Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Decreased production ofpara-hydroxypenicillin V in penicillin V fermentations

Summary Penicillin V (phenoxymethyl penicillin) is produced by industrial strains ofPenicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain ofP. chrysogenum produces an undesirable penicillin byproduct,para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, viapara-hydroxylation of POAc and subsequent incorporation of thep-OH phenoxyacetic acid into the penicillin molecule. Most of thep-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level ofp-OH penicillin V produced by the control strain ranges up to 10–15% of the total penicillins produced. 3-Phenoxypropionic acid andp-bromophenylacetic acid partially inhibit the formation ofp-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels ofp-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which producep-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.     

Mutants of a lovastatin-hyperproducingAspergillus terreus deficient in the production of sulochrin

Summary A lovastatin-hyperproducing culture ofAspergillus terreus was shown to produce several co-metabolites extracted from whole broth. The predominant co-metabolite was the benzophenone, sulochrin, reported to arise from a polyketide biosynthetic pathway. This compound was targeted for elimination by classical mutagenesis and screening. A surface culture method employing microtiter, plates was used to ferment mutants for the primary screen. Qualitative determinations of lovastatin and sulochrin production were achieved by high-performance thin-layer chromatography. A mutant, strain AH6, which produced lovastatin titers equivalent to the parent culture and no detectable sulochrin was isolated. In addition, a lovastatin-hyperproducing mutant designated CB4 was capable of producing 16% more lovastatin and 30% less sulochrin than the parent culture in shake flask fermentations. In a pilot-scale 250-gallon fermentation, strain CB4 gave a 20% increase in lovastatin titer while producing 83% less sulochrin than the parent culture.     

Extractive lactic acid fermentation using ion-exchange resin

Lactic acid fermentation is an end-product-inhibited reaction. The restriction imposed by lactic acid on its fermentation can be avoided by extractive fermentation techniques. Studies were performed by attaching an ion-exchange resin packed column with a 2-L fermentor for separation of lactic acid. The fermentation, in a conventional batch mode, resulted in a lactic acid yield of 0.828 g . g(-1) and a lactic acid productivity of 0.313 g . L(-1) . h(-1). However, these could be further enhanced to 0.929 g . g(-1) and 1.665 g . L(-1) . h(-1) by extractive fermentation techniques. The effect of temperature on extractive fermentation was remarkable and has been included in this work.     

Effect of feed zone in fed-batch fermentations of Saccharomyces cerevisiae

In production-scale, fed-batch fermentations, feed is often added to a single point at the top of the fermentor, which, combined with poor mixing, results in formation of a "feed zone" rich in nutrients. Frequent exposure of the culture to high concentrations of nutrients in the feed zone for sufficient duration can produce unexpected effects on its performance. The effect of the feed zone was evaluated by conducting aerobic fed-batch fermentations of Saccharomyces cerevisiae with both complex and defined media. The broth was recirculated between a recycle loop and a bench-scale fermentor, and feed was intermittently added into the recycle loop to simulate the circulation of cells through the feed zone. Experiments were carried out for a range of residence times in the recycle loop from 0.5 to 12 min. Biomass yields from the complex-media fermentations were not affected by exposure to high nutrient levels in the recycle loop for residence times up to 12 min. Ethanol consumption was reduced by as much as 50% for residence time in the loop up to 3 min. Very long exposure of yeast cells to excess nutrient levels (12 min) gave acetic acid formation. In a defined medium, the simulated feed zone effect increased biomass yield by up to 10%, but had no effect on ethanol levels. This study indicates that the feed zone effect on biomass yield in yeast fermentation, using complex substrates, will be negligible under fully aerobic conditions.     

Direct measurement of the yield stress of filamentous fermentation broths with the rotating vane technique

The existence of a yield stress in filamentous fermentation broths has important transport phenomena implications in the design and operation of bioreactors. In this study, the constant shear rate vane method was assessed for directly measuring the yield stress of filamentous Aspergillus niger fermentation broths, as well as model fluids (ketchup, yogurt, and pulp suspensions). The method involved rotating 4-, 6-, and 8-bladed vanes (7.2 cm 相似文献   

Continuous butanol/isopropanol fermentation in down-flow column reactor coupled with pervaporation using supported liquid membrane

Continuous butanol/isopropanol fermentation with immobilized Clostridium isopropylicum was performed in a downflow column reactor using molasses as the substrate. In order to prevent product inhibition and at the same time obtain high concentration of the products, the column reactor was coupled with a pervaporation module using a supported liquid membrane. The liquid membrane was prepared with oleyl alcohol nontoxic to the microorganism. In comparison with the continuous fermentation without product removal, the specific butanol production rate was 2 times higher. The butanol concentration in the permeate was 230 kg/m(3), which was about 50 times higher than that in the culture broth. A numerical investigation suggested a further increase in the productivity by improving the module construction.     

Regulation of a potassium-selective current in rabbit corneal epithelium by cyclic GMP,carbachol and diltiazem

Summary The effects of cyclic GMP (cGMP), carbachol and diltiazem on a potassium-selective, delayed-rectifier current in freshly dissociated rabbit corneal epithelial cells were studied using a modified perforated-patch-clamp technique. The current was stimulated by both 500 m cGMP (2.3–4.5-fold, mean = 2.9) and 250 nm carbachol, a muscarinic agonist (1.12–7.04-fold, mean = 3.8), and the stimulated current was totally blocked by diltiazem (10 m). The effects of cGMP appeared to be, at least in part, different from those of carbachol as they required the presence of external calcium. Single-channel data suggest that cGMP and carbachol activate the potassium current by increasing the open probability of the channel via a second-messenger system and that the action of diltiazem is probably through a direct blocking effect on the open channel.We are grateful to Erika Wohlfiel for secretarial help, Helen Hendrickson for cell preparation, and Joan Rae for software development. The work was supported by NIH grants EY06005 and EY03282 and an unrestricted award from Research to Prevent Blindness.     

Modulation of inwardly rectifying Na+-K+ channels by serotonin and cyclic nucleotides in salivary gland cells of the leech,Haementeria

Summary The electrically excitable salivary cells of the giant Amazon leech, Haementeria, display a time-dependent inward rectification. Under voltage clamp, hyperpolarizing steps to membrane potentials negative to about –70 mV were associated with the activation of a slow inward current (I _h) which showed no inactivation with time. The time course of activation of I _hwas described by a single-exponential function and was strongly voltage dependent. The activation curve of_hranged from –72 to –118 mV, with half-activation occurring at –100 mV. Ion-substitution experiments indicated that I _his carried by both Na⁺ and K⁺ ions. 5-Hydroxytryptamine (5-HT) increased the amplitude of I _hand its rale of activation. It also produced a positive shift of the activation curve of the conductance underlying I _h G_hwithout altering the slope factor, thus indicating that the voltage dependence of I _hwas modulated by 5-HT. Cs⁺ blocked both I _hand the 5-HT-polentiated current in a voltage-independent manner, whereas Ba²⁺ had little effect. It is concluded that 5-HT increases I _hby modulating the inwardly rectifying Na⁺-K⁺ channels in the salivary cells. The effect of 5-HT may be mediated by an increase in adenylate cyclase activity since I _hwas increased by 8-bromocyclic AMP and by the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine. In contrast, I _hwas reduced by 8-bromo-cyclic GMPand by zaprinast (an inhibitor of cyclic GMP-scnsitive phosphodieslerase). Cyclic GMP itself also reduced I _h, and the effect was specific to the 3,5 form; 2,3-cyclic GMP was inactive. The results suggest that the inward-rectifier channel may be modulated in opposite directions by cyclic AMP and cyclic GMPThis work was supported by a grant from the Science and Engineering Research Council (no. GR/F/17087). We are grateful to the SmithKline (1982) Foundation for provision of a pulse generator     

Effect of alteration of the acetic acid synthesis pathway on the fermentation pattern of escherichia coli

The glucose metabolism of an Escherichia coli strain bearing mutations abolishing both acetyl phosphotransferase (PTA) and acetate kinase (ACK) activities was studied under aerobic and anaerobic conditions. These studies were conducted in a complex medium with the mutant carrying no plasmid, the mutant carrying the common cloning vector pUC19, and the mutant carrying a plasmid bearing the "pet" operon that encodes Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase activities. The mutant carrying no plasmid showed lower specific growth and glucose uptake rates relative to the parent wild-type strain (K-12), Lactic acid was produced at higher levels than the wild type, and considerable amounts of pyruvic acid were secreted as an unusual byproduct. Analysis of other fermentation products showed low but significant amounts of acetic acid, no accumulation of formic acid, and lower secretion of succinate and ethanol. The maintenance of the plasmid pUC19 in the mutant negatively affected metabolism. Expression of the pet operon overcame the metabolic stress caused by the plasmid, enhancing growth and glucose uptake rates to the values observed in the plasmidfree mutant. Also, expression of the pet operon allowed consumption of pyruvate accumulated during the first hours of fermentation.