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PubMed2002

Mesophilic acidification of gelatinaceous wastewater.

The influence of hydraulic retention time (HRT) and gelatin concentration on the acidification of gelatinaceous wastewater in an upflow anaerobic reactor was investigated at pH 5.5 and 37 degrees C. The degree of gelatin degradation increased with the HRT, from 84.1% at 4 h to 89.6% at 24 h, but decreased with the increase of the gelatin concentration in the influent from 65.2% at 2 g-CODl(-1) to 51.9% at 30 g-CODl(-1). The degradation of gelatin followed the Monod kinetics with a maximum rate of 1.10 g (g-VSS x d)(-1) and a half-rate constant of 0.23 gl(-1). The overall production rate of VFA and alcohols decreased with HRT, from 0.33 g (g-VSS x d)(-1) at 4 h to 0.15 g (g-VSS x d)(-1) at 24 h, but increased with gelatin concentration in the influent, from 0.10 g (g-VSS x d)(-1) at 4 g-CODl(-1) to 0.58 g (g-VSS x d)(-1) at 30 g-CODl(-1). The key acidification products were acetate, propionate and butyrate, plus i-butyrate, valerate, i-valerate, caproate and ethanol in smaller quantities. Formate, methanol, propanol and butanol were found only in certain runs. Only 4.5-7.8% of COD in wastewater was converted to hydrogen and methane. The sludge yield was estimated as 0.320+/-0.014 g-VSS (g-COD)(-1).

Alcohols↗
PubMed1975

17 beta-Hydroxysteroid dehydrogenase of the sheep ovary : purification, properties and substrate binding site.

Sheep ovarian 17 beta HSDH has been purified about 1000 fold to a specific activity of 0.5 IU/mg protein, using DEAE cellulose chromatography, affinity chromatography on estrone-amino caproate-Sepharose and a second DEAE cellulose chromatography. The molecular weight is 70,000 ; the pH optimum for activity is 9.2 and the energy of activation is 16.5 Kcal/mole. The kinetics of the oxidation of estradiol and many analogues have been studied at various concentrations and in the presence of different amounts of coenzyme. The data are in agreement with a compulsory order mechanism with the binding of NAD+ as the first substrate. Sheep ovarian 17 beta HSDH accepts subtituents in position C3, C11, C13 ; the substrate binding site is open in this region. On the contrary, the binding requirements are strict for the region of C10 since the presence of a C19 methyl group impairs binding and (or) oxidation of the steroid. Sheep ovarian and human placental 17 beta HSDH have close analogies : molecular weight, pH optimum, substrate binding site requirements. Their reaction mechanisms are different : random for the placental 17 beta HSDH, compulsory order for the ovarian 17 beta HSDH : this can be explained by the effect of the coenzyme upon the binding of the substrate : without effect on placental enzyme, the coenzyme fixation enhances the affinity of the ovarian 17 beta HSDH for any substrate.

Animals↗
PubMed2003

Synthesis and neuroprotective activity of bergenin derivatives with antioxidant activity.

Norbergenin, which is the O-demethyl derivative of bergenin, the main component of Mallotus japonicus, has been found to show moderate antioxidant activity (IC(50) 13 microM in DPPH radical scavenging; 32 microM in superoxide anion scavenging). Modification of sugar part on norbergenin by coupling with a variety of fatty acids was employed for increasing its antioxidant activity. Selective esterification of hydroxyl groups on the sugar part enhanced greatly antioxidant activity. The most potent one is norbergenin 11-caproate, which not only exhibits stronger antioxidant activity than that of catechin but also prevents neuronal death at 10 microM on the primary culture of rat cortical neurons in DMEM supplemented with N2.

Animals↗
PubMed1999

Construction of recombinant sake yeast containing a dominant FAS2 mutation without extraneous sequences by a two-step gene replacement protocol.

