GLUCOSE AND AMINO ACID METABOLISM IN OCTOPUS OPTIC AND VERTICAL LOBES IN VITRO

(1) The metabolism of glucose and amino acids in vitro was compared in the rat cerebral cortex and the optic and vertical lobes of the octopus brain. (2) Specific activities and pool sizes of the five amino acids, glutamate, aspartate, glutamine, alanine and γ-aminobutyric acid (GABA), were determined in octopus and rat brain slices after 2 hr incubation with 10 mm -[U-¹⁴C]glucose, 10 mm -L-[U-¹⁴C]glutamate, and 10mm -^L-[U-¹⁴C]glutamate with added 10 mM-glucose. Amino acid pool sizes were similar in rat and octopus brain, with the exception of alanine, which was higher in the octopus. Generally specific activities were from four- to 20-fold higher in rat brain. With [U-¹⁴C]glucose as substrate, specific activities of GABA and glutamate were highest in rat; those of alanine and glutamine highest in octopus brain. With ^L-[U-¹⁴C]glutamate the specific activities of GABA and aspartate were highest in rat, that of aspartate highest and GABA lowest in octopus. The addition of glucose to ^L-[U-¹⁴C]glutamate as substrate had little effect on the specific activities of any of the amino acids. (3) The uptake of some amino acids was determined by incubation with [U-¹⁴C]amino acids for 2 hr, and ¹⁴CO₂ formation was also measured. The amount of label taken up by octopus was uniformly 20-25 per cent of that found for rat brain. The amount of ¹⁴CO₂, however, differed according to the amino acid. Four times as much ¹⁴CO₂ was generated from alanine by octopus optic lobe and twice as much by the vertical lobe than rat cortex, but from glutamate, only 24 per cent in the optic and 15 per cent in the vertical lobe. No ¹⁴CO₂ was generated from [U-¹⁴C]GABA in the octopus, by contrast with the rat. (4) Activity of some of the enzymes involved in amino acid metabolism was determined in homogenates of rat cortex and octopus optic and vertical lobes, with and without activation by Triton X-100. Enzymic activities in the octopus, with the exception of alanine aminotransferase, were lower than in the rat, and glutamate decarboxylase could not be detected in octopus brain, in the absence of detergent.     

Properties of epithelial cells cultured from human carcinomas and nonmalignant tissues

Human epithelial cell cultures were examined for expression of plasminogen activator and fibronectin matrix. All of the cells examined showed ultrastructural evidence suggesting their epithelial origin, including microvilli and specialized junctions. The nonmalignant cells were also negative for endothelial cell markers (ie, they lacked factor VIII antigen, a nonthrombogenic surface and Weibel-Palade bodies). The nonmalignant lines all produced large amounts of plasminogen activator, whereas the tumor-derived lines showed a gradation of activities, ranging from lines having as much activity as the nonmalignant lines to lines having little or no activity above background. For both normal and malignant cells, addition of dexamethesone only slightly decreased the levels of plasminogen activator. By immunofluorescence microscopy, normal bladder and fetal intestine epithelial cells showed fibronectin in a globular and fibrillar matrix. In contrast, normal mammary epithelial cells had a much diminished amount of fibronectin with a punctate distribution.     

Fat formation in preformed fungal mats of penicillium notatum

A fat-forming fungus:Penicillium notatum was grown on a physiologically balanced medium for 4 days. The old medium was then replaced by a fat-promoting medium and the fungal cultures were reincubated for a further period of 10 days. Active growth was resumed. Protein synthesis also continued actively at the expense of the low nitrogen supply in the replacement medium and soon stopped when proteolysis started. Fat formation, on the other hand, started and proceeded for a longer period at a very high rate leading to a very high fat yield.When the fungal mats were replaced on the fat-promoting medium at different nitrogen concentrations, the medium with the lowest nitrogen supply gave rise to weak growth and low fat yield as well. The highest nitrogen supply gave rise to active growth with high protein content and low fat yield. At a certain concentration of nitrogen, there was a high growth rate involving the highest fat yield. It is concluded that replacement medium deficient in nitrogen is not the most favorable for fat formation. The nitrogen level in the replacement medium should be adjusted so as to afford good growth and consequently good utilization of excess carbohydrates in fat syntheses.     

Clonal propagation of kurrat (Allium ampeloprasum var. kurrat)

Summary Two protocols for clonal propagation of kurrat (Allium ampeloprasum var.kurrat) using explants from the basal plates of mature plants are described. In direct formation, explants were cultured in Murashige and Skoog (MS) medium and supplemented with either benzyladenine at 0.0 or 4.4 μM, or supplemented with 7.0 μM benzyladenine and 0.1 μM naphthaleneacetic acid. Shoots appeared after 4 wk of culture. In the two-step procedure, explants were cultured first on MS medium supplemented with 1.4 μM 2,4-dichlorophenoxyacetic acid and 1.4 μM kinetin, and incubated in the dark for 4 wk. They were then transferred to MS medium supplemented with 4.4 μM benzyladenine for shoot formation. All shoots were rooted on MS medium containing 5 g·liter⁻¹ activated charcoal. Normal viable plants were successfully established in soil.     

Glycolysis inhibition by palmitate in renal cells cultured in a two-chamber system

A major shortcoming of renalproximal tubular cells (RPTC) in culture is the gradual modification oftheir energy metabolism from the oxidative type to the glycolytic type.To test the possible reduction of glycolysis by naturally occurringlong-chain fatty acids, RPTC were cultured in a two-chamber system,with albumin-bound palmitate (0.4 mM) added to the basolateral chamberafter confluency. Twenty-four hours of contact with palmitate decreasedglycolysis by 38% provided that carnitine was present;lactate production was decreased by 38%, and the decrease inglycolysis resulted from a similar decrease of basolateral and apicalnet uptake of glucose. In contrast to the previously described effectof the nonphysiological oxidative substrate heptanoate, palmitatepromoted a long-term decrease in lactate production and sustainedexcellent cellular growth. After 4 days of contact, decreasedglycolysis was maintained even in the absence of carnitine and resultedfrom a decrease of basolateral uptake only, suggestive of long-term regulation different from the earlier effects. Thus, although culturedRPTC lost their oxidative phenotype, they exhibited a type ofregulation (Randle effect) that is found in the oxidative-type but notin the glycolytic-type tissues, therefore unmasking a regulativecapacity barely detectable in fresh RPTC. LowPO₂ (50 mmHg in the apical chamber) could be amajor cause of elevated glycolysis and could hinder the effects ofpalmitate.

     

Site-specific intercalation at the triplex-duplex junction induces a conformational change which is detectable by hypersensitivity to diethylpyrocarbonate.

Using site-specific intercalation directed by intermolecular triplex formation, the conformation of an intercalation site in DNA was examined by footprinting with the purine-specific (A much greater than G) reagent diethylpyrocarbonate. Site specific intercalation was achieved by covalently linking an intercalator to the 5' end of a homopyrimidine oligodeoxynucleotide, which bound to a homopurinehomopyrimidine stretch in a recombinant plasmid via intermolecular triplex formation. This directs intercalation to a single site in 3kb of DNA at the 5' triplex-duplex junction. Footprinting with diethylpyrocarbonate and dimethylsulphate revealed strong protection from modification of adenine residues within the triple-helix in concordance with their Hoogsteen pairing with the third strand, and a strong hypersensitivity to diethylpyrocarbonate at the first adenine of the duplex. This result indicates that intercalation at this site induces a conformational change at the 5' triplex-duplex junction. Furthermore, the same diethlypyrocarbonate hypersensitivity was observed with an unmodified triple-strand forming oligonucleotide and a range of intercalating molecules present in solution. Thus the 5' triplex-duplex junction is a strong binding site for some intercalating molecules and the junction undergoes a conformational change which is sensitive to diethylpyrocarbonate upon insertion of the planar aromatic chromophore. This conformational change can be used to direct a single-strand cut in duplex DNA to a defined site.     

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine—the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfection of Gs-Enbrel and Aspg-Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5-fold higher specific productivity and ~3-fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.     

Prevention of ischemia-reperfusion lung injury by sulfated Lewisa pentasaccharide

Reignier, Jean, Hassan Sellak, Rémy Lemoine,André Lubineau, Guy Michel Mazmanian, Hélène Detruit,Alain Chapelier, Philippe Hervé, and The Paris-Sud UniversityLung Transplantation Group. Prevention of ischemia-reperfusionlung injury by sulfated Lewis^apentasaccharide. J. Appl. Physiol.82(4): 1058-1063, 1997.Inhibition of polymorphonuclearneutrophil (PMN) adhesion to the pulmonary endothelium attenuatesischemia-reperfusion (I/R) lung injury. We hypothesized that3-sulfated Lewis^a (SuLa), apotent ligand for the selectin adhesion molecules, may have abeneficial effect on I/R lung injury, as measured by the filtrationcoefficient(K_fc),and reduce pulmonary sequestration of PMN as assessed by the lungmyeloperoxidase (MPO) activity. Blood-perfused rat lungs were subjectedto 30 min of perfusion, 60 min of warm ischemia, and 90 min ofreperfusion after treatment with either SuLa (200 µg) or saline.Effects of SuLa on PMN adhesion to cultured human umbilical veinendothelial cells (HUVEC) stimulated with tumor necrosis factor- andcalcium ionophore were also investigated. Compared with preischemiaconditions, I/R induced a significant increase inK_fc,which was attenuated with SuLa (80 ± 8 vs. 30 ± 5%; P < 0.001). SuLa reduced lungMPO and PMN adhesion to stimulated HUVEC. These results indicate thatSuLa reduces I/R-induced lung injury and PMN accumulation in lung. Thisprotective effect might be related to inhibition of PMN adhesion toendothelial cells.