Induction of T4 DNA ligase in a recombinant strain of Escherichia coli

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed-batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature-sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30 degrees C followed by an induction phase which was initiated by shifting the temperature to 42 degrees C. In the fed-batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42 degrees C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed-batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.     

TGF-beta 1-induced inhibition of mouse mammary ductal growth: developmental specificity and characterization

TGF-beta 1, implanted into growing mouse mammary glands, was previously shown to inhibit ductal growth in an apparently normal and fully reversible manner. In this report we extend these findings to show that TGF-beta 1 inhibition is highly specific. In pregnant or hormone-treated mice, doses of TGF-beta 1 that were capable of fully inhibiting ductal elongation had little effect on the proliferation of lobuloalveolar structures. Additionally, the inhibitory action of TGF-beta 1 on ducts is epithelium-specific, resulting in cessation of DNA synthesis in the rapidly proliferating epithelium of mammary end buds, but does not inhibit DNA synthesis in the stroma surrounding the end buds. At the cellular level, transplant studies showed that TGF-beta 1 inhibited the regeneration of mammary ductal cells when implanted into mammary gland-free fat pads by suppressing the formation of new end buds, without inhibiting maintenance DNA synthesis in ductal lumenal epithelium; this observation indicates the potential of TGF-beta 1 to maintain patterning by suppressing adventitious lateral branching. The time-course of TGF-beta 1 inhibition of end buds was rapid, with cessation of DNA synthesis by 12 hr, followed by loss of the stem cell (cap cell) layer. The question of glandular exposure to TGF-beta 1 administered in EVAc implants was also investigated. Incorporation of TGF-beta 1 into EVAc was found not to degrade the hormone, while the release kinetics of the ligand from implants, its retention in the gland, and the demonstrable zone of exposure were consistent with observed inhibitory effects. These results support the hypothesis that TGF-beta 1 is a natural regulator of mammary ductal growth.     

Determination of [U-13C]glucose turnover into various metabolite pools for the differential diagnosis of lactic acidemias

Stable-isotope tracer experiments performed in vitro are evaluated for their utility in differentiating between pyruvate dehydrogenase and cytochrome oxidase deficiencies, two of several enzyme defects commonly associated with the lactic acidemias. Fibroblasts of enzyme-deficient individuals and of age-matched controls are grown in medium containing [U-13C]glucose. Direct analysis of cells and conditioned culture medium provides only minor differences in the organic acid/amino acid GC-MS profiles, making differentiation of enzyme defects difficult by this method. However, differences have been found in the glucose turnover into various cell metabolites, making differentiation of these two enzyme defects possible. The cellular pool of glutamic acid experiences 13C-enrichment in both the control and cytochrome oxidase deficient lines, but not in the pyruvate dehydrogenase-deficient line. The cellular pool of an unknown, possibly an aminopentose sugar, on the other hand, experiences 13C-enrichment in the pyruvate dehydrogenase and control lines, but not in the cytochrome oxidase line. These observations, as well as other differences in the extent of enrichment into various metabolite pools, suggest that this stable-isotope approach, in vitro, is feasible and may allow these two enzyme defects to be differentiated in a definitive manner. Such stable-isotope experiments are easy to carry out with cultured cells and are inexpensive. Applications of the technique to other genetic disorders might be appropriate.     

The preferential uptake of very-low-density lipoprotein cholesteryl ester by rat liver in vivo.

The removal from the blood and the uptake by the liver of injected very-low-density lipoprotein (VLDL) preparations that had been radiolabelled in their apoprotein and cholesteryl ester moieties was studied in lactating rats. Radiolabelled cholesteryl ester was removed from the blood and taken up by the liver more rapidly than sucrose-radiolabelled apoprotein. Near-maximum cholesteryl ester uptake by the liver occurred within 5 min of the injection of the VLDL. At this time, apoprotein B uptake by the liver was only about 25% of the maximum. Maximum uptake of the injected VLDL apoprotein B label was not achieved until at least 15 min after injection, by which time the total uptakes of cholesteryl ester and apoprotein B label were very similar. The results suggest that preferential uptake of the lipoprotein cholesteryl ester by the liver occurred before endocytosis of the entire lipoprotein complex. The fate of the injected VLDL cholesteryl ester after its uptake by the liver was also monitored. Radiolabel associated with the hepatic cholesteryl ester fraction fell steadily from its early maximum level, the rate of fall being faster and more extensive when the fatty acid, rather than the cholesterol, moiety of the ester was labelled. By 30 min after the injection of VLDL containing [3H]cholesteryl ester, over one-third of the injected label was already present as [3H]cholesterol in the liver. When VLDL containing cholesteryl [14C]oleate was injected, a substantial proportion (about 25%) of the injected radiolabelled fatty acid appeared in the hepatic triacylglycerol fraction within 60 min: very little was present in the plasma triacylglycerol fraction at this time.     

Light-dependent GTP-binding proteins in squid photoreceptors.

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5'-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.     

