bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modification of the Enzyme Enantioselectivity by Product Inhibition
Authors: Rakels J. L. L. , Caillat P. , Straathof A. J. J. , Heijnen J. J. 
Published: March 2005

AbstractA kinetic explanation of solvent effects on the enantioselective hydrolysis of racemic methyl and ethyl 2‐chloropropionate by carboxylesterase NP has been studied. After a general ping‐pong model for the kinetic resolution of chiral esters was defined, the progress curves of the reactions were analyzed, and a simplified uni‐uni four‐parameter model was selected. First‐order rate constants and alcohol inhibition constants were determined for both enantiomers. Enantiomeric excess values were correctly predicted from the model. According to the kinetic description, the enantioselectivity could be enhanced or decreased by adding the product alcohol as a cosolvent. It appeared that these changes could be predicted quantitatively by enantioselective product inhibition for alcohol concentrations <10%. At higher alcohol concentrations additional changes in enantioselectivity occurred, which may be due to well‐known changes in enzyme conformation, alteration of the solvent properties, or enzyme‐solvent interactions. Similar results were found for hydrolyses catalyzed by α‐chymotrypsin and Candida cylindracea lipase.
Turbidimetric Titration Study of the Interaction of Proteins with Acrylic Polyampholytes
Authors: Nath Sunil , Patrickios Costas S. , Hatton T. Alan 
Published: March 2005

AbstractA study of the interaction of proteins [soybean trypsin inhibitor (0.3–1 mg/mL)] with dilute solutions of synthetic polyampholytes (0.05–0.4 mg/mL) composed of acrylic (or methacrylic) acid, (dimethylamino)ethyl methacrylate, and methyl methacrylate was undertaken experimentally using turbidimetric titration. The polyampholytes had a number‐average molecular weight of 4000 (block polyampholytes) or 70000 (random polyampholytes) and a polydispersity index of only 1.3. Alone, the polyampholytes showed a tendency to self‐aggregate around their isoelectric point. Therefore, polyampholyte‐polyampholyte interactions (self‐aggregation) and polyampholyte‐protein complexation behavior were studied as a function of pH (3–9), ionic strength (0–0.75 M) and polymer dosage (50–400 mg of polymer/g of protein). Large increases in turbidity (>500%) were observed for protein‐polyampholyte mixtures (compared to polyampholyte alone) in the case of both block and random copolymers. The observed turbidity increases could be due to protein‐polymer interactions or to enhanced precipitation of polymer in the presence of protein. To differentiate between these alternatives, turbidimetry experiments were carried out at different dosages by (a) keeping the protein concentration constant and varying the polymer concentration and (b) keeping the polymer concentration constant and varying the protein concentration in small steps; they demonstrated that the turbidity increases were indeed due to protein‐polymer interactions. These experiments offer a general methodology to distinguish between intermolecular protein‐polymer and intermolecular polymer‐polymer interactions.
Constitutive Overexpression of Secreted Heterologous Proteins Decreases Extractable BiP and Protein Disulfide Isomerase Levels in Saccharomyces cerevisiae
Authors: Robinson Anne Skaja , Wittrup K. Dane 
Published: March 2005

AbstractHigh‐level gene expression does not always lead to corresponding high‐level secretion of heterologous proteins in yeast. The rate‐limiting step in many cases has been shown to exit from the endoplasmic reticulum (ER). Within the ER, the correct folding of secreted proteins is required for export competence; hence, the cellular proteins involved in these events are likely to be important for efficient secretion. We have found that the extractable levels of two ER‐resident proteins involved in folding—heavy chain binding protein (BiP) and protein disulfide isomerase (PDI)‐‐are significantly reduced by prolonged constitutive overexpression of human granulocyte colony stimulating factor (GCSF), human erythropoietin, or Schizosaccharomyces pombe acid phosphatase. However, the rate of BiP synthesis measured in pulse‐chase radiolabeling experiments is not reduced by GCSF overexpression, and galactose‐directed transcription of the BiP gene does not restore normal BiP protein levels once they have been depleted. The observed loss of lumenal resident proteins, either by proteolysis or irreversible aggregation, is expected to contribute significantly to the inefficiency of foreign protein secretion in yeast.
Potential for Bioremediation of Xenobiotic Compounds by the White‐Rot Fungus Phanerochaete chrysosporium
Authors: Paszczynski Andrzej , Crawford Ronald L. 
Published: March 2005

