The reduction of anti-tumour diaziridinyl benzoquinones

The properties of the semiquinone radicals produced for 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ) and 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), have been investigated. AZQ semiquinone radicals can be produced from the reduction of AZQ by superoxide radicals, whereas BZQ semiquinone radicals are unstable in the presence of oxygen. The one-electron reduction potentials of the couples Q/Q-. at pH 7.0 were determined as -70 +/- 10 mV for AZQ and -376 +/- 15 mV for BZQ. The difference in these potentials is explained. As a consequence of ESR studies on the enzymatically produced radicals, we have considered the factors which determine the detection of ESR signals for reduced quinones produced in a biological system.     

The spliceosomal snRNAs of Caenorhabditis elegans.

Nematodes are the only group of organisms in which both cis- and trans-splicing of nuclear mRNAs are known to occur. Most Caenorhabditis elegans introns are exceptionally short, often only 50 bases long. The consensus donor and acceptor splice site sequences found in other animals are used for both cis- and trans-splicing. In order to identify the machinery required for these splicing events, we have characterized the C. elegans snRNAs. They are similar in sequence and structure to those characterized in other organisms, and several sequence variations discovered in the nematode snRNAs provide support for previously proposed structure models. The C. elegans snRNAs are encoded by gene families. We report here the sequences of many of these genes. We find a highly conserved sequence, the proximal sequence element (PSE), about 65 bp upstream of all 21 snRNA genes thus far sequenced, including the SL RNA genes, which specify the snRNAs that provide the 5' exons in trans-splicing. The sequence of the C. elegans PSE is distinct from PSE's from other organisms.     

Control of follicular epithelium development and vitelline envelope formation in the mosquito; role of juvenile hormone and 20-hydroxyecdysone

Using microsurgical manipulations, hormone applications, and transmission electron microscopy we have investigated the regulation of differentiation of the follicular epithelium and formation of the vitelline envelope (VE) in primary follicles in the ovary of the mosquito, Aedes aegypti. During the first 3 days after eclosion, the primary follicle grows, and cells of the follicular epithelium differentiate, their content of mitochondria, rough endoplasmic reticulum, and Golgi complexes increases significantly. Growth and differentiation of the follicular epithelium appear to be under the control of juvenile hormone (JH), because they are blocked by removal of corpora allata in newly closed adult females and can be restored by either implantation of corpora allata or application of JH III. In insects, including mosquitoes, VE is the first layer of the eggshell to be deposited. It is formed from the secretory products of the follicle cells and its deposition coincides with yolk accumulation by developing oocytes. Only follicle cells adjacent to the oocyte deposit VE. In decapitated females, given a blood meal by enema and injected with picogram doses of 20-hydroxyecdysone (20-HE), follicle cells synthesize the VE precursors and deposit morphologically normal VE, in contrast to saline injected controls which deposit no VE. We conclude that 20-HE, as well as factors originating from the blood meal and the oocyte, are required for the normal formation of VE in the mosquito follicles.     

Volume comparisons in the cerebellar complex of primates. II. Cerebellar nuclei

Volumes of medial, interposed, and lateral cerebellar nuclei (MCN, ICN, and LCN) were measured in Insectivora, Scandentia, and Primates, including man. The relative size of the nuclei was expressed in size indices. Insectivora had by far the smallest cerebellar nuclei. The simians, in general, had larger cerebellar nuclei than the prosimians, but there was considerable overlap. From Insectivora to man, the MCN was the least progressive and the LCN the most progressive. The indices are expected to reflect the relative size of the three longitudinal zones of the cerebellum (vermis/MCN, pars intermedius/ICN, hemisphere/LCN). They, together with those of the ventral pons and cerebellum (part I), are discussed in relation to the predominant locomotor pattern of a species, and with reference to evolutionary trends in primate phylogeny.     

