A model for glucose metabolism in energy-sufficient bacterial cultures

Summary A model for uncoupled glucose uptake under energy-sufficient conditions is presented. The model is derived from glucose catabolic pathways. The resulting model predicts specific glucose uptake rate as a function of both growth rate and extracellular glucose concentration. This prediction is consistent with reported literature data.     

Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.     

Effect of bacterial growth-inhibiting ingredients on the Ames mutagenicity of medicinal herbs

A solvent fractionation method was introduced to screen for mutagenicity in 10 medicinal herbs being consumed in Korea. The Ames mutagenicity test result of Scutellariae and Rhei was significantly increased by eliminating growth-inhibiting substances through solvent fractionation of the crude extract. It is suggested that a physicochemical pretreatment should reduce the false-negative results which are caused by the presence of growth-inhibiting substances in complex mixtures.     

Dietary induction of hepatic glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme in lean and obese female Zucker rats

Responses of the hepatic lipogenic enzymes, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and malic enzyme (ME) to starvation refeeding and diet shifting were determined in lean and obese female Zucker rats. Rats were either fed nonpurified diet, starved 48 hr, and then refed nonpurified diet or one of the refined carbohydrate diets containing either glucose, fructose, cornstarch, or sucrose for 72 hr, or shifted from nonpurified diet directly to one of the refined carbohydrate diets for 72 hr. Initial activities were greater in obese than lean rats for all three enzymes studied. Similar to other strains of female rats, lean Zucker rats failed to demonstrate a starve-refeed response when refed nonpurified diet. Obese female littermates showed a statistically significant increase in enzymes when refed a nonpurified diet. Both lean and obese female Zucker rats demonstrated increases in enzyme activities above controls when starved and refed any of the refined carbohydrate diets. The greatest responses were observed when female rats were starved and refed sucrose; activities increased 2.6- to 3.5-fold in lean and 3.0- to 4.3-fold in obese Zuckers. In lean females 50-70% of the starve-refeed response observed with G6PDH and ME can be accounted for by simply shifting from a nonpurified diet to the respective refined carbohydrate diet, whereas in obese females only 33-55% of the increase could be attributed to diet shifting. Plasma testosterone/estrogen ratios were consistently 1.5 times higher in obese than in lean female rats. This phenotypic difference may potentiate the heightened starve-refeed overshoot response observed in obese rats.     

Polyphosphate Formation from Pyrophosphate in Intact Cells of a Photosynthetic Bacterium, Rhodospirillum rubrum

The hypothesis that the photosynthetic bacterium, Rhodospirillumrubrum, may synthesize polyphosphate using pyrophosphate asa precursor has been tested. The cells were cultured in thepyrophosphate media, and the cell yield, cell shape, and thepolyphosphate content were determined and compared with thatgrown in the orthophosphate media. In the control experiment, the total amount of polyphosphateformed by the cultures grown in the pyrophosphate media wasfound to be up to 70% less than that grown in orthophosphatemedia. When the ATP formation was blocked by using the phosphorylationinhibitor, antimycin A, however, the cells grown in the pyrophosphatemedia formed up to 50% more polyphosphate than that grown inthe orthophosphate media. The results seem to support the hypothesis that Rhodospirillumrubrum may use pyrophosphate as the precursor of polyphosphate. (Received May 26, 1986; Accepted February 21, 1987)     

Electrical injury mechanisms: electrical breakdown of cell membranes

Electric fields are capable of damaging cells through both thermal and nonthermal mechanisms. While joule heating is generally recognized to mediate tissue injury in electrical trauma, the possible role of electrical breakdown of cell membranes has not been thoroughly considered. Evidence is presented suggestive that in many instances of electrical trauma the local electrical field is of sufficient magnitude to cause electrical breakdown of cell membranes and cell lysis. In theory, large cells such as muscle and nerve cells are more vulnerable to electrical breakdown. To illustrate the significance of cell size and orientation, a geometrically simple model of an elongated cell is analyzed.     

Site-directed mutagenesis to determine essential residues of ribulose-bisphosphate carboxylase ofRhodospirillum rubrum

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.     

Demonstration of structural differences between the two subunits of human-plasma fibronectin in the carboxy-terminal heparin-binding domain

Structural differences between the two subunits of human plasma fibronectin were studied by analyzing the carboxy-terminal heparin-binding domain (Hep-2). Two fragments (29 kDa and 38 kDa) derived from the Hep-2 domain were purified from thermolysin-digested human plasma fibronectin. Identical NH2-terminal sequences were obtained for both fragments through 16 Edman cycles. Neither domain contained the 90-amino-acid extra domain which is predicted by cDNA analysis of the cellular form of fibronectin. We have examined the primary structures of the 29-kDa and 38-kDa Hep-2 domains produced from the two chains of plasma fibronectin by analyzing the tryptic peptides by fast atom bombardment/mass spectrometry and comparison with the predicted fragments deduced from the corresponding cDNA-derived peptide sequences. Peptides that were unique to each domain were further characterized by microsequence analysis. The two domains showed identical amino acid sequences through 274 residues, followed by a region of variability. The 29-kDa domain contains 279 amino acids with an estimated relative molecular mass (Mr) of 30,460. This domain is located in the heavy chain of plasma fibronectin and contains three repeats of type III sequences plus a portion of the connecting segment (IIICS) region. The 38-kDa domain contains 359 amino acids and one O-linked glycosyl unit for an estimated Mr of 39,263. This domain is from the light chain of plasma fibronectin and contains four repeats of type III sequences with the deletion of the entire 120-amino-acid IIICS area. Secondary structure analysis by Chou/Fasman and circular dichroism reveals extensive beta-sheet structure for these domains. Key sulfhydryl and glycosylation sites are located near the mRNA splice junctions for the two chains. It is postulated that the splice junctions are adjacent to a flexible domain joining two regions of extensive beta-sheet structure.     

Reciprocal effects of epidermal growth factor and transforming growth factor beta on the anchorage-dependent and -independent growth of A431 epidermoid carcinoma cells

The effects of epidermal growth factor (EGF) and transforming growth factor beta (TGF beta) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGF beta. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGF beta on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGF beta. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture.     

Molecular cloning of Clostridium difficile toxin A gene fragment in lambda gt11

Toxin A of Clostridium difficile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. difficile chromosomal DNA was developed which allowed for the construction of a genomic library in lambda gt11. Approx. 35,000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, lambda cd19. Verification of the immunological identity of the isolated toxin A gene fragment in lambda cd19 was determined by affinity purifying toxin A antibodies specific for lambda cd19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in lambda cd19 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. difficile. The peptide coded for by the toxin A gene fragment in lambda cd19 was not cytotoxic for 3T3 mammalian tissue culture cells.