and C-26-tumor-immune lymphocytes given during rIL-2 treatment significantly increased the effects of rIL-2 at the low but not at the high-dose, with tumor-immune effectors resulting in the highest percentage (63%) of cures. When mice bearing 3-day artificial lung metastases of C-26 cells were treated with low- or high-dose rIL-2, in combination with or without LAK or tumor-immune lymphocytes, a highly significant reduction or abrogation of the number of lung foci was observed with all treatments, including those involving or tumor-immune lymphocytes alone. Assessment of survival benefit in these mice, however, showed survival prolongation, with 20% cures achieved by low-dose rIL-2 alone and up to 65% cures by LAK in combination with low-dose rIL-2. In this system of artificial metastasis high-dose rIL-2 alone increased the survival time but failed to cure the animals, and the addition of LAK was ineffective whereas that of tumor-immune lymphocytes led to 80% cure. These results suggest that tumorimmune lymphocytes are more effective than LAK when combined with rIL-2 and that caution is necessary in extrapolating findings obtained in artificial metastasis models.     

Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.     

Spectroscopic and kinetic properties of the oxidized intermediates of lignin peroxidase from Phanerochaete chrysosporium

Stopped-flow rapid scan techniques were used to obtain a spectrum of nearly homogeneous lignin peroxidase compound I (LiPI) under pseudo-first order conditions at the unusually low pH optimum (3.0) for the enzyme. The LiPI spectrum had a Soret band at 407 nm with approximately 60% reduced intensity and a visible maximum at 650 nm. Under steady-state conditions a Soret spectrum for lignin peroxidase compound II (LiPII) was also obtained. The Soret maximum of LiPII at 420 nm was only approximately 15% reduced in intensity compared to native LiP. Transient state kinetic results confirmed the pH independence of LiPI formation over the pH range 3.06-7.39. The rate constant was (6.5 +/- 0.2) x 10(5) M-1 S-1. Addition of excess veratryl alcohol to LiPI resulted in its reduction to LiPII with subsequent reduction of LiPII to the native enzyme. Reactions of LiPI and LiPII with veratryl alcohol exhibited marked pH dependencies. For the LiPI reaction the rate constants ranged from 2.5 x 10(6) M-1 S-1 at pH 3.06 to 4.1 x 10(3) M-1 S-1 at pH 7.39; for the LiPII reaction, 1.6 x 10(5) M-1 S-1 (pH 3.06) to 2.3 x 10(3) M-1 S-1 (pH 5.16). These single turnover experiments demonstrate directly that the pH dependence of these reactions dictates the overall pH dependence of this novel enzyme. These results are consistent with the one-electron oxidation of veratryl alcohol to an aryl cation radical by LiPI and by LiPII.     

Esterase-6 polymorphism inDrosophila melanogaster:Effects of temperature and methyl malonate on genotypic trajectories in polymorphic populations set up with highly inbred lines

It is generally difficult to identify possible effects of selection at a specific locus because of the heterogeneity of the genetic background. Geographical patterns ofEst-6 gene frequencies suggest that there is selection at this locus but selection on loci closely linked to it cannot be excluded. Differences in catalytic properties between allozymes have been shownin vitro; further, several laboratory studies have shown apparent fitness differences between allozymes. Our study used inbred lines highly homogeneous in the genetic background. Four populations were set up fromEst-6s andEst-6F homozygous females inseminated by males of the same genotype at each combination of three factors: temperature (18 and 25°C); methyl malonate (presence or absence); input gene frequencies [p(S) = 0.2 and 0.8]. The populations were sampled periodically for about 28 generations. Methyl malonate was chosen to exert pressure in the enzymatic function of esterase-6. Statistical analyses show that: there are no sex differences; gene frequencies change from input values to those of the first sampling, when only individuals of the first generation are present at 18^oC or individuals of the second generation just begin to appear at 25°C; gene frequencies do not change thereafter and Hardy-Weinberg equilibrium is established. The changes in gene frequencies observed in the first generations suggest thatEst-6 can under certain conditions be a target of selection. Such conditions may not, however, occur in natural populations.     

Purification of human plasma retinol-binding protein by hydrophobic interaction chromatography

Human plasma retinol-binding protein has been purified to homogeneity by a simple method that requires an ammonium sulfate fractionation, a hydrophobic interaction chromatography on phenyl-Sepharose, which dissociates the complex between retinol-binding protein and its carrier, transthyretin, and a gel filtration on Sephadex G-50. The yield of pure protein is comparable or higher than that obtained with the more complex procedures previously reported.