AbstractThe white‐rot fungi produce an unusual enzyme system, characterized by a specialized group of peroxidases, that catalyzes the degradation of the complex plant polymer lignin. This ligninolytic system shows a high degree of nonspecificity and oxidizes a very large variety of compounds in addition to lignin. Among these compounds are numerous environmental pollutants. Thus, the white‐rot fungi show considerable promise as bioremediation agents for use in the restoration of environments contaminated by xenobiotic molecules. One white‐rot fungus, Phanerochaete chrysosporium, has been studied in great detail with regard to ligninolytic enzymes and the degradation of anthropogenic chemicals. It has been widely promoted as a bioremediation agent. This article examines literature concerning the degradation of xenobiotic compounds by Phanerochaete chrysosporium and attempts to critically assess this organism's real potential as a bioremediation tool.
Suppressed Acid Formation By Cofeeding of Glucose and Citrate in Bacillus Cultures: Emergence of Pyruvate Kinase as a Potential Metabolic Engineering Site
Authors: Goel Akshay , Lee Jinwon , Domach Michael M. , Ataai Mohammad M. 
Published: March 2005

AbstractMicrobial cultures typically produce acids when metabolizing the common carbon source, glucose. Acid production not only represents a waste of carbon, but its accumulation can limit cell concentration and culture stability, thereby reducing productivity. On the basis of prior work, acid production was attributed to be due to a mismatch between glycolytic and tricarboxylic acid (TCA) cycle capacities. To suppress acid production, a strategy entailing adding citrate to glucose minimal medium proved extremely effective. The effect of citrate on in‐vivo flux distribution was quantified using a detailed flux‐model. When the molar glucose—citrate ratio was varied between 3 and 6, a significant reduction in glycolytic flux and essentially complete suppression of acid formation was found as compared to chemostat cultures grown solely on glucose. Adding other biosynthetic precursors such as glutamine did not invoke the same suppression, thus indicating that citrate's effect is at the regulatory level. We hypothesized that the reduction of glycolytic flux in the presence of citrate results from its transport being coupled with the uptake of divalent metal ions. Citrate transport alters the intracellular balance of metal ions which in turn could trigger a sophisticated series of metabolic events leading to reduction of the activities of the pyruvate kinase and phosphofructokinase (PFK), the regulatory enzymes of glycolysis. On the basis of this scenario and other regulatory information, pyruvate kinase has emerged as a potential metabolic engineering site. It's deactivation in Bacillus subtilis or Escherichia coli strains is expected to yield constructs with a much lower tendency for making acid byproducts.
Effects of Varying Media, Temperature, and Growth Rates on the Intracellular Concentrations of Yeast Amino Acids
Authors: Martínez‐Force Enrique , Benítez Tahía 
Published: March 2005

AbstractVariations of the yeast free amino acid pool under different culture conditions were studied in two Saccharomyces strains, the laboratory haploid strain S288C and the industrial fermentative yeast IFI256. The internal amino acid pool of both strains was measured when grown in laboratory (minimal and complete) versus semiindustrial (molasses with or without added biotin and/or diammonium phosphate) media, in fermentable (glucose, fructose, sucrose) versus respirable (glycerol) carbon sources, in different temperatures (22, 30, and 37 °C), pHs (2. 0–4.75), and growth rates (0. 018–0.24 h−1) in continuous culture, and at different phases of the growth curve in batch culture (lag, exponential, early and late stationary). Results indicated that environmental conditions, particularly the presence of amino acids in the media, enormously influenced the intracellular amino acid concentration. Higher values were detected in molasses than in laboratory media and in fermentable carbon sources (glucose, fructose, sucrose) than in glycerol. Variations in the amino acid pool along the growth curve were greater at 37 °C than at other temperatures; in all cases, the highest values were measured at the begining of the exponential phase. In continuous culture and at different growth rates, intracellular free amino acid concentrations increased by 3–10‐fold when the growth rate was lower than 0.05 h−1, representing 20–35% of the total (free plus protein) amino acid content and indicating that amino acid yield was a partly growth‐linked parameter.
RNA Transcription from Immobilized DNA Templates
Authors: Marble H. Anthony , Davis Robert H. 
Published: March 2005