A novel two-step gene replacement protocol was developed to construct a recombinant industrial yeast free of bacterial and drug-resistant marker sequences. A yeast strain exhibiting cerulenin resistance conferred by a dominant mutation of FAS2 was previously shown to produce high levels of a flavor component of Japanese sake. A N- and C-terminally truncated portion of the mutant FAS2 gene was subcloned to an integrating plasmid containing an aureobasidin A-resistant transformation marker and a galactose-inducible growth inhibitory sequence (GAL10p::GIN11). The plasmid was targeted into the chromosomal FAS2 locus of sake yeast Kyokai no. 7, resulting in a tandem repeat of inactive FAS2 sequences surrounding the integrated plasmid sequences. Cells containing the integrated plasmid were unable to grow on galactose medium due to the inhibitory effect of GAL10p::GIN11. This growth inhibition allowed efficient counter-selection for cells that had undergone homologous recombination between the FAS2 repeats by their growth on galactose medium. This recombination event resulted in loss of the integrated plasmid sequences and the resulting strains should contain a single copy of either wild-type or cerulenin-resistant FAS2. The selected cerulenin-resistant strains produced approximately 3.7-fold more ethyl caproate, a flavor component, than the Kyokai no. 7 strain. Southern blot and sequence analyses confirmed the presence of the FAS2 mutation and the absence of integrated plasmid sequences in the genome of the selected strain. This gene replacement method provides a straightforward approach for the construction of recombinant industrial yeasts free of undesirable DNA sequences.

Journal Article↗
PubMedDate not supplied

Preparation and characterization of anti-tenascin monoclonal antibody-streptavidin conjugates for pretargeting applications.

Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclide and most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with the extracellular matrix protein tenascin, in surgically created glioma resection cavity patients. To be able to investigate pretargeting in this setting, the synthesis of 81C6 mAb-SA conjugates was required. In the current study, we have evaluated five methods for preparing both murine 81C6 (m81C6) and human/mouse chimeric 81C6 (c81C6) SA conjugates with regard to yield, biotin-binding capacity, immunoreactivity, and molecular weight. The 81C6 mAb and SA were coupled by covalent interaction between sulfhydryl groups generated on the mAb via N-succinimidyl-S-acetylthioacetate, dithiothreitol or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-succinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotin caproate, and SA also was studied. The evaluation criteria were yield of mAb-SA 215 kDa monomer, as well as conjugate biotin-binding capacity and immunoreactive fraction. The optimal procedure involved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCC and yielded a conjugate with excellent biotin-binding capacity and immunoreactivity. The ((125)I-labeled m81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity after a 72 h incubation in human glioma cyst fluid in vitro. Although the conjugate was stable in murine serum in vivo, its biotin-binding capacity declined rapidly, consistent with high endogenous biotin levels in the mouse. After injection of the radioiodinated conjugate into athymic mice with subcutaneous D-54 MG human glioma xenografts, high tumor uptake (36.0 +/- 10.7% ID/g at 3 days) and excellent tumor:normal tissue ratios were observed.

Animals↗
PubMed1992

Isolation and characterization of the three polypeptide components of 4-chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS-3.

The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudomonas sp. CBS-3 DNA fragment and separately cloned and expressed in Escherichia coli. The three enzymes were purified from the respective subclones by using an ammonium sulfate precipitation step followed by one or two column chromatographic steps. The 4-chlorobenzoate:coenzyme A ligase was found to be a homodimer (57-kDa subunit size), to require Mg2+ (Co2+ and Mn2+ are also activators) for activity, and to turn over MgATP (Km = 100 microM), coenzyme A (Km = 80 microM), and 4-chlorobenzoate (Km = 9 microM) at a rate of 30 s-1 at pH 7.5 and 25 degrees C. Benzoate, 4-bromobenzoate, 4-iodobenzoate, and 4-methylbenzoate were shown to be alternate substrates while 4-hydroxybenzoate, 4-aminobenzoate, 2-aminobenzoate, 2,3-dihydroxybenzoate, 4-coumarate, palmate, laurate, caproate, butyrate, and phenylacetate were not substrate active. The 4-chlorobenzoate-coenzyme A dehalogenase was found to be a homotetramer (30 kDa subunit size) to have a Km = 15 microM and kcat = 0.3 s-1 at pH 7.5 and 25 degrees C and to be catalytically inactive toward hydration of crotonyl-CoA, alpha-methylcrotonyl-CoA, and beta-methylcrotonyl-CoA. The 4-hydroxybenzoate-coenzyme A thioesterase was shown to be a homotetramer (16 kDa subunit size), to have a Km = 5 microM and kcat = 7 s-1 at pH 7.5 and 25 degrees C, and to also catalyze the hydrolyses of benzoyl-coenzyme A and 4-chlorobenzoate-coenzyme A. Acetyl-coenzyme A, hexanoyl-coenzyme A, and palmitoyl-coenzyme A were not hydrolyzed by the thioesterase.