Biochemistry and physiology of monoamine oxidase (MAO) activity in the ring dove (Streptopelia risoria). II. Distribution of MAO in different tissues

Monoamine oxidase (MAO) activity was measured fluorometrically in liver, kidney, intestine and brain of adult male and female ring doves. Liver MAO was inhibited in a concentration-related fashion by clorgyline and harmaline (MAO type A inhibitors) where a plateau in the inhibition curve occurred with about 15% activity remaining, and also by the type B inhibitor deprenyl, which produced a plateau when about 85% activity remained. Kidney, intestine and brain MAO were inhibited in a biphasic manner by harmaline. Results with inhibitors suggest that 85% of liver MAO, 86% of kidney MAO, 88% of intestine and 75% of brain MAO is type A. Using 10(-6) M harmaline to differentiate between MAO-A and MAO-B type activities, the apparent maximal velocities (Vmax) and Michaelis constants (Km) were determined in different tissues. Most activity occurred in the intestine, with proportionally lesser amounts of kidney, liver and brain. The majority of MAO present was in the A form. Except for kidney, Km of MAO-B was higher than that of MAO-A. Both MAO-A and -B activities were higher in the intestines of male birds, although sex differences in content and type of MAO activity were not observed in other tissues of the ring dove.     

Optimization of fetal lung organ culture for surfractant biosynthesis

Summary Lung organ culture has been a widely used system for studying differentiation and maturation of alveolar epithelium through various culture conditions. The purpose of this work was to carefully characterize in vitro lung biochemical diffeentiation through isolation of surfactant fraction from tissue and to search for optimal culture conditions. Fetal rat lung was explanted on the 18th gestational day for studying glycogen storage, and on the 20th gestational day for studying surfactant accretion, and cultivated for 48 h. Morphologic differentiation was studies byelectron microscopy tissue explanted on the 17th or 18th gestational days and cultivated for various times. Glycogen storage was greater on fluid medium, although less than occurring in vivo. Cellular integrity and surfactant accumulation were maximal on a semisolid medium containing 0.5% agar. Use of O₂-CO₂ instead of air-CO₂ for gassing the explants slighlty decreased phospholipid accumulation. Among media used in previous lung culture studies, Waymouth MB 752/1 was the only one to allow net glycogen accumulation in vitro. The most favorable media for surfactant phospholipid accretion were Waymouth MB 752/1, Eagle’s minimum essential and its Dulbeccco’s modification, CMRL 1066, and NCTC 109. They allowed a 12- to 14-fold increase of surfactant fraction phospholipids in vitro, which is similar to the increase occurring in vivo during the same peiod. Ham’s F10 and F12 media allowed a six fold increase. RPMI 1640 and medium 199 (M199) allowed only a three fold increase. Phospholipid concentration in nonsurfactant fraction only doubled during culture, and differences between various media were much less marked. DNA concentration changed little during culture. Morphologic differentiation of epithelial cells was advanced as compared with in vivo timing in a medium allowing maximal surfactant accretion (Waymouth MB 752/1) but not in a medium allowing low surfactant increase (RPMI 1640). The possible role of compositional differences between media is discussed.     

Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells.

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.     

The Reductive Deglycosylation Of Adriamycin In Aqueous Medium: A Pulse Radiolysis Study

The free radical (II) produced by one-electron reduction of adriamycin (I) exists in aqueous solution at pH 7.0 in equilibrium with the parent and the two-electron reduced form (III). Over some hundreds of milliseconds deglycosylation takes place yielding an aglycone (IV) which subsequently rearranges to form a more stable aglycone. 7-deoxyadriamycinone (V). The changes in the optical absorption spectrum accompanying these processes are reported. The rate constant for III + IV is 1.1 s^-1 and for IV + V is 1.5 × 10^--² s.^-1. At pH 4.0 the two electron reduced form of adriamycin exists predominantly in a different tautomeric form (VII). It is suggested that this deglycosylates via a free radical mechanism involving the acidic form of the semiquinone free radical (VI)     

Lateral diffusion in planar lipid bilayers: a fluorescence recovery after photobleaching investigation of its modulation by lipid composition, cholesterol, or alamethicin content and divalent cations.

In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the study of reconstituted ion channels and receptors, data dealing with their physical characterization, especially as regards dynamics, are scanty. A combined electrical and optical chamber was designed and allowed fluorescence recovery after photobleaching recovery curves to be recorded from stable virtually solvent-free bilayers. D, the lateral diffusion coefficient of N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, was found to be relatively insensitive to the phospholipid composition (headgroup, chain unsaturation, etc.), whereas inclusion of 33-50% cholesterol in the membrane reduced D by a factor of 2. Divalent cations significantly reduced D of negatively charged bilayers. These results compare well with data gathered on other model and natural systems. In addition, the incorporation of the voltage-dependent pore-former alamethicin did slightly reduce lipid lateral mobility. This study demonstrates the feasibility of such experiments with planar bilayers, which are amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.