AbstractWe describe an RNA transcription protocol based on the multiple reuse of solid‐phase synthetic DNA templates. The templates are assembled onto streptavidin‐coated agarose beads via a single 5′‐terminal biotin located on the noncoding template strand. Transcription occurs in an aqueous buffered suspension containing solid‐phase DNA, dissolved enzyme (T7 RNA polymerase), and nucleoside triphosphate substrates (NTPs). A direct comparison of solution and solid‐phase templates under standard transcription conditions reveals similar initial reaction rates and overall yields. Immobilized templates store stably for periods of several months and are easily recovered by mild centrifugation. We demonstrate the successive reuse of these templates throughout 15 rounds of transcription. The templates remained active, although an incremental decay in transcription was observed beyond five rounds. Template activity was partially restored by supplementing the support‐bound oligonucleotide with fresh coding‐strand DNA. These findings indicate that multiple reuse of template is a viable strategy for reducing the amount of DNA template required in RNA transcription.
Eucalyptus Kraft Black Liquor Enhances Growth and Productivity of Spirulina in Outdoor Cultures
Authors: Chauhan Vikas S. , Singh Gurbaksh , Ramamurthy V. 
Published: March 2005

AbstractMass cultivation of microalgae for commercial applications suffers from poor productivities when measured against laboratory results or theoretical projections. In an effort to reduce this gap it was discovered that addition of eucalyptus kraft black liquor (BL) enhanced biomass productivity in outdoor cultures of Spirulina by increasing growth rate by 38% and biomass yield by 43%. BL treatment resulted in elevation of nitrogen assimilating enzyme activities and efficiency of phosphate utilization. Analyses of forenoon and afternoon oxygen production rates (OPRs) indicated higher photosynthetic and respiratory activity in BL‐treated cultures compared to untreated cultures.
Optimization of Rice α‐Amylase Production Using Temperature‐Sensitive Mutants of Saccharomyces cerevisiae for the PHO Regulatory System
Authors: Uchiyama Keiji , Ohtani Tomoko , Morimoto Masaaki , Shioya Suteaki , Suga Ken‐Ichi , Harashima Satoshi , Oshima Yasuji 
Published: March 2005

AbstractA typical example of rational system synthesis for bioproduction by the cultivation of microorganisms harboring a recombinant plasmid was studied. First, two temperature‐controllable expression systems for a foreign gene in Saccharomyces cerevisiae were constructed. The promoter of the PHO84 gene, which encodes an inorganic phosphate (Pi) transporter of S. cerevisiae and is controlled by Pi concentration through the PHO regulatory system, was used. And two temperature‐sensitive mutants of S. cerevisiae for the PHO regulatory system were used as the host with rice α‐amylase expression vector. One was temperature‐sensitive pho81 mutant, SH3337, and the other was temperature‐sensitive pho80 mutant, YKU107. When the strain SH3337 is cultivated at a lower temperature, the rice α‐amylase gene connected downstream of the PHO84 promoter is expressed, but at a higher temperature, it is not. Conversely, with the strain YKU107, the transcription of the rice α‐amylase gene is induced at a higher temperature and repressed at a lower one. The optimal cultivation temperature strategies for maximum production of rice α‐amylase in batch cultures of these two recombinant strains were then determined by the Maximum Principle using the relationships of the specific growth and specific production rates to the cultivation temperature. The optimal strategies were easy to realize and were shown to be effective for maximum product. Finally, under these maximum α‐amylase production strategies, the α‐amylase activities and α‐amylase yields in terms of glucose consumption for these recombinant strains were calculated on the basis of experimental data and compared for various operation times. The study demonstrates how a foreign gene expression system can be synthesized using a temperature‐sensitive mutant with a given objective and under given constraints.
Fluctuating Shear Stress Effects on Stress Fiber Architecture and Energy Metabolism of Cultured Renal Cells
Authors: Bhat Vinayak D. , Windridge Paula A. , Cherry Robert S. , Mandel Lazaro J. 
Published: March 2005