The reaction mechanism of Na, K-ATPase

To help characterize the Na,K-ATPase active site with enzyme incorporated into phospholipid vesicles, the activities with alternative substrates were compared, 22Na/Na-transport was equivalent with ATP, CTP, carbamylphosphate and acetylphosphate, but slower with CTP, 3-O-methylfluoresceinphosphate (3-O-MFP), nitrophenylphosphate and umbelliferonephosphate. It indicates a slower rate of formation of phosphorylating enzyme complex in conformation position of E1 (E1P) when the second group of substrates is bound with enzyme active center. 22Na/K-transport was half as effective with CTP as with ATP and was far slower with the other substrates. It indicates a more stringent selectivity at the low-affinity site of enzyme in conformation E2 that accelerates the slow step of this transport mode. Although enzyme modification with fluoresceinisothiocyanate blocks the high-affinity site to ATP, the K-phosphatase reaction catalyzed by E2 is retained, even with a substrate, 3-O-MFP, that binds to the adenine pocket. Dimethylsulfoxide inhibits hydrolysis of the nucleotides and of the carboxylic phosphate substrates of the K-phosphatase reaction, but stimulates hydrolysis of the phenolic phosphate substrates (nitrophenylphosphate and umbelliferone phosphate) which normally are hydrolyzed more slowly than the other substrates. On the basis of these data the authors propose the model of Na,K-ATPase active center.     

Visualization of collagenase-sensitive acetylcholinesterase in isolated cardiomyocytes and in heart tissue

Summary Previous studies have indicated that the asymmetric form of acetylcholinesterase (collagen-tailed) is localized in the basal lamina of the neuromuscular junction of skeletal muscle. The present study shows localization of the asymmetric acetylcholinesterase in the heart of the rat. Antiserum to 14+18 S acetylcholinesterase of the electric eel was raised in rabbits. The purified antibody did not react with collagen type I or laminin. Collagenase reduced the immunoreactivity of the enzyme with the purified antibody. Isolated cardiomyocytes and frozen sections of the heart were stained for acetylcholinesterase with the antibody. Diffuse immunofluorescence appeared over the surface of the cardiomyocytes. In the frozen sections, the immunofluorescence was most intense at the cell boundaries. These data suggest that collagenase-sensitive acetylcholinesterase in the heart is present in the myocytes and occurs in the vicinity of the basal lamina.Abbreviations AChE acetylcholinesterase - BSA bovine serum albumin - PBS phosphate-buffered saline - DME Dulbecco's Modified Eagle Medium     

Kinetics of phosphorus uptake by immobilizedChlorella

Summary Alginate-entrappedChlorella demonstrate rapid uptake of phosphorus from synthetic growth medium in batch culture. Rates of phosphorus uptake demonstrated by immobilized algae were found to be much lower than those of non-immobilized cells. Uptake was dependent upon matrix stocking density, cell preculture conditions and cell viability, but not upon cell growth.     

The effect of prolonged hypobaric hypoxia on growth of fetal sheep

The effect of prolonged hypobaric hypoxia on fetal sheep was studied. Pregnant ewes were subjected to an atmospheric pressure of 429 torr from 30 days to 135 days gestation (long-term study). Average fetal weight for the hypoxaemic group (3.35 +/- 0.53 kg; n = 4; mean +/- SD) was significantly lower than for the controls (4.23 +/- 0.29 kg; n = 7; P less than 0.05). A short-term study was undertaken with fetuses (n = 8) which were catheterized at 110 days gestation and whose dams were subjected to hypobaric hypoxia from 120 to 141 days gestation. The mean carotid PO2 of fetuses in the hypoxic group was 12.7 +/- 0.7 torr compared to 22.7 +/- 0.7 torr for the control group (n = 9; P less than 0.001) throughout the period of treatment. Fetal arterial oxygen content fell from 6.5 +/- 1.7 to 4.9 +/- 0.4 ml/dl (P less than 0.05), but rose to control values after 7 days due to an increase in fetal haemoglobin concentration (9.6 +/- 1.1 to 13.0 +/- 1.9 g/dl, P less than 0.001) and packed cell volume (33 +/- 3 to 45 +/- 4%, P less than 0.001). In the hypoxaemic fetuses, pH fell initially from 7.34 +/- 0.02 to 7.28 +/- 0.03 (P less than 0.05) and then recovered to 7.32 +/- 0.03 within 24 h. Mean fetal weight of the short-term hypoxic group was 3.46 +/- 0.72 kg compared to 4.15 +/- 0.51 for the control group (P less than 0.05). Both long- and short-term hypoxia produced a similar reduction in fetal body weight. The adrenal glands were significantly heavier in the hypoxic fetuses than in controls. Placental weight was not effected by hypoxia, but exposure from 30 days gestation reduced the average size of cotyledons (P less than 0.05). It is concluded that the fetal sheep increases its ability to acquire and transport oxygen in response to chronic hypoxia, but this compensation is not sufficient to prevent growth retardation or changes to the pattern of tissue growth.     

Magnetic properties of tunicate blood cells. I. Ascidia nigra

The magnetic properties of intact and freeze-dried blood cells of the tunicate Ascidia nigra and of model vanadium(III) and (IV) compounds as polycrystalline solids and in aqueous solution have been measured up to 50 kOe with a SQUID susceptometer. Corrections for the samples' diamagnetism were extracted from the temperature dependence of the data without any further assumptions. For vanadium(IV), measured values of the magnetic moment at different values of the applied magnetic field over the temperature range 2-100 K obey a Brillouin function with spin 1/2. For vanadium(III), the magnetic moment data did not obey a Brillouin function and were analyzed in terms of a spin Hamiltonian with S = 1. Measurements on both whole and freeze-dried blood samples give consistent results with vanadium(III) the predominant species. These results are discussed in terms of the mechanisms of vanadium accumulation and the use of vanadium oxidation states as criteria of ascidian taxonomy.