Hippocampal brain amines in methotrexate-induced learning and memory deficit

Intrathecal methotrexate in children with leukemia is known to cause seizures, dementia, leukoencephalopathy, and cognitive dysfunction after long-term treatment. To investigate the cognitive dysfunction, male Wistar rats were given multiple intracerebroventricular injections of methotrexate. Its effect on behaviour was tested in the two-compartment conditioned avoidance task and dark-bright arena test. Levels of brain amines in the hippocampal region of the brain were estimated by HPLC. The qualitative and quantitative histopathological changes in the different regions of the hippocampus were studied by cresyl violet staining. Multiple injections (1 or 2 mg/kg) produced convulsions and learning and memory impairment but did not induce anxiolytic activity. They also reduced concentrations of all three brain amines (norepinephrine, dopamine, and serotonin) and the serotonin metabolite 5-hydroxyindoleacetic acid. The CA4 region of the hippocampus was severely affected by intraventricular methotrexate. Disruption of brain monoamines has been proposed as a cause of brain dysfunction from this chemotherapy, and that disruption may in turn involve cytotoxic effects of methotrexate on brain tissue. The outcomes of this study may have therapeutic implications in the management of cancer conditions, particularly in childhood lymphoblastic leukemia.     

Multiple apoptotic defects in hematopoietic cells from mice lacking lipocalin 24p3

The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis, iron trafficking, development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3, we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid, myeloid, and erythroid cells, which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead, apoptotic defects were unique to many mature hematopoietic cell types, including neutrophils, cytokine-dependent mast cells, thymocytes, and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells, thus explaining their resistance to the ensuing cell death. The results of these studies, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly, these functions are limited to relatively mature blood cell compartments.     

Translation and phosphorylation of wheat germ lysate: phosphorylation of wheat germ initiation factor 2 by casein kinase II and in N-ethylmaleimide-treated lysates

Previously, we observed that N-ethylmaleimide (NEM), a thiol-alkylating agent, was found to stimulate the phosphorylation of several proteins in translating wheat germ (WG) lysates, including the phosphorylation of alpha, the p41-42 doublet subunit, and beta, the p36 subunit, of the WG initiation factor 2 (eIF2). We find now that NEM increases phosphorylation of several proteins significantly in lysates which are moderate or low in their translation compared to optimally active lysates. Heat treatment, which stimulates oxidation of protein sulfhydryls, decreases the translation and phosphorylation ability of WG lysates. The decrease in phosphorylation, but not translation, that occurs in heat-treated lysates is prevented very efficiently by NEM and partially by reducing agents such as dithiothreitol (DTT) and GSH. DTT prevents, however, completely the loss of sulfhydryl content of heat-treated WG lysates and does not at all prevent heat-induced inhibition of translation. In contrast, DTT prevents completely the diamide-induced translational inhibition and also the loss of sulfhydryl content. These findings therefore suggest that in addition to the maintenance of sulfhydryl groups, heat-labile proteins and their interactions with other proteins play an important role in overall translation and phosphorylation. It is also observed here that heat treatment stimulates the phosphorylation of rabbit reticulocyte eIF2 alpha but not the alpha subunit (p41-42 doublet) of WG eIF2. A phosphospecific anti-eIF2 alpha antibody recognizes the WG eIF2 alpha(P) that is phosphorylated by an authentic eIF2 alpha kinase such as double-stranded RNA-dependent protein kinase, but it is unable to recognize the eIF2 alpha that is phosphorylated in NEM-treated lysates. These findings therefore suggest that phosphorylation of WG eIF2 alpha in NEM-treated lysates occurs on a site different from the serine 51 residue that is phosphorylated by authentic eIF2 alpha kinases. In addition, it also suggests that WG eIF2 alpha, unlike reticulocyte eIF2 alpha, is phosphorylated by eIF2 alpha kinases and also by other kinases. Consistent with this idea, it has been observed here that casein kinase II (CKII) phosphorylates WG eIF2 alpha and the phosphorylation is enhanced by NEM in vitro and in lysates. The phosphopeptide analysis suggests that WG eIF2 alpha has separate phosphorylation sites for CKII and heme-regulated eIF2 alpha kinase (a well-characterized mammalian eIF2 alpha kinase), and NEM-induced phosphorylation in WG lysates resembles CKII-mediated phosphorylation.     

A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake

The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis due to interleukin-3 (IL-3) deprivation and iron transport. Here we report cloning of the 24p3 cell-surface receptor (24p3R). Ectopic 24p3R expression confers on cells the ability to undergo either iron uptake or apoptosis, dependent upon the iron content of the ligand: Iron-loaded 24p3 increases intracellular iron concentration without promoting apoptosis; iron-lacking 24p3 decreases intracellular iron levels, which induces expression of the proapoptotic protein Bim, resulting in apoptosis. Intracellular iron delivery blocks Bim induction and suppresses apoptosis due to 24p3 addition or IL-3 deprivation. We find, unexpectedly, that the BCR-ABL oncoprotein activates expression of 24p3 and represses 24p3R expression, rendering BCR-ABL(+) cells refractory to secreted 24p3. By inhibiting BCR-ABL, imatinib induces 24p3R expression and, consequently, apoptosis. Our results reveal an unanticipated role for intracellular iron regulation in an apoptotic pathway relevant to BCR-ABL-induced myeloproliferative disease and its treatment.     

New diterpenoids from Caesalpinia species and their cytotoxic activity

Chemical investigation on Caesalpinia crista afforded two new diterpenoids, 6β-cinnamoyloxy-7β-acetoxyvouacapen-5α-ol and 6β,7β-dibenzoyloxyvouacapen-5α-ol and on Caesalpinia pulcherrima another new diterpenoid, 12-demethyl neocaesalpin F along with several known constituents. The structures of the new compounds were settled from their 1D and 2D NMR spectral data. The cytotoxicity of these compounds was measured on two different cancer cell lines.     

2,5-Dimethylphenacyl carbamate: a photoremovable protecting group for amines and amino acids.

2,5-Dimethylphenacyl (DMP) carbamates (1a-c) released the corresponding free amines or amino acids in high chemical yields, albeit with quantum yields Phi of only 0.04-0.09, upon irradiation in either aprotic or protic solvents. The photoreaction proceeded principally from the triplet excited state via the E-photoenol. The lifetimes of the triplet enol and the E- and Z-enols in the ground state were determined by laser flash photolysis. The primary photoinitiated transformation liberated a carbamic acid derivative, which subsequently decarboxylated to the amino group-containing compound. Exhaustive irradiation of a DMP-protected aniline (1a) in acetonitrile did not provide aniline in quantitative chemical yields, because it was involved in reductive cleavage of the starting material as an electron donor, thereby decreasing the overall deprotection yield (86%). Phenylalanine methyl ester, liberated from 1c, was, however, obtained in excellent chemical yield (97%). It was also found that the carbamates, while thermally stable, released amines with higher quantum yields in acidic methanol solutions. The DMP chromophore is proposed as an excellent photoremovable protecting group for amino acids and, under specific conditions, for amines in organic synthesis and biochemistry.     

Stereoselective total synthesis of a potent natural antifungal compound (6S)-5,6,dihydro-6-[(2R)-2-hydroxy-6-phenyl hexyl]-2H-pyran-2-one

A practical stereoselective synthesis of (6S)-5,6,dihydro-6-[(2R)-2-hydroxy-6-phenyl hexyl]-2H-pyran-2-one (1), a potent natural antifungal compound, is described. The sequence involves diastereoselective iodine-induced electrophilic cyclization, epoxide ring opening with a vinyl Grignard reagent and ring closing metathesis (RCM) as the key steps.