AbstractThe project investigates the relationship between the external shear force and the actin cytoskeleton along with the metabolic changes occurring inside the cells due to this force. Anchorage‐dependent Madin Darby canine kidney (MDCK) cells were placed in spinner flasks with paddle‐type stirrers agitated at 20 rpm, where they experienced shear stress fluctuations from 0.02 to 0.27 dyn/cm2 in magnitude. Following fixation, permeabilization, and staining with rhodamine—phalloidin, the relative amounts and distribution of F‐actin stress fibers in the 1 μm basal layer of the cells were visualized by confocal microscopy. These structures disappeared after 12–15 h of exposure to shear stress. Previous results showed that the stress fibers disappear, leading to loss of epithelial attachment, after only 1 h of starvation‐induced energy depletion. Therefore, in this study, the energy metabolism of the cells was established by measuring adenosine triphosphate (ATP) levels at different time intervals. No statistical difference in ATP content was found between the shear‐stressed cells and the controls, showing that shear stresses cause cytoskeletal reorganization by a mechanism other than ATP depletion.
Derivation of Kinetic Equations for Growth on Single Substrates Based on General Properties of a Simple Metabolic Network
Authors: Heijnen J. J. , Romein B. 
Published: March 2005

AbstractResearch on microbial growth has resulted in a large number of different equations to describe growth kinetics, the most famous of which is that of Monod. In the present work, the case of single substrate limitation in the absence of substrate inhibition is studied. Metabolism can then be regarded as a simple network consisting of a single reaction for anabolism and another for catabolism. These reactions are interconnected by cofactors of rather constant concentration. Growth is described as a combination of substrate uptake kinetics and growth kinetics. The notion that enzymatic activities of anabolism and catabolism depend on growth rate leads to a number of substrate uptake models, which fall into a few families. Each of them can be described by a general and flexible three‐parameter equation. Well‐known equations like that of Monod and Blackman turn out to be special cases. It could be concluded that the presented equations show more or less the same behavior in a scaled uptake rate versus substrate concentration plot, although the (powered) Monod equation might in general be preferred because its overall shape seems to resemble experimental observations most.
Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli
Authors: BENABDELHAK Houssain , SCHMITT Lutz , HORN Carsten , JUMEL Kornelia , BLIGHT Mark A. , HOLLAND I. Barry 
Published: March 2005

The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers. The ATPase activity of the isolated ABC domain clearly demonstrated positive co-operativity, with a Hill coefficient of 1.7. Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately decreased binding of 8-azido-ATP. Inhibition of activity with increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence. Finally, ATPase activity was sensitive towards orthovanadate, with an IC50 of 16 μM, consistent with the presence of transient dimers during ATP hydrolysis. Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC ATPase was only detected as a monomer, as measured by ultracentrifugation or gel filtration. In contrast, in the absence of salt, the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers. Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration, even in the presence of salt. These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis.
Light‐chain‐restricted plasmacellular infiltrates in necrobiosis lipoidica – a clue to an underlying monoclonal gammopathy
Authors: Cioc Adina M. , Frambach Gwyn E. , Magro Cynthia M. 
Published: March 2005

Background:  Necrobiosis lipoidica (NL) is a member of the palisading granulomatous dermatitides that is associated, in most cases, with diabetes mellitus. However, there are an increasing number of cases of NL associated with other forms of systemic disease. We describe a novel case of NL associated with a light‐chain‐restricted plasmacellular infiltrate; subsequent investigations established an underlying monoclonal gammopathy.Methods:  Skin biopsy material was obtained and was processed in the usual fashion for hematoxylin and eosin (H&E) examination. Immunohistochemical staining was performed by utilizing kappa and lambda monoclonal antibodies (Dako Corporation, Carpentiera, CA, USA). Kappa and lambda in situ hybridization was also performed (Ventana Medical Systems, Tucson, AZ, USA).Results:  A 55‐year‐old woman with a 5‐year history of bilateral thigh subcutaneous nodules underwent a skin biopsy, showing typical changes of NL; there was a concomitant prominent perivascular plasmacellular infiltrate. Kappa light chain restriction was observed amid the plasmacellular infiltrate. Bone marrow biopsy and immunophenotyping studies revealed a clonal plasmacytosis with kappa light chain restriction.Conclusions:  Granulomatous inflammation, including NL, may be a cutaneous paraneoplastic expression of low‐grade B‐cell lymphoproliferative disease in the context of an underlying plasma cell dyscrasia.
The importance of mitotic rate as a prognostic factor for localized cutaneous melanoma
Authors: Barnhill Raymond L. , Katzen Jason , Spatz Alain , Fine Judith , Berwick Marianne 
Published: March 2005

Background:  Tumor ulceration (TU) is considered the second most important prognostic factor after Breslow thickness for localized cutaneous malignant melanoma (CMM). However, many studies have not included mitotic rate (MR) with TU in these analyses. When both TU and MR are included in the same analysis, MR appears to be the more important than TU and TU loses its significance as an independent prognostic factor.Methods:  The relative importance of TU and MR as prognostic factors in localized CMM were compared in a population‐based series of 650 consecutive invasive CMM cases ascertained from the Connecticut tumor registry and reviewed by a single dermatopathologist (RLB), during the period between January 15, 1987 and May 15, 1989. Seventeen clinical and histopathological variables including tumor thickness measured in mm, TU recorded as present or absent, and MR recorded as number per mm2 were included in an unconditional logistic regression model and selected for inclusion using a backward stepwise algorithm with death as an endpoint or at least five‐years follow‐up.Results:  In the multivariate regression, the independent prognostic factors included: 1. tumor thickness in millimeters (OR = 1.5, 95% CI = 1.3−1.9) 2. moderate mitotic index (between 1 and 6): (OR = 8.3, 95% CI 2.4–28.7), 3. mitotic index (>6): (OR = 11.6, 95% CI = 3.0−44.6), 4. solar elastosis: (inversely associated with mortality)(OR = 0.4, 95% CI = 0.2−8). After adjustment for MR, TU lost its significance. When MR was left out of the analysis, ulceration then became an independent prognostic factor. The model with ulceration only (excluding MR) showed a relative risk (RR) of 2.4 (95%CI: 1.1–5.1). In the model with MR only, MR had a RR of 14.5 (95% CI3.9–53.7). Finally, regression analysis including both TU and MR yielded an RR of 11.6 for MR and 1.7 for TU.Conclusions:  Our results suggest that MR as a proxy for tumor proliferation is a more important prognostic factor than TU.
Cutaneous lipomatous neurofibroma: characterization and frequency
Authors: Val‐Bernal J. Fernando , González‐Vela M. Carmen 
Published: March 2005

Background:  There are numerous variants of cutaneous neurofibroma reflecting its manner of growth and histologic composition. Lipomatous neurofibroma is the latest described variant with only eight cases reported.Methods:  A systematic study based on 320 consecutive specimens diagnosed of cutaneous neurofibroma was carried out. Conventional microscopy, immunohistochemistry, and statistical methods were used to determine the presence of fat cells, their amount, distribution, and frequency.Results:  Intratumoral fat was observed in 22 (6.9%) neurofibromas. All these were dermal neurofibromas. Intraneoplastic fat was divided into two groups: focal and diffuse (regularly interspersed). Eighteen tumors (5.6%) presented adipocytes focally intermingled with the spindle cells. There were four (1.3%) neurofibromas showing spindle cell proliferation with regularly scattered adipocytes. Lipomatous neurofibroma was more frequent located on head and neck than non‐lipomatous neurofibroma ( p = 0.04). Neurofibromas without mature adipocytes were more frequently immunoreactive for CD34 compared with tumors showing intratumoral fat ( p = 0.02).Conclusions:  We suggest that both metaplasia and aberrant adipose differentiation from multipotential cells may result in lipomatous neurofibroma. Focal presence of adipose cells may be attributable to metaplasia as the pathogenic mechanism. The fatty tissue being intrinsic to the tumor structure in its diffuse form, the lesion represents a distinctive tumor of the peripheral nerve sheath.