Control of mixed-substrate utilization in continuous cultures of Escherichia coli

The chemostat culture technique was used to study the control mechanisms which operate during utilization of mixtures of glucose and lactose and glucose and l-aspartic acid by populations of Escherichia coli B6. Constitutive mutants were rapidly selected during continuous culture on a mixture of glucose and lactose, and the beta-galactosidase level of the culture increased greatly. After mutant selection, the specific beta-galactosidase level of the culture was a decreasing function of growth rate. In cultures of both the inducible wild type and the constitutive mutant, glucose and lactose were simultaneously utilized at moderate growth rates, whereas only glucose was used in the inducible cultures at high growth rates. Catabolite repression was shown to be the primary mechanism of control of beta-galactosidase level and lactose utilization in continuous culture on mixed substrates. In batch culture, as in the chemostat, catabolite repression acting by itself on the lac enzymes was insufficient to prevent lactose utilization or cause diauxie. Interference with induction of the lac operon, as well as catabolite repression, was necessary to produce diauxic growth. Continuous cultures fed mixtures of glucose and l-aspartic acid utilized both substrates at moderate growth rates, even though the catabolic enzyme aspartase was linearly repressed with increasing growth rate. Although the repression of aspartase paralleled the catabolite repression of beta-galactosidase, l-aspartic acid could be utilized even at very low levels of the catabolic enzyme because of direct anabolic incorporation into protein.     

Mycoside G, a Specific Glycolipid in Mycobacterium marinum (Balnei).

Navalkar, R. G. (University of Wisconsin, Madison), E. Wiegeshaus, E. Kondo, H. K. Kim, and D. W. Smith. Mycoside G, a specific glycolipid in Mycobacterium marinum (Balnei). J. Bacteriol. 90:262-265. 1965.-A new specific glycolipid in extracts prepared from strains designated Mycobacterium marinum and M. balnei has been demonstrated by use of the techniques of column chromatography and infrared spectroscopy. Since there is now agreement among many workers that M. marinum and M. balnei are identical, the demonstration of the same specific glycolipid in both species is not surprising. This substance, which we have designated mycoside G, is chemically similar to mycosides A and B, and apparently differs only in the sugar moiety. In addition, the lipids extracted from these cultures contain phthiocerol dimycocerosate, a wax component found also in M. tuberculosis and M. bovis.     

Light microscopic localization of hepatic fatty acid binding protein mRNA in jejunal epithelia of rats using in situ hybridization, immunohistochemical, and autoradiographic techniques

An in situ hybridization technique using a [35S]-labeled oligonucleotide probe was employed, in combination with immunohistochemistry and autoradiography, to examine gene expression for hepatic fatty acid binding protein (FABP) in the jejunal epithelia from both fed and fasted rats. In rats fed ad libitum, immunoreactivity and mRNA signal for FABP were localized to the absorptive epithelial cells lining the villus, whereas they were absent in the crypt epithelial cells. The level of FABP mRNA was relatively low in the tip of the villus, although FABP immunoreactivity remained high in this area. Animals fasted for 3 days exhibited a downward shift of the lower boundary of the FABP-expressing cell population into the middle portion of the crypt, in terms of the immunoreactivity and the mRNA signal. The proliferative cell compartment of the crypt, as revealed by [3H]-TdR incorporation, showed no substantial change in size between the fed and fasted states. The present results provided evidence that (a) during the differentiation and upward migration of the absorptive epithelial cells, the expression of FABP gene begins at the crypt-villus junction and declines before the cells reach the villus tip, and (b) fasting induces an earlier expression of the FABP gene in the maturing crypt epithelial cells.     

Pathway for the synthesis of triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated spinach leaves

When the upper leaf surface of spinach (Spinacia oleracea L.) plants was treated with [1-(14)C]acetate and grown for 2 days, (14)C was effectively incorporated into acyl moieties of leaf lipids in ratios approximately their composition by mass. Fumigation of the plants with ozone (0.5 microliter per liter) caused a redistribution of (14)C among lipid classes, i.e. a marked increase of (14)C content in triacylglycerol (TG) and 1,2-diacylglycerol (1,2-DG) and a decrease of label in monogalactosyldiacylglycerol (MGDG) without affecting (14)C distribution in leaf fatty acids. Label in both TG and 1,2-DG was found predominantly in their polyene molecular species. Since MGDG consists of similar polyene molecular species, the results indicate the synthesis of TG from MGDG via 1,2-DG. Label was also accumulated in tri- and tetragalactosyldiacylglycerol, products of galactolipid:galactolipid galactosyltransferase (GGGT). Moreover, there was a close relation between increases in the amounts of TG and the oligogalactolipids in ozonetreated leaves. These results indicate that MGDG was converted to 1,2-DG by GGGT and then to TG. In intact chloroplasts isolated from ozone-treated leaves, there was an enhanced production of free fatty acid (FFA), which was diminished by the addition of coenzyme A (CoA) and ATP, indicating that ozone stimulated the hydrolysis of MGDG to liberate FFA, which was in turn converted to acyl-CoA. The final step of TG synthesis, acylation of 1,2-DG with acyl-CoA, was confirmed by feeding with [1-(14)C]linolenic acid in leaf discs excised from ozone-fumigated leaves; (14)C was effectively incorporated into TG but not into 1,2-DG. These results demonstrate the synthesis of TG from 1,2-DG and FFA which were liberated from MGDG in ozone-fumigated spinach leaves.     

Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.     

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.     

Interaction between endothelium-derived relaxing factors, S-nitrosothiols, and endothelin-1 on Ca2+ mobilization in rat vascular smooth muscle cells.

S-Nitrosothiols (S-nitrosocysteine, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine), which belong to the group of endothelium-derived relaxing factors (EDRFs), caused decreases of cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMCs). The endothelin-1 (ET-1)-induced sustained increase of [Ca2+]i in rat VSMCs was completely abolished by preaddition of at least an equal molar quantity of S-nitrosocysteine (Cys-SNO). Also exposure of VSMCs to a mixture of Cys-SNO and ET-1 at the same time resulted in the transient increase only. These results suggest that S-nitrosothiols may have no significant effect on ET-1-induced Ca2+ release from intracellular stores via inositol 1,4,5-triphosphate production but do affect Ca2+ influx through Ca2+ channels in the plasma membrane.     

Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis.

To delineate domains essential for G-protein coupling in angiotensin II type 1 receptor (AT1), we mutated the receptor cDNA in the putative cytosolic regions and determined consequent changes in the effect of GTP analogs on angiotensin II (Ang II) binding and in inositol trisphosphate production in response to Ang II. Polar residues in targeted areas were replaced by small neutral residues. Mutations in the second cytosolic loop, carboxy terminal region of the third cytosolic loop or deletional mutation in the carboxyl terminal tail simultaneously abolished both the GTP-induced shift to the low affinity form and Ang II-induced stimulation of inositol trisphosphate production. These results suggest that polar residues in the second cytosolic loop, the carboxy terminal region of the third cytosolic loop, and the carboxy terminal cytosolic tail are important for G-protein coupling of AT1 receptor.     

Influence of sleep on the cardiovascular and metabolic responses to a decrease in ambient temperature in lambs

Experiments were done on seven lambs between the ages of 10 and 24 days to investigate the effects of sleep on the cardiovascular and metabolic responses to a decrease in ambient temperature. Each lamb was anesthetized and instrumented for recordings of electrocorticogram, electro-oculogram, and nuchal electromyograms and measurements of cardiac output, systemic and pulmonic pressures and hemoglobin oxygen saturations as well as body core temperature. No sooner than three days after surgery, measurements were made during periods of quiet wakefulness, quiet sleep and active sleep at ambient temperatures of 25 degrees C and 18 degrees C. Decreasing the environmental temperature from 25 degrees C to 18 degrees C elicited a similar thermogenic response during quiet wakefulness, quiet sleep and active sleep as evidenced by an increase in total body oxygen consumption. The increased metabolic oxygen demand was met by an increase in systemic oxygen transport as well as by an increase in total body oxygen extraction. Since shivering was absent during active sleep, it is likely that nonshivering thermogenesis played a major role in the metabolic response. Our data provide evidence that sleep does not significantly alter the cardiovascular and metabolic responses to a modest decrease in ambient temperature in young lambs.