Bacterial Proteins↗
PubMed1985

Identification of the site of cross-linking in 16S rRNA of an aromatic azide photoaffinity probe attached to the 5'-anticodon base of A site bound tRNA.

The site of Escherichia coli 16S ribosomal RNA cross-linked to the 5'-anticodon base of A site bound E. coli valyl-tRNA was identified. Cross-linking was via the affinity probe 6-[(2-nitro-4-azidophenyl)amino]caproate (NAK) or 3-[[2-[(2-nitro-4-azidophenyl)amino]ethyl]dithio]propionate (SNAP) attached to the carboxyl group of the 5'-anticodon base 5-(carboxyethoxy)uridine via an ethylenediamine spacer [Gornicki, P., Ciesiolka, J., & Ofengand, J. (1985) Biochemistry (preceding paper in this issue)]. With both probes, RNase T1 digestion of the isolated 16S RNA-tRNA covalent complex, 5'-32P postlabeling, and gel electrophoresis yielded two oligonucleotides larger than any fragments from non-cross-linked tRNA or rRNA. Appearance of the oligomers was dependent on the presence of the probe on the tRNA. Unmodified tRNA in the A and/or P sites did not yield any product. The presence of elongation factor Tu in the incubation mixture was also required. Dithiothreitol (DDT) treatment of the SNAP-induced covalent complex prior to electrophoresis also abolished the oligomers. Only the larger of the two oligomers (present in a 3:1 ratio) was sequenced. The SNAP dimer was cleaved with DTT, and the rRNA and tRNA oligomers were separated and sequenced as monomers. The NAK dimer was sequenced without cleavage by taking advantage of the differences in electrophoretic mobility among sequence and/or composition isomers of the same length. In both cases, the rRNA oligomer was identified as UACACACCG1401, and the nucleotide cross-linked was shown to be the C1400 residue. The expected tRNA modification site was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1976

Effects of the aliphatic carboxylate series of salts on the conformation of proteins.

The effects of the aliphatic acid series of salts, formate, acetate, propionate, butyrate, valarate, and caproate, on the conformation of sperm whale myoglobin, human hemoglobin A, and horse heart cytochrome c were investigated by spectral measurements in the Soret region, optical rotation, and intrinsic viscosity measurements. The effectiveness of the aliphatic acid salts as unfolding reagents for proteins is found to increase with increasing hydrocarbon content of the alkyl chains of the salts, which is analogous in behavior to effects of the urea, amide, and alcohol series of protein denaturants. The denaturation midpoints, Sm, as a function of the unfolding reagent were analyzed using the equations of Peller (Peller, L. (1959), J. Phys, Chem- 63, 1199) and Flory (Flory, P.J. (1957), J. Cell. Comp. Physiol. 49, 175) with binding constants based in part on the Scherage-Nemethy theory of hydrophobic bonding or evaluated from free-energy transfer data of nonpolar amino acid side chains from aqueous to nonaqueous solvents. The summation of the polar KP and hydrophobic KHphi contributions of solvent to protein amino acid side chain interactions were found to give best account of the protein denaturation data. Intrinsic viscosity and optical rotation data obtained on hemoglobin and myoglobin at high salt concentrations, above the unfolding transition regions, indicate that the product of denaturation by the aliphatic acid salts is less unfolded than in 6 M guanidine hydrochloride solutions. Residual elements of the helical regions of the proteins seem to either escape unfolding or are reformed at high concentrations of the denaturing salts.

