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PubMed2026

A study on the directed engineering and multiple transformations of cannabidiolic acid synthase to enhance the expression level of the recombinant enzyme.

To increase the activity of cannabidiolic acid synthase (CBDAS) and its expression levels in yeast, this study focused on the CBDASG183V-N482W mutant. Using computer-aided techniques and literature reviews, four mutation sites were further identified, resulting in the mutant CBDASH114E-S116A-C176Y-G183V-N328Q-N482W. The CBDAS gene was integrated into the Pichia pastoris genome via multiple transformation rounds, and relative enzyme activity was analyzed using high-performance liquid chromatography. The results of the molecular docking analysis revealed factors such as increased intermolecular forces, shorter bond lengths, and an increased number of amino acid-substrate interaction sites, which may have contributed to the enhanced catalytic activity of the mutant. The concentrations of CBDA and CBD produced by the CBDASH114E-S116A-C176Y-G183V-N328Q-N482W mutant were 71.543 ng/mL and 75.163 ng/mL, respectively, which were 11.87% and 11.53% greater than those produced by the CBDASG183V-N482W mutant. The recombinant CBDAS strain obtained after two consecutive transformations of the CBDASH114E-S116A-C176Y-G183V-N328Q-N482W vector presented the highest CBDAS expression levels and CBD and CBDA yields; compared with those obtained after a single transformation, the CBDA and CBD yields increased by 9.77% and 12.65%, respectively. In addition, the tolerance of the recombinant strain to induction culture conditions was analyzed, revealing that the strain could be induced to express the protein at temperatures ranging from 20 to 45 °C and at pH values ranging from 3 to 9, with optimal expression observed at 30 °C and pH 6. These findings provide theoretical and technical support for the production of enzyme preparations for the in vitro-directed biosynthesis of cannabidiol.

Molecular Docking Simulation↗
PubMed2004

Regularly alternating spin-1/2 anisotropic XY chains: the ground-state and thermodynamic properties.

Using the Jordan-Wigner transformation and continued fractions we calculate rigorously the thermodynamic quantities for the spin-1/2 transverse Ising chain with periodically varying intersite interactions and/or on-site fields. We consider in detail the properties of the chains having a period of the transverse field modulation equal to 3. The regularly alternating transverse Ising chain exhibits several quantum phase transition points, where the number of transition points for a given period of alternation strongly depends on the specific set of the Hamiltonian parameters. The critical behavior in most cases is the same as for the uniform chain. However, for certain sets of the Hamiltonian parameters the critical behavior may be changed and weak singularities in the ground-state quantities appear. Due to the regular alternation of the Hamiltonian parameters the transverse Ising chain may exhibit plateaulike steps in the zero-temperature dependence of the transverse magnetization vs transverse field and many-peak temperature profiles of the specific heat. We compare the ground-state properties of regularly alternating transverse Ising and transverse XX chains and of regularly alternating quantum and classical chains. Making use of the corresponding unitary transformations we extend the elaborated approach to the study of thermodynamics of regularly alternating spin-1/2 anisotropic XY chains without field. We use the exact expression for the ground-state energy of such a chain of period 2 to discuss how the exchange interaction anisotropy destroys the spin-Peierls dimerized phase.

Journal Article↗
PubMed2002

Survivin as a cell cycle-related and antiapoptotic protein in granulosa cells.

Survivin is a relatively unique member of the inhibitor of apoptosis protein (IAP) family in that it contains a single baculovirus IAP repeat (BIR) domain combined with a COOH-terminal alpha-helix coiled-coil domain instead of the more common zinc-binding RING finger. Results from a variety of transformed or continuous mammalian cell lines suggest that, due to the combination of these features, Survivin is capable of regulating both cell proliferation and apoptotic cell death. However, to date there is essentially no information regarding Survivin expression, regulation or function within the ovary, or in any nonmammalian vertebrate species. In the present studies, cDNAs for chicken (ch) Survivin-142 (homologous to human Survivin-142) plus three alternatively spliced variants (ch Survivin-short, -gamma, and -delta) are described, and of these, transcripts for ch Survivin-142 and -short are expressed in granulosa cells from the hen ovary. Highest levels of Survivin mRNA during follicle development occur in mitotically active granulosa cells from undifferentiated, prehierarchal follicles. Cell cycle analysis determined that Survivin mRNA expression is elevated specifically during the G2/M phase of mitosis. Significantly, transient transfection with ch Survivin-142 in primary cultures of hen granulosa cells attenuates taxol- and N-octanoylsphingosine- (C8-ceramide-) induced caspase-3 activity, whereas overexpression of ch Survivin-short (a truncated variant that lacks much of the functional BIR domain plus the entire alpha-helix coil domain) lacks this antiapoptotic activity. Taken together, these data provide evidence for Survivin in granulosa cells acting as a bifunctional protein associated with regulation of the cell cycle and the inhibition of apoptosis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

The role of capillarization in hepatic failure: studies in carbon tetrachloride-induced cirrhosis.

During the cirrhotic process, the hepatic microvascular phenotype is transformed from sinusoids (discontinuous capillaries) into continuous capillaries. This transformation has been termed capillarization. Many hepatic functions depend on the rapid, bidirectional exchange of macromