Monoamine Oxidase-Inhibiting Properties of SR 95191, a New Pyridazine Derivative, in the Rat: Evidence for Selective and Reversible Inhibition of Monoamine Oxidase Type A In Vivo but Not In Vitro

In rodents, SR 95191 [3-(2-morpholinoethylamino)-4-cyano-6-phenylpyridazine] has been shown to be active in animal models of depression. The profile of activity of SR 95191 suggests that the compound is a selective and short-acting type A monoamine oxidase (MAO) inhibitor (MAOI) in vivo. In the present study, the interaction of SR 95191 with MAO-A and MAO-B activity was further examined in vivo and in vitro. In brain, liver, and duodenum of pretreated rats, SR 95191 selectively inhibited MAO-A (ED50 = 3-5 mg/kg, p.o.), whereas MAO-B was only weakly inhibited for doses as high as 300 mg/kg, p.o. In vivo, SR 95191 (1-100 mg/kg, p.o.) antagonized, in a dose-dependent fashion, the irreversible inhibition of brain and liver MAO-A induced by phenelzine. Finally, dopamine and 5-hydroxytryptamine depleted from their striatal stores by tetrabenazine were able to displace SR 95191 from the active site of MAO-A. However, ex vivo, kinetic studies showed that the inhibitory effect of SR 95191 (1-10 mg/kg) towards MAO-A was noncompetitive and was unchanged after dilution or dialysis. In vitro, the inhibition of brain MAO-A, but not MAO-B, by SR 95191 was time dependent, with a 19-fold decrease in the IC50 values being observed over a 30-min incubation period (140 to 7.5 microM). At this time, the SR 95191-induced inhibition of MAO-A was not removed by repeated washings. When the reaction was started by adding the homogenate without prior preincubation with SR 95191, the inhibition of brain MAO-A was fully competitive (Ki = 68 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence implicating heterozygous deletion of chromosome 7 in the pathogenesis of familial leukemia associated with monosomy 7.

Complete or partial monosomy 7 is a recurring cytogenetic abnormality in acute myelogenous leukemia (AML) and myeloproliferative syndromes (MPS) and is particularly common in patients with Fanconi's anemia and in secondary AML. A familial form of monosomy 7 has been recognized in which two or more siblings develop MPS or AML before age 20. We tested the hypothesis that a recessive cancer susceptibility locus on chromosome 7 was important in the pathogenesis of leukemia in familial monosomy 7 by determining the parental origins of the chromosome 7 retained in the bone marrows of three pairs of affected siblings. We found no overlapping region where all three pairs retained DNA derived from the same paternal or maternal chromosome. These data suggest that inactivation of a single allele of a putative tumor-suppressor gene may be sufficient to contribute to leukemic transformation in familial monosomy 7.     

Augmentation of therapeutic effect of adoptive immunotherapy through a synergy between transferred killer cells and host's fresh lymphocytes]

Among several approaches to augment the therapeutic effect of adoptive immunotherapy, we focused the antitumor synergy between transferred killer cells and host's fresh lymphocytes. Immunotherapy models using murine tumors or clinical experiments revealed that preadministration of immunostimulator such as OK-432, followed by chemotherapeutic agents such as cyclophosphamide, can induce host's non-cytotoxic fresh lymphocytes that act synergistically with cultured killer cells against autologous tumor cells. Immuno-chemo-lymphocytotherapy (a sequential treatment with OK-432, chemotherapy and adoptive immunotherapy) is useful to treat the patients with advanced cancer even if the number of transferred lymphocytes is limited.     

Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis

The interaction between the fungal pathogen Cladosporium fulvum and tomato is supposed to have a gene-for-gene basis. Races of C. fulvum which have 'overcome' the resistance gene Cf9 of tomato, lack the avirulence gene avr9 which encodes a race-specific peptide elicitor. Races avirulent on tomato genotypes carrying the resistance gene Cf9 produce the race-specific peptide elicitor, which induces the hypersensitive response (HR) on those genotypes. The causal relationship between the presence of a functional avr9 gene and avirulence on tomato genotype Cf9 was demonstrated by cloning of the avr9 gene and subsequent transformation of C. fulvum. A race virulent on tomato genotype Cf9 was shown to become avirulent by transformation with the cloned avr9 gene. These results clearly demonstrate that the avr9 gene is responsible for cultivar specificity on tomato genotype Cf9 and fully support the gene-for-gene hypothesis. The avr9 gene is the first fungal avirulence gene to be cloned.     

Nuclear magnetic resonance studies of cis-syn, trans-syn, and 6-4 photodimers of thymidylyl(3'-5')thymidine monophosphate and cis-syn photodimers of thymidylyl(3'-5')thymidine cyanoethyl phosphotriester

Three out of four possible photodimers of thymidylyl(3'-5')thymidine monophosphates (i.e., cis-syn, 6-4, and one of the trans-syn) and two structural isomers (i.e., R and S forms) of cis-syn-thymidylyl(3'-5')thymidine cyanoethyl phosphotriester have been isolated and purified from the reaction mixtures after UV irradiation and studied by multinuclear magnetic resonance Spectroscopy. All five inter thymine base linked photodimers have grossly similar structures which are quite different from those of the parent thymidylyl(3'-5')thymidine. The base of Tp- is in the syn conformation, and that of -pT it is in the anti conformation. The sugar puckering of Tp- is dominated by the 2E conformer, but in -pT it is in 4E; except for the conformer around C5'-O5' bond, the 6-4 isomer is very similar to those of cis-syn and trans-syn conformation. As expected, there are sugar-phosphate backbone distortions in the phosphotriesters, due to the neutralization of the negative charge of the phosphate. In general the structures of all five photodimers are very close to those of the cis-syn photodimer of thymidylyl(3'-5')thymidine monophosphate cyanoethyl ester as studied by X-ray diffraction [Cadet, J., Voituriez, L., Hruska, F. E., & Grand, A. (1985) Biopolymers 24, 897-903; Hruska, F. E., Voituriez, L., Grand, A., & Cadet, J. (1986) Biopolymers 25, 1401-1417]. While the trans-syn photodimer has two structural isomers, only one [C6(of Tp-)-R] was produced by the UV irradiation and studied.     

Fc receptors on monocytes cause OKT3-treated lymphocytes to internalize T3 and to secrete IL-2

Monocytes cause OKT3-treated T cells to secrete IL-2 and to lose cell surface T3. We have studied the fate of the "lost" T3. Immunofluorescence microscopy on permeabilized cells showed that monocytes induce T cells to internalize T3. Furthermore, experiments with radioiodinated T cells showed that the internalized T3 was not degraded and exhibited an unaltered polypeptide composition for at least 16 hr. The role of Fc receptors in inducing internalization was also investigated. Fc receptors were depleted from monocytes by allowing the phagocytes to spread on immune complexes. Such depleted monocytes exhibit a fourfold reduction in their ability to promote both internalization of T3 and production of IL-2. A comparable reduction is seen if F(ab')2 fragments of OKT3 were employed in place of intact IgG. Furthermore, monocyte Fc receptors that have been blocked by heat-aggregated human IgG also show much reduced capability for induction of OKT3-mediated T-cell proliferation. We therefore conclude that Fc receptors bind to the Fc domain of OKT3 and thereby cause OKT3-treated T cells to internalize T3 and become activated.     

epsilon-Crystallin, a novel avian and reptilian eye lens protein

Gel filtration of Peking duck eye lens proteins reveals a component eluting just behind delta-crystallin and comprising approximately 10% of the total soluble protein. The native Mr of this additional component is estimated to be 120000; it appears to be composed of three identical chains of Mr 38000 and pI 7.5. Circular dichroic spectroscopy showed a relatively high alpha-helical content. No immunological cross-reactivity is found with alpha-, beta-, gamma- or delta-crystallins, and partial amino acid sequence determinations likewise failed to reveal any similarity with other known crystallins. We conclude that this protein represents another and novel family of eye lens proteins, for which we propose the designation epsilon-crystallin. epsilon-Crystallin is translated from a 1450-base mRNA, which has been partially purified. epsilon-Crystallin is found scattered among avian and reptilian taxa, but not in other vertebrates. Its rate of evolutionary change seems to be as slow as that of alpha- and beta-crystallins.     

Molecular cloning of cDNA coding for human preprourokinase

A cDNA library was constructed in pBR322 from 18S to 20S mRNA that was extracted from human kidney cells, fractionated on oligo(dT)-cellulose column and sucrose-density gradient, and confirmed for urokinase production in Xenopus laevis oocytes. The Escherichia coli RR1 transformants were hybridized to synthetic oligonucleotide probe prepared according to the known amino acid sequence, Glu 73 to Glu 77 of human urinary urokinase chain B. The entire cloned cDNA covers a 2250-bp region, wherein the 1293-bp sequence codes for preprourokinase consisting of 431 amino acids, with the first 20 residues being a signal peptide. The 5'-untranslated region is at least 80 bp long and the 3'-untranslated region is longer than 850 bp.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Isolation and purification of deoxyribonucleosides from 90% 13C-enriched DNA of algal cells and their characterization by 1H and 13C NMR.

13C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The 1H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive 1H-13C couplings. Similarly, the 13C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz 13C NMR spectrum, due to 13C-13C couplings. The 1H-13C and 13C-13C coupling constants were also measured and tabulated. The isotopic enrichment of 13C these deoxyribonucleosides was obtained by integration of the 1H and/or 13C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% 13CO2 during metabolism pathways. This protocol provides experimentally useful quantities of 13C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis.