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PubMed2004

Optimization of Brewery's spent grain dilute-acid hydrolysis for the production of pentose-rich culture media.

Dilute-acid hydrolysis of brewery's spent grain to obtain a pentose-rich fermentable hydrolysate was investigated. The influence of operational conditions on polysaccharide hydrolysis was assessed by the combined severity parameter (CS) in the range of 1.39-3.06. When the CS increased, the pentose sugars concentration increased to a maximum at a CS of 1.94, whereas the maximum glucose concentration was obtained for a CS of 2.65. The concentrations of furfural, hydroxymethylfurfural (HMF), as well as formic and levulinic acids and total phenolic compounds increased with severity. Optimum hydrolysis conditions were found at a CS of 1.94 with >95% of feedstock pentose sugars recovered in the monomeric form, together with a low content of furfural, HMF, acetic and formic acids, and total phenolic compounds. This hydrolysate containing glucose, xylose, and arabinose (ratio 10:67:32) was further supplemented with inorganic salts and vitamins and readily fermented by the yeast Debaryomyces hansenii CCMI 941 without any previous detoxification stage. The yeast was able to consume all sugars, furfural, HMF, and acetic acid with high biomass yield, 0.68 C-mol/C-mol, and productivity, 0.92 g/(L.h). Detoxification with activated charcoal resulted in a similar biomass yield and a slight increase in the volumetric productivity (11%).

Alcoholic Beverages↗
PubMed1991

Aerobic growth and respiration of a delta-aminolevulinic acid synthase (hemA) mutant of Bradyrhizobium japonicum.

Oxygen-dependent growth of the Bradyrhizobium japonicum hemA mutant MLG1 (M.L. Guerinot and B.K. Chelm, Proc. Natl. Acad. Sci. USA 83:1837-1841, 1986) was demonstrated in cultured cells in the absence of exogenous delta-aminolevulinic acid (ALA), but growth of analogous mutants of Rhizobium meliloti or of Escherichia coli was not observed unless ALA was added to the yeast extract-containing media. No heme could be detected in extracts of strain MLG1 cells as measured by the absorption or by the peroxidase activity of the heme moiety, but the rates of growth and endogenous respiration of the mutant were essentially identical to those found in the parent strain. A role for ALA in the viability of strain MLG1 could not be ruled out since the ALA analog levulinic acid inhibited growth, but neither ALA synthase nor glutamate-dependent ALA synthesis activity was found in the mutant. The data show that the cytochromes normally discerned in wild-type B. japonicum cultured cells by absorption spectroscopy are not essential for aerobic growth or respiration.

5-Aminolevulinate Synthetase↗
PubMed2000

Gabaculine does not inhibit cytokinin-stimulated biosynthesis of chlorophyll in Pinus nigra seedlings in the dark.

Chlorophyll (Chl) accumulation was monitored during black pine (Pinus nigra L.) seed germination for 14 days in the light and in the dark in the presence of gabaculine (GAB) and cytokinin in order to elucidate the regulation of gymnosperm seedling greening in the dark, primarily at the level of aminolevulinic acid formation. In the light, GAB inhibited chlorophyll accumulation in a manner dependent on concentration and developmental stage, and in the dark it showed no effect. Cytokinin, 10(-5) M benzyl adenine (BA) partly overcame GAB-induced inhibition in the light, mainly during earlier developmental stages. In the seedlings grown in the dark, an equal quantity of Chl accumulated in the presence of cytokinin with and without GAB and it was approximately 20-40% higher than in the control seedlings or in the seedlings grown only in the presence of GAB. 5-Amino-levulinic acid (ALA) synthesis was equal in the light and in the dark in seedlings of the same age and seedlings treated with GAB grown in the dark. In the light, GAB inhibited ALA synthetic activity. The results indicate that ALA synthesis is not a rate-limiting step within Chl biosynthesis in pine seedlings grown in the dark.

Journal Article↗
PubMed1967

Control of chlorophyll production in rapidly greening bean leaves.

The possible involvement of nucleic acid and protein synthesis in light-regulated chlorophyll formation by rapidly greening leaves has been studied.Removing leaves from illumination during the phase of rapid greening results in a reduction in the rate of pigment synthesis; cessation occurs within 2 to 4 hours. Etiolated leaves which exhibit a lag in pigment synthesis when first placed in the light do not show another lag after a 4 hour interruption of illumination during the phase of rapid greening.Actinomycin D, chloramphenicol, and puromycin inhibit chlorophyll synthesis when applied before or during the phase of rapid greening. Application of delta-amino-levulinic acid partially relieves the inhibition by chloramphenicol.It is suggested that light regulates chlorophyll synthesis by controlling the availability of delta-aminolevulinic acid, possibly by mediating the formation of an enzyme of delta-aminolevulinate synthesis. This process may result from gene activation or derepression; the involvement of RNA synthesis of some sort is suggested by the inhibitory effect of actinomycin D on chlorophyll production by rapidly greening leaves.

Journal Article↗
PubMed1988

Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase.

The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid. A hemA- mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor. The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid. Porphobilinogen, the substrate, interacts with the free alpha-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes. Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex. The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate. Specific labeling of the dipyrromethane cofactor by growth of the E. coli in the presence of 5-amino[5-14C]levulinic acid has confirmed that the cofactor is not subject to catalytic turnover. Experiments with the alpha-substituted substrate analogue alpha-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site. On the basis of these cumulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH3 or H2O. The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed.

Aminolevulinic Acid↗
PubMed1976

Effect of cobalt on synthesis of heme and cytochrome P-450 in the liver. Studies of adult rat hepatocytes in primary monolayer culture and in vivo.

Synthesis of heme and cytochrome P-450 have been studied in adult rat hepatocytes in primary monolayer culture. Incubation of cells with delta-aminolevulinic acid increases both cellular heme and cytochrome P-450 relative to that found in cells incubated under control conditions. Formation of heme is proportional to the concentration of delta-aminolevulinic acid in the culture medium and is not saturable. By contrast, formation of cytochrome P-450 is saturable; excess intracellular heme appears as a new absorption band at 420 nm in the carbon monoxide-reduced difference spectrum. We have studied the effect of cobalt on heme and hemoprotein formation in this cell system. The metal blocks formation of cytochrome P-450 but fails to affect heme synthesis. In contrast to previous findings with isolated mitochondria, no cobalt protoporphyrin formed in hepatocytes cultured in the presence of the metal. In studies of rats in vivo, it was confirmed that cobalt acts to reduce the amount of [14C]heme in the liver after administration of delta-amino[14C]levulinic acid. However, the present findings suggest that this effect of cobalt represents accelerated breakdown of newly labeled hepatic heme rather than inhibition of synthesis. We conclude that cobalt interferes with formation of cytochrome P-450 not by direct inhibition of heme synthesis but most likely by blocking the association of heme and apocytochrome.

Aminolevulinic Acid↗
PubMed1994

DcrA, a c-type heme-containing methyl-accepting protein from Desulfovibrio vulgaris Hildenborough, senses the oxygen concentration or redox potential of the environment.

The amino acid sequence of DcrA from Desulfovibrio vulgaris Hildenborough, a strictly anaerobic, sulfate-reducing bacterium, indicated homology with the methyl-accepting chemotaxis proteins from enteric bacteria (A. Dolla, R. Fu, M. J. Brumlik, and G. Voordouw, J. Bacteriol. 174:1726-1733, 1992). The homology is restricted to the cytoplasmic C-terminal signaling domain. The periplasmic N-terminal sensor domain was found to contain a unique sequence, CHHCH, corresponding to a consensus c-type heme binding site. A pretreated, DcrA-specific polyclonal antiserum, generated against DcrA protein overproduced in Escherichia coli, was used for immunoprecipitation of 35S-labeled DcrA from D. vulgaris and Desulfovibrio desulfuricans G200(pJRFR2), a transconjugant that overexpresses functional DcrA. Labeling of the latter with the heme precursor 5-amino-[4-14C]levulinic acid, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography, confirmed the presence of c-type heme, while labeling with L-[methyl-3H]methionine in the absence of protein synthesis confirmed that DcrA is a methyl-accepting protein. The base liability of the incorporated radioactivity indicated methyl ester formation like that occurring in the methyl-accepting chemotaxis proteins of enteric bacteria. L-[methyl-3H]methionine labeling of D. desulfuricans G200(pJRFR2) under different conditions indicated that methyl labeling of DcrA decreased upon addition of oxygen and increased upon subsequent addition of the reducing agent dithionite. These results indicate that DcrA may serve as a sensor of oxygen concentration and/or redox potential.

Amino Acid Sequence↗
PubMed1997

Laser Doppler perfusion imaging: new technique for determination of perfusion and reperfusion of splanchnic organs and tumor tissue.

BACKGROUND AND OBJECTIVE: Several investigations indicated that laser Doppler flowmetry on the liver surface reflects relative changes of the total liver blood flow. In this study, Laser Doppler Perfusion Imaging (LDI), monitoring the surface only, was used for measurements of tissue perfusion of normal and/or impaired liver, pancreas, spleen, stomach and intestines, and the blood flow of hepatic tumors in rats. STUDY DESIGN/MATERIALS AND METHODS: Eighty Wistar/Furth rats were divided into five groups. Group I served as controls. Groups II and III underwent ischemic injury of the liver and intestine with or without the administration of WEB2170, a platelet-activating factor receptor antagonist. Laser-induced photodynamic therapy (PDT) utilizing delta-amino levulinic acid sensitization was performed in Groups IV and V. RESULTS: Normal pancreas and intestine had a high LDI perfusion value and the liver and stomach exhibited a medium perfusion value whereas the perfusion value from the spleen was low. WEB2170 improved the reperfusion of the postischemic liver and intestine. An immediate decrease in surface blood flow of hepatic tissue treated by laser-induced PDT and a decreased blood flow in large tumors were observed. CONCLUSION: LDI is a useful technique for the measurement of tissue perfusion of various splanchnic organs or tumor tissues.

Aminolevulinic Acid↗
PubMed1999

Comparative study on the ALA photodynamic effects of human glioma and meningioma cells.

BACKGROUND AND OBJECTIVE: The purpose of this study was to compare the differential susceptibility to photodynamic therapy (PDT) mediated damage in human U-105MG glioma cells and CH-157MN meningioma cells in vitro using 5-amino-levulinic acid (ALA) as photosensitizer, and to determine if growth factors would enhance PDT-mediated damage of these cells. STUDY DESIGN/MATERIALS AND METHODS: U-105MG or CH-157MN cells were irradiated with polychromatic light in the presence of ALA. A Xenon lamp (150 W) was used as the light source. For the study on the effect of growth factor on ALA-PDT, cells were cultured in serum free medium for 24 hours. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet derived growth factor BB (PDGF-BB) was added to achieve a final concentration of 50 ng/ml. 30 minutes later, cells were incubated with ALA (100 microg/ml) for 24 hours, washed, and irradiated with light (11 J/cm2). MTT tetrazolium assays were performed 24 hours after light irradiation. RESULTS: The inhibition of metabolic cellular function in U-105MG cells by ALA depended on both light energy density and ALA concentration. The susceptibility to ALA-PDT was profoundly lower for CH-157MN meningioma cells than U-105MG glioma cells. When incubated with ALA (100 microg/ml), U-105MG cells exhibited an LD50 around 8 J/cm2 of light irradiation, whereas that of CH-157MN cells was more than 25 J/cm2. EGF, bFGF, or PDGF-BB did not have any effects on the susceptibility of these two cell lines to ALA-PDT. CONCLUSION: ALA-PDT was more effective in killing U-105MG glioma cells than CH-157MN meningioma cells. The differential susceptibility was likely due to differential accumulation of PpIX in these cells. EGF, bFGF, or PDGF-BB did not have stimulatory or inhibitory effect on the efficiency of ALA-PDT.

Aminolevulinic Acid↗
PubMed1985

Identification of proteins involved in the regulation of yeast iso- 1-cytochrome C expression by oxygen.

On the basis of a gel electrophoresis retardation assay, protein(s) which interact specifically with the upstream activating site (UASc) of the yeast iso-1-cytochrome C (CYC1) gene were identified and separated by heparin ultrogel chromatography. DNase I protection experiments indicate that these factors protect a 23-bp sequence overlapping the UASc site previously defined. The specific binding activity is strongly reduced in extracts prepared from a wild-type strain grown anaerobically. It is absent in a mutant strain blocked in the biosynthesis of heme but it is restored upon the addition of the missing precursor, delta amino levulinic acid (dALA) to the growth medium. In contrast, the binding activity does not differ significantly in extracts form a wild-type strain grown in either glucose or glycerol as carbon source. These data strongly argue that the CYC1 UAS binding protein(s) that we have identified mediate the oxygen and heme control of cytochrome C biosynthesis.

Anaerobiosis↗
PubMed1990

Urinary N-acetyl-beta-D-glucosaminidase activity in lead-exposed workers.

The adverse effects of long-term exposure to lead dust on renal tubular functions were studied in 39 male and 7 female workers in a secondary lead refinery. Laboratory examinations showed low or moderate lead absorption levels. The N-acetyl-beta-D-glucosaminidase (NAG) activity in urine showed significant correlation with each of the lead exposure indices. NAG activity increased remarkably in lead workers with blood lead (PbB) above 80 micrograms 100g-1. Blood urea nitrogen (BUN) showed significant correlation with PbB and logarithmic urine delta-amino-levulinic acid (log(ALAU)), but not with urine lead (PbU) and logarithmic urine coproporphyrin (log(CPU)). All the other indices of renal function were almost within the normal range and showed no correlation with lead indices. This study suggests that moderate lead absorption may cause renal tubular impairment, and NAG can be one of the most sensitive indices for estimating renal dysfunction due to lead poisoning.

Acetylglucosaminidase↗
PubMed1982

Stability of phenylpropanolamine hydrochloride in liquid formulations containing sugars.

Stability studies with a decongestant syrup formulation containing phenylpropanolamine hydrochloride in a sugar vehicle indicated a loss of phenylpropanolamine hydrochloride. To ascertain the biological significance of this indicated chemical loss, the degraded formulation was administered to human volunteers and the urinary excretion of phenylpropanolamine hydrochloride was determined. The excretion and chemical assay patterns were in good agreement, indicating that the sugar vehicle was both chemically and biologically incompatible with phenlypropanolamine hydrochloride. A control formulation made with phenylpropanolamine hydrochloride in a sorbitol vehicle showed good chemical stability and urinary excretion patterns. Further studies showed chemical losses with phenylpropanolamine hydrochloride in the presence of fructose, dextrose, and 5-(hydroxymethyl)-2-furaldehyde but not with levulinic acid. Possible mechanisms for the sugar-phenylpropanolamine hydrochloride interaction are discussed.

Carbohydrates↗
PubMed2005

Photodynamic therapy on keloid fibroblasts in tissue-engineered keratinocyte-fibroblast co-culture.

BACKGROUND AND OBJECTIVES: Keloids are disfiguring, proliferative scars that are a pathologic response to cutaneous injury. An organotypic tissue culture system (the Raft model 1-10) was used to investigate the feasibility of using photodynamic therapy (PDT) as an adjunctive therapy to treat keloids following surgical excision. The Raft co-culture system mimics skin by layering keratinocytes on top of fibroblasts embedded in a collagen matrix. PDT uses drugs that produce singlet oxygen in situ when irradiated by light, and may lead to a number of effects in living tissues varying from the modulation of growth to apoptosis. PDT is already used to treat several benign and malignant diseases in organs such as the skin, retina, and esophagus. STUDY DESIGN/MATERIALS AND METHODS: Normal adult, neonatal, and keloid fibroblasts and keratinocytes were isolated from skin obtained from patients undergoing elective procedures and used to construct the Rafts. Mature Rafts (after 4 days) were incubated with 5-amino levulinic acid (5-ALA), a photosensitizer, for 3 hours and were laser-irradiated (635 nm) for total energy delivery of 5 J/cm2, 10 J/cm2, or 20 J/cm2. Rafts were examined 24 hours and 14 days later. Cell viability was determined using confocal imaging combined with live-dead fluorescent dyes. Multi-photon microscope (MPM) imaged collagen structure and density. As Rafts contract over time, surface area was measured using optical micrometry daily. RESULTS: At 10 and 20 J/cm2, near-total cell death was observed in all constructs, while at 5 J/cm2 cell viability was comparable to controls. Cell viability in keloid and neonatal Rafts was greater than that observed in normal adult Rafts. Treated Rafts contracted less over the 14-day period compared to controls. Contraction and collagen density were greatest in keloid and neonatal Rafts. CONCLUSIONS: A PDT dosimetry range was established, which reduces tissue contraction and collagen density while minimizing injury to fibroblasts.

Adult↗
PubMed1999

Enantiomeric determination of D- and L-lactate in rat serum using high-performance liquid chromatography with a cellulose-type chiral stationary phase and fluorescence detection.

A method is described for the quantitative determination of d- and L-lactate in 10 microl of rat serum, which includes fluorescence derivatization of D- and L-lactate with 4-(N, N-dimethylaminosulfonyl)-7-piperazino-2,1,3- benzoxadiazole (DBD-PZ) followed by O-acetylation. The derivatives are separated by HPLC on an octadecylsilica, and, via column switching, on a cellulose-type chiral column. Levulinic acid was used as the internal standard. The enantiomers of lactate were separated with the separation factor (alpha) of 1.27 and the resolution (Rs) of 2.72, while the linearity for the detection was over the range of 10 nmol/ml to 20 micromol/ml (r = 0.999). Interday precision values for D-lactate in rat serum were 5.8, 5.3, and 4.1% for 10, 100, and 1000 nmol/ml, and accuracy values were 109.6, 98.2, and 103.1%, respectively (n = 5). The reduction of d-lactate concentration in rat serum by fasting was observed with the method.

Acetylation↗
PubMed1978

Increased ALA dehydratase activity and spleen weight in lead-intoxicated rats. A consequence of increased blood cell destruction.

Lead was given in the diet (1%) to rats from birth and at different times the animals were studies for delta amino levulinic acid dehydratase (ALAD) activity, spleen weight, 59Fe incorporation in erythrocytes and 51Cr-labeled erythrocytes survival. The increased ALAD and spleen weight found after lead treatment is explained as a consequence of a shortened survival, which results in a younger age of circulating erythrocytes with higher ALAD activity.

Animals↗
PubMed1980

Evaluation of the effect of lead exposure on the liver in Egyptian lead tank welders.

In a group of Egyptian lead tank welders who were exposed to lead fumes for periods to 22 years the changes in serum lipids and some of the liver function tests which may elucidate the effect of lead on the liver were investigated. The results revealed increased blood lead level associated with decreased blood haemoglobin and increased urinary excretion of delta amino levulinic acid. However, no clinical abnormalities were recorded in the exposed group of the present work. Thus the increase in serum triglycerides and B-lipoprotein together with the lowering of the phospholipid/cholesterol ratio which were found may indicate premature development of atherosclerosis. Indirect evidence of the beginning of liver fattening was also provided by the increase in serum GOT, GPT, LDH enzymes and decreased albumin/globulin ratio besides the changes in serum lipid values. It is concluded therefore that lead poisoning may have a vascular as well as hepato-toxic action.

Adult↗