Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5′-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5′-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5′-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O₂ concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O₂. 5′-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O₂ concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O₂ concentration.     

Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.     

Modifications in repair and expression of potentially lethal damage (alpha-PLD) as measured by delayed plating or treatment with beta-araA in plateau-phase Ehrlich ascites tumor cells after exposure to charged particles of various specific energies

The ability of Ehrlich ascites tumor cells (EAT cells) to repair potentially lethal damage (alpha-PLD) as demonstrated by either an increase in survival after delayed plating or a decrease in survival after treatment with beta-arabinofuranosyladenine (beta-araA) was investigated after exposure to protons, deuterons, 3He, 4He, and heavy ions of various specific energies. A significant amount of repair or fixation was observed after delayed plating or treatment with beta-araA, respectively, in cells that were exposed to protons of 6-21 MeV energy, reflecting mainly variations in the survival curve shoulder width. Four-hour treatment with 80 microM/liter beta-araA resulted in an exponential survival curve for all proton energies tested. A decrease in particle energy increased killing and caused a reduction in Dq without a significant change in D0. The survival curve obtained after exposure of cells to 3.4 MeV protons had only a small shoulder and was only slightly modified by either delayed plating or treatment with beta-araA, suggesting a decrease in the induction rate of alpha-PLD. Similar results were also obtained after exposure to deuterons and 4He ions. The results are interpreted as indicating the importance of the specific particle energy and the delta-electron spectrum in the induction of alpha-PLD. When the results of delayed plating of cells exposed to protons, deuterons, or helium ions were pooled, an exponential relationship between Dq and penumbra radius was indicated. After exposure to 40Ar ions of 18 MeV specific energy, a shouldered survival curve was obtained, and beta-araA significantly enhanced killing by modifying Dq as well as D0, a result that also suggests induction of repairable damage by the delta particles produced and interaction of lesions induced within the core of the ion path with penumbra lesions. Based on these results a model is proposed assuming that alpha-PLD results from interaction, during the course of repair, of pairs of DNA lesions induced within a distance di. The model assumes the existence of a critical separation distance dic, with the property that pairs of lesions induced with separation distance shorter than dic (expressed as number of base pairs) will always be expressed as lethal, and the existence of a maximum separation distance dim, with the property that pairs of lesions induced with separation distance larger than dim will not interact.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytoskeletal elements in cotton seed hair developmentin vitro: Their possible regulatory role in cell wall organization

Summary The localization and orientation of cytoskeletal elements in developing cotton fibres were studied by the indirect immunofluorescence and the dry cleaving technique. Microtubules are transversely arranged to the cell axis, most probably in a flat helix, in the cortex of expanding fibres. Since the innermost deposited cellulose microfibrils always show primarily the same orientation it is postulated that the microtubules control the transverse deposition of the cellulose fibrils. Little further cell expansion takes place during secondary wall formation and the microfibril pattern corresponds to that of the cortical microtubules,e.g., in the steepness of their helicoidal turns. Microtubules with a length of 7–20 m were observed, probably they are longer. The importance of microtubule length on microfibril deposition is discussed. The density of microtubule packing is in the range of 8–14 m^-1 as in other comparable cell types. In contrast to the microtubules, actin filaments are most likely longitudinally oriented during different phases of fibre development. The dry cleaving technique reveals numerous coated pits in the plasma membrane which are not crossed by microtubules. They seem to be linked to the latter by filamentous structures.     

Effect of microinjected amine and diamine oxidases on the ultrastructure of eukaryotic cultured cells

Diamine oxide and serum amine oxidase, which catalyse the oxidation of diamines and polyamines, respectively, were trapped within reconstituted Sendai virus envelopes. These loaded envelopes were incubated with cultured normal chick fibroblasts or with fibroblasts transformed by Rous sarcoma viruses. The binding of the reconstituted envelopes to the cultured cells was confirmed by scanning electron microscopy. It has been shown that the reconstituted envelopes (1-3 microns diameter) were attached to the eukaryotic cells. No significant changes in the morphology of the normal chick embryo fibroblasts were noted upon treatment with enzyme-loaded envelopes. On the other hand, chick embryo fibroblasts transformed by Rous sarcoma virus were affected by the microinjected amine oxidases. Scanning electron microscopy demonstrated the formation of holes in the microinjected cells. Similar morphological changes were also observed when diamine oxidase was microinjected into cultured glioma cells. These holes may be the result of the ejection of the nucleus. These findings are in line with the observed effect of the injected amine oxidases on macromolecular synthesis in normal and transformed chick embryo fibroblasts.     

The effect of repeated administration of insulin on pain threshold and gamma-aminobutyric acid level in mouse brain

In experiments on male Albino-Swiss mice weighing 18-22 g insulin given in doses of 2 i.u./kg caused no change in the time of reaction to pain, while the same dose administered daily for 7 days potentiated the analgesic action of morphine (3 mg/kg s.c.). Glucose caused no change in this effect of insulin. After 14 days of insulin treatment the time of reaction to pain in the animals subjected to the action of morphine returned to its initial value. Twenty-four hours after the last administration of morphine the level of gamma-aminobutyric acid (GABA) was found to be decreased in the animals receiving insulin with glucose. These results suggest that the central action of insulin is dependent not only on hypoglycaemia produced by it, but may be due also to its direct action on the central structures and an indirect action mediated by its effect on other neurotransmitter systems.     

Orientation and molecular map position of the complement genes in the mouse MHC.

Over the past few years six gene clusters have been isolated from the major histocompatibility complex (MHC) of the BALB/c mouse encompassing a total of 1600 kb of DNA and 48 genes. The molecular distances between these gene clusters and the orientation of four of the six clusters on chromosome 17 is not known. Here we use pulse-field gradient gels and Southern blot hybridization to establish large-scale genomic restriction maps covering several hundreds of kb surrounding the three gene clusters located in the K, I, S, and D regions of the MHC. Comparison of the maps orients the complement gene clusters in the S region with the 21-OHB gene pointing towards the K end and the C2 gene pointing towards the D end of the MHC. The distances between the E alpha and 21-OHB genes is 430 kb and between the C2 and TNF-alpha genes at least 420 kb.     

High affinity uptake of L-glutamate and γ-aminobutyric acid inDrosophila melanogaster

Preparations having properties resembling those of synaptosomes have been isolated from whole fly homogenates ofDrosophila melanogaster using ficoll gradient floatation technique. These have been characterized by marker enzymes and electron microscopy and binding of muscarinic antagenist³H Quinuclidinyl benzilate. An uptake system for neurotransmitter, ã-Aminobutyric acid has been demonstrated in these preparations. A high affinity uptake system for L-glutamate has also been studied in these subcellular fractions. This uptake of glutamate is transport into an osmotically sensitive compartment and not due to binding of glutamate to membrane components. The transport of glutamate has an obligatory requirements for either sodium or potassium ions. Kinetic experiments show that two transport systems, withK _m values 0.33 X 10^-6M and 2.0 X 10^-6M, respectively, function in the accumulation of glutamate. ATP stimulates lower affinity transport of glutamate. Inhibition of glutamate uptake by L-aspartate but not by phenylalanine and tyrosine indicates that a common carrier mediates the transport of both glutamate and aspartate. β-N-oxalyl-L-β β-diamino propionic acid and kainic acid, both inhibitors of glutamate transport in mammalian brain preparations, strongly inhibited transport of glutamate inDrosophila preparations Comparison with uptake of ã-aminobutyric acid and glutamate in isolated larval brain is presented to show that the synaptosome-like preparations we have isolated are rich in central nervous system derived structures, and presynaptic endings from neuromuscular junctions.     

Binding of a nuclear factor to a consensus sequence in the 5' flanking region of zein genes from maize

The genomic organization of the zein structural genes and of regulatory loci influencing their expression suggests that control of zein gene expression will involve interactions between cis elements in the flanking DNA sequences and products from trans-acting genes. The interaction between fragments from the 5' flanking region of a zein gene and specific, double-stranded oligonucleotides with crude nuclear extracts from maize endosperm have been studied by nitrocellulose filter binding, gel retention and DNase I footprinting assays. Specific binding of a nuclear factor was observed and the exact position of the protein binding site was determined. The 22-nt binding site included 14 bp of a 15-bp sequence conserved in all zein genes.