Binding Sites↗
PubMed2006

Functionalized surface arrays for spatial targeting of immune cell signaling.

The effect of surface topography and chemistry on cellular response is of fundamental importance, especially where living systems encounter device surfaces as in medical implants, tissue engineering, and cell-based sensors. To understand these biological processes on surfaces, there is a widespread interest in tailored surface-active materials produced by a combination of surface chemistry coupled to advanced patterning processes. We utilize self-assembled monolayers (SAMs) as molecular templates with submicrometer-scale spatial resolution to engage and cluster IgE receptors on rat basophilic leukemia (RBL) mast cells. Bioactive templates consisted of gold arrays on silicon with patterns from 1 mum down to 45 nm. These gold arrays served as molecular tethering sites, enabling covalent binding of functionalized self-assembled monolayers of alkanethiols. The free ends of the monolayers were functionalized with 2,4-dinitrophenyl(DNP)-caproate-based ligands which interact specifically with anti-DNP IgE bound to its high affinity cell surface receptor, FcepsilonRI on RBL mast cells. Present results on structures 1 mum down to 600 nm in size indicate that these ligand-immobilized patterned arrays can function as a powerful tool for visualization and systematic characterization of cell membrane involvement in IgE receptor-mediated immune cell signaling.

Microscopy, Confocal↗
PubMed2002

Fermentation of cottonseed and other feedstuffs in cattle rumen fluid.

Bovine rumen fluid was fermented anaerobically over 48 h with cottonseed, corn, alfalfa, or a mixture of these substrates in anaerobic mineral buffer. Samples taken at different incubation times were derivatized with n-butanol and subjected to gas chromatography and mass spectroscopy. No unusual fermentation end-products from the cottonseed substrate were detected. Cottonseed supported rumen fermentation at levels comparable to those of the other substrates. Major components were usually found in the decreasing order of acetate, propionate, butyrate, and valerate, although acetate and propionate concentrations decreased late in the alfalfa and mixed-feed fermentations, eventually allowing butyrate concentrations to exceed those of propionate. As expected, lactate was produced in high concentrations when corn was fermented. The minor components 2-methylpropionate, 2- and 3-methylbutyrate, phenylacetate, phenylpropionate, and caproate also accumulated, with their relative concentrations varying with the substrate. Succinate was produced in substantial amounts only when corn and alfalfa were fermented; it did not accumulate when cottonseed was the substrate. Samples containing cottonseed were derivatized and subjected to reversed-phase high-performance liquid chromatography, revealing that gossypol concentrations did not change during fermentation.

Acetates↗
PubMed1999

Effect of sorbed oil on food aroma loss through packaging materials.

Mass transport of six food aroma components, D-limonene, n-decane, ethyl caproate, phenylethanol, 1-hexanol, and hexanal, through three sealable polymer films suitable for direct food contact, ultra-low-density polyethylene (ULDPE), ionomer (ION), and modified polyester (mPET), was analyzed by permeation experiments. Transport was characterized by the permeability coefficient and its two contributing factors, the diffusion coefficient (related to kinetics) and the solubility coefficient (related to equilibrium). The results show that ULDPE is more permeable to aromas that ION, which is more so than mPET. Differences in diffusivity are mainly responsible for barrier improvement. With aromas, nonpolar compounds permeate faster than polar ones through ULDPE. The effect of sorbed oil on the behavior of these materials as food aroma barriers was investigated. The sorption of oil apparently resulted in polymer swelling, increasing the solubility of aromas into the polymer matrixes. Little or no effect was found on the values of the diffusion coefficient. The permeability coefficient was affected as a consequence of changes in solubility. When aromas were compared, the transport of nonpolar penetrants showed a considerable increase while permeability values for the polar ones were either unaffected or even reduced. This behavior has been discussed in terms of polymer/oil/aroma compatibility.

Food Packaging↗
PubMed2004

Quantification of intra- and extra-cellular thermophilic lipase/esterase production by Thermus sp.

Four Thermus strains produced lipolytic activity when grown in liquid medium for 30 h at 70 degrees C. The highest total lipase/esterase activity (57 U l(-1)) was in Thermus aquaticus YT-1, followed by Thermus thermophilus HB27 and HB8 (33 and 25 U l(-1), respectively), and finally by Thermus sp. (16 U l(-1)). Extra-cellular activity was detected in T. aquaticus YT-1 and T. thermophilus HB27 (33 and 17 U l(-1)). All enzymes were stable at 80 degrees C over 30 min, and their activity towards fatty acid esters increased as substrate chain-length diminished (i.e. hydrolysis rate was up to 6-fold higher on p-nitrophenyl caproate than on laurate).

Biotechnology↗
PubMed2004

Regulation of microglial inflammatory response by sodium butyrate and short-chain fatty acids.

1. Recent studies have shown that sodium butyrate and other short-chain fatty acids (SCFAs) can prevent inflammation in colon diseases. Our aim was to elucidate whether sodium butyrate and SCFAs regulate the inflammatory responses in different neural inflammation models in cell cultures. 2. Inflammatory responses to LPS-induced microglial activation were recorded by the secretion of nitric oxide (NO) and cytokines IL-6 and TNF-alpha and related to the changes in the DNA-binding activities of NF-kappaB complex. 3. We observed that sodium butyrate is strongly anti-inflammatory against LPS-induced responses in rat primary microglia as well as in hippocampal slice cultures and in neural cocultures of microglial cells, astrocytes and cerebellar granule neurons. 4. In murine N9 microglial cell line, instead, sodium butyrate and other SCFAs (propionate, valerate and caproate) enhanced the LPS-induced inflammatory response. 5. The pretreatment with butyrate before LPS exposure induced an equal or more enhanced response than simultaneous exposure with butyrate and LPS. This indicates that butyrate induces an adaptative response against microglial activation. 6. We also observed that butyrate treatment both in transformed N9 cells and in hippocampal slice cultures downregulates the NF-kappaB-binding capacity induced by LPS stimulation. 7. Our results show that butyrate is anti-inflammatory in primary, brain-derived microglial cells, as observed recently in colon diseases, but proinflammatory in transformed, proliferating N9 microglial cells, which may be related to the anticancer properties of butyrate observed in tumor cells.

Animals↗
PubMed2006

Avidin and streptavidin ligands based on the glycoluril bicyclic system.

Glycoluril derivatives with a carboxylic acid side chain have been synthesized and shown to bind to both avidin and streptavidin. Introduction of a valerate side chain in glycoluril led to an increased binding to both proteins only when the valerate group was bound to a N atom and with the proper stereochemistry [(+)-enantiomer]. On the other hand, introduction of the valerate side chain either on the bridgehead carbon or on the N atom with the opposite stereochemistry [(-)-enantiomer] led to a decrease in binding constant compared with unsubstituted glycoluril. Direct spectrophotometric competitive titration of each protein with a racemic ligand allowed measurement of the enantioselectivity of the ligand-protein complexation, together with the binding constant of the two enantiomers. In the case of the N-substituted glycoluril, the extension of the side chain by one methylene group, from valerate to caproate, led to an increase in the binding constant to both proteins. Docking studies using AutoDock 3.05 have been performed in order to predict the binding modes of these ligands to streptavidin. The effect of the stereochemistry and the position of the side chain on the binding constant to streptavidin is discussed in view of the predicted binding modes.

Alkynes↗
PubMed2000

Biochemical properties of Streptococcus macedonicus strains isolated from Greek Kasseri cheese.

A total of 32 Streptococcus macedonicus strains, isolated from Greek Kasseri cheese, were screened for biochemical properties of technological importance in milk fermentation processing, such as acid production, proteolytic and lipolytic activity, citrate metabolism, exopolysaccharide production, antimicrobial activity and biogenic amines production. All strains were found to be moderate acidifiers in milk. Only four strains could hydrolyse milk casein, while 11 strains showed lipolytic activity against tributyrin. Using amino acid derivatives of 4-nitroaniline as substrates, the highest peptidase activities were determined against phenylalanine- and glycine-proline-4-nitroanilide. Using fatty acid derivatives of 4-nitrophenol, it was shown that all strains exhibited esterase activities up to caprylate, with highest values against butyrate and caproate. Only one showed activity up to palmitate; this was also the most active strain against tributyrin. Five of the 32 strains could metabolize citrate but none of them produced exopolysaccharides. Nine strains displayed antimicrobial activity towards Clostridium tyrobutyricum, while no antimicrobial activity was detected against Listeria innocua and Propionibacterium freudenreichii subsp. shermanii. Finally, none was able to decarboxylize ornithine, histidine or lysine, and only four strains produced tyramine from tyrosine.

Animals↗
PubMed2002

The occurrence of malolactic fermentation in brandy base wine and its influence on brandy quality.

AIMS: In this study we determined the extent to which lactic acid bacteria (LAB) occurred in brandy base wines, their ability to catalyse the malolactic fermentation (MLF) and the effect of MLF on the quality of the base wine and the brandy distillate. METHODS AND RESULTS: Lactic acid bacteria were isolated and enumerated from grape juice, experimental and commercially produced brandy base wines. Spontaneous MLF occurred in approximately 50% of the commercial base wines. The occurrence of MLF had an influence on the quality of the base wines and the resulting distillates. In samples where MLF occurred there was a loss of fruitiness and in the intensity of aroma. Volatile compounds like iso-amyl acetate, ethyl acetate, ethyl caproate, 2-phenethyl acetate and hexyl acetate decreased in samples having undergone MLF, while ethyl lactate, acetic acid and diethyl succinate increased in the same samples. CONCLUSIONS: Spontaneous malolactic fermentation does occur in commercial brandy base wines and it has an influence on base wine and brandy quality. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that MLF influences the quality of the base wine and the resulting distillate and with this in mind commercial base wine producers should be able to produce brandy of higher quality.

Fermentation↗
PubMed1996

The effect of Saccharomyces cerevisiae and Aspergillus oryzae on fermentations in the rumen of faunated and defaunated sheep; protozoal and probiotic interactions.

We measured the effect of the direct addition to the rumen of Saccharomyces cerevisiae (SC 50 mg/day) and Aspergillus oryzae (AO 3 g/day) on the fermentation processes in fistulated sheep. The measurements were carried out on animals whose rumens were first defaunated and then refaunated. The animals received a ration composed of hay (600 g/day), barley (600 g/day) and soybean meal (150 g/day), fed twice daily in two equal meals. The number of fungi and total, viable or cellulolytic bacteria were lower after the inoculation of protozoa in defaunated rumens. The probiotics stimulated the development of total bacteria but reduced the population of cellulolytic bacteria. The addition of the probiotics and the presence of protozoa each incurred a decrease in the redox potential values. The association of both treatments had an additive effect on this parameter. The two probiotics and the protozoa stabilized the rumen pH after the meal, maintaining it above the value of 6 for a longer period of time. The positive effects on pH were accumulated in the refaunated animals receiving probiotics. The ammonia nitrogen concentration was considerably increased by the presence of the protozoa; the probiotics increased the ammonia concentration only in the refaunated sheep. The methane and hydrogen proportions in the fermentation gases were invariably higher in the refaunated animals. The probiotics had no clear effect either on the gas composition or the concentration and the composition of the mixture of volatile fatty acids; only the concentration of isovalerate was significantly increased by probiotics and only in refaunated animals. The protozoa did, however, considerably increase the concentrations of acetate, butyrate and isoacids and decreased the concentration of caproate.

Ammonia↗
PubMed2004

[Prevention of preterm delivery with gestagens].

Preterm delivery with its short-term and long-term sequealae constitutes a serious problem in terms of morbidity, disability and mortality of the newborn and cost to the society. The incidence of premature deliveries persists and according to the latest national and international reports a tendency for an increase has been observed. Therefore, besides screening programmes for the detection of vaginal infections one has to look for additional therapeutic concepts. According to previous data and recent publications, progesterone vaginally and 17alpha-hydroxyprogesterone caproate intramuscularely should be considered as possible treatment options for the prevention of preterm delivery in high-risk women, with therapy starting in the second trimester.

17-alpha-Hydroxypregnenolone↗