Chemical characterization of two lipopolysaccharide species isolated from Rhizobium loti NZP2213

Phenol-water extraction of Rhizobium loti NZP2213 cells allowed a simultaneous isolation of two structurally different lipopolysaccharides, from the aqueous (LPS-W) and phenol (LPS-P) phase that differed in their sodium doexycholate-PAGE pattern and composition. LPS-W showed a profile indicating an R-type LPS; LPS-P had a cluster of poorly resolved bands in the high-molecular-weight region. LPS-P contained large amounts of 6-deoxy-l-talose (6dTal), and a small amount of 2-O-methyl-6-deoxy-talose (molar ratio 30:1), both of which were completely absent in LPS-W. Methylation analysis gave only one major product, 2,4-di-O-methyl-6dTal, indicating that the O-chain is composed of a homopolymer of 1,3-linked 6dTal, having the methylated 6dTal (2-O-me-6dTal) probably localized at the non-reducing end of the O-chain. This homopolymeric O-chain was additionally O-acetylated, as evidenced by GC-MS and by ¹³C NMR analysis. The lipid A moieties of both LPS-W and LPS-P showed almost identical composition, with six, different 3-OH fatty acids and with two, so far not described, long-chain 4-oxo-fatty acids, all being amide-linked, and with 27-OH-28:0 as the main ester-linked fatty acid. Lipid A was of the lipid A_DAG-type, i.e., having a (phosphorylated) 2,3-diamino-2,3-dideoxy-d-glucose-containing lipid A backbone. Lipid A_DAG is widespread among species of the -2 group of Proteobacteria, but has so far not been encountered in any other rhizobial or agrobacterial species.     

Changes in metabolism and hormone trafficking during exposure of endocrine cells to elevated ammonium

Endocrine cell cultures have potential in bioprocessing, for the production of biologically active hormones, and in tissue engineering, for the development of implantable artificial tissues for long-term restoration of endocrine function. To optimize such systems, it is necessary to develop a thorough understanding of how inherently present environmental stresses, such as nutrient depletion and metabolite accumulation, affect the cells. This work focuses on the effects of the metabolite ammonium on indicators of endocrine cell metabolism and on the processing, storage and secretion of regulated secretory proteins. Experiments were conducted on recombinant insulin-producing mouse pituitary AtT-20 cells and mouse insulinoma TC3 cells. Exposure for 24–48 hours to 6 mM of exogenous ammonium resulted in higher rates of glucose consumption by both AtT-20 and TC3 cells, while the formation of additional ammonium generally decreased relative to ammonium-free controls. When TC3 cells were discharged of their intracellular insulin stores, the presence of ammonium during a subsequent recharge completely inhibited addition of new insulin-related peptides to the stores, as we had observed previously for both cell lines. There was a correlation between insulin-related peptides stored in TC3 cells during recharging and the amount that could be released upon secretagogue stimulation. Using a combination of radioimmunoassay and high performance liquid chromatography, we found that intracellular insulin and insulin-related peptides changed in the same fashion. Intracellular mechanisms that may be producing the observed results are discussed.Abbreviations IRP insulin-related peptides - HPLC high performance liquid chromatography - DAMP 3-(2,4-dinitroanilino)-3 amino-N-methyldipropylamine     

Inositol (1,4,5)trisphosphate-promoted Ca2+ release from microsomal fractions of rat liver

Crude mitochondrial fractions containing a substantial amount of microsomes accumulate Ca2+ in the presence of ATP, ruthenium red and oligomycin. A proportion of this accumulated Ca2+ is released by the addition of low concentrations (ca. 1 microM) of inositol (1,4,5) trisphosphate . Under some conditions the release is transient, and evidence is presented which suggests that this is due to inhomogeneity in the vesicle population. (1,4,5)inositol trisphosphate -induced Ca2+ release can also be demonstrated, under appropriate experimental conditions, in a more purified microsomal fraction essentially free of mitochondria.     

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.     

Distribution of repeated DNA families in the human genome

By means of restriction enzymes analysis and molecular hybridization, the distribution of repeated DNA families has been studied in the different DNA components into which the human genome can be fractionated by density gradient techniques. Three classes of DNA molecules have been analyzed: i) an homogeneous DNA component (satellite-like sequences; Q = 1.696 g/cm3, 3% of total DNA, AT repeated), ii) AT rich (Q = 1.698 g/cm3, 30% of total DNA, AT main-band) and GC rich (Q = 1.708 g/cm3, 6% of total DNA, GC main-band) DNA components. By this approach we have observed that Sau3A digestion of GC main-band gives rise to two bands of 75bp and 150bp, absent or under-represented in both AT rich DNA components. A preliminary characterization of these DNA fragments suggests that they contain one or more families of repeated sequences which fail to hybridize to EcoRI, HindIII and AluI families of repeats. In addition, we have observed that EcoRI sequences (alpha-RI DNA) are under-represented in GC main-band and show the same clustered organization in both AT rich DNA components.     

Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903

Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS). To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (L-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol. Several ARS-containing plasmids were selected from a K. lactis recombinant DNA library in K. lactis and in Saccharomyces cerevisiae. Two of four ARS clones selected in K. lactis promoted transformation frequencies of 5-10 X 10(2) G418-resistant cells/micrograms of plasmid DNA. This frequency of transformation was at least twice as high as with ARS clones selected in S. cerevisiae. The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug. In the absence of selection pressure less than 5% of the cells retained the drug-resistance phenotype. Plasmids containing the ARS1 or 2 mu replicon of S. cerevisiae failed to transform K. lactis for G418 resistance. Inclusion of S. cerevisiae centromere, CEN4, in a K. lactis ARS recombinant plasmid did not increase the stability of the plasmid in K. lactis, and marker genes on the vector segregated predominantly 4-:0+ through meiosis. We conclude that neither the ARS sequences or the centromere of S. cerevisiae was functioning in K. lactis.     

Les ammonites boréales des formationsnéocomiennes du Sud-Est français (province subméditerranéenne)

The boreal Ammonites (and the more or lessphylogenetically related ones) of the southeast France (Tethyan Realm) are described and figured. 24 forms are refered to, or compared with, previously identified species. 3 are left under open nomenclature, and 7 are new ones. Two new subgenus: Julianites (Paquiericeras) and Lemurostephanus (Olcostephanus) are also introduced.Their biostratigraphic position in the Frenchzonal scheme is detailed. Their contribution to the establishment of correlation between the Boreal and Tethyan provinces is stressed.The most interesting feature is the identification of a Prodichotomites horizon just below the Verrucosum zone, allowing comparison of the definition of the Lower-Upper Valanginian boundary in these two paleobiogeographic realms.     

Hormonal control of uric acid storage in the fat body during last-larval instar of Manduca sexta

In the last-larval instar of the tobacco hornworm, Manduca sexta, a switch from excretion of uric acid to storage in the fat body occurs during transition from the feeding to the wandering stage. Neuroendocrine control of this change from excretion to storage was demonstrated by neck-ligation experiments with synchronously reared larvae. Results indicate that a neurohormone is released from the head 24–30 hr before the initiation of wandering and coincident with the first release of ecdysone that initiates metamorphosis. Direct involvement of the moulting hormone was shown by the effects of multiple injections of 20-hydroxyecdysone into the abdomen of larvae that had been ligated before the release of hormone. Urate levels in the fat body were 20- to 100-fold higher from hormone-injected larvae as from saline inject controls. Topically applied juvenile hormone or methoprene reversed the 20-hydroxyecdysone-induced storage of urate. Increased levels of uric acid in the haemolymph during pupal development result from the presence of juvenile hormone, and the abrupt decrease in uric acid concentration in the haemolymph just prior to pupal ecdysis results from a decreased titre of juvenile hormone. Applications of methoprene prevented the decrease in uric acid levels in the haemolymph.     

Progestin metabolism in the rhesus monkey corpus luteum

For the purpose of describing the pathway by which estrogens are synthesized in the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) corpus luteum (CL), CL were obtained during the midluteal phase of the menstrual cycle and fragments incubated with equimolar amounts of [7-³H]pregnenolone plus [4-¹⁴C]progesterone. Metabolites including ³H-progesterone, ³H, ¹⁴C-20α-dihydroprogesterone, ³H, ¹⁴C-17-hydroxyprogesterone, ³H-estrone and ³H-estradiol-17β appeared in the medium during the first 20 minutes of incubation, ³H, ¹⁴C-Androstenedione was not consistently noted until after 60 minutes. Despite the fact that the ¹⁴C/³H-17-hydroxyprogesterone ratio quickly approached a constant value in the medium, ¹⁴C-estrogens were not detected in the medium or tissue fragments suggesting that progesterone was not a principal precursor for estrogen synthesis. As evidenced by the observation that the ¹⁴C/³H-progesterone ratio was significantly higher in luteal fragments than the 17-hydroxyprogesterone ratio, 17-hydroxyprogesterone appeared to be synthesized from pregnenolone both by way of progesterone and by another route which did not include progesterone. C₂₁- and C₁₈-Steroids were more concentrated in tissue fragments after 120 minutes of incubation than in the medium indicating that these steroids were sequestered by luteal tissue.     

Identification of newly synthesized wing imaginal disc proteins induced by 20-hydroxyecdysone inGalleria mellonella

Summary Wing imaginal discs from 7th instarGalleria mellonella L. larvae evaginate and exhibit tracheolar elongation when exposed to 20-hydroxyecdysone in vitro. This response was elicited within 24 h of treatment as was a greater than fourfold stimulation of the incorporation of [³H]leucine into disc proteins. Autoradiographic analyses of [³⁵S]methionine labeled polypeptides separated on two-dimensional gels, however, revealed no differences in protein profiles between control and treated discs until 48 h following exposure to molting hormone. At this time, wing imaginal discs exposed to 1 μg/ml 20-hydroxyecdysone synthesized four unique polypeptides not detected either in controls or in discs treated for 24 h. These four new proteins were also found to be synthesized by imaginal discs that had evaginated in vivo. These results suggest that these proteins are normally synthesized subsequent to evagination and do not play a role in the morphological events necessary for evagination. Mention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA. S. G. M. is employed through a cooperative agreement between the Insect Attractants, Behavior and Basic Biology Laboratory and the Department of Entomology, University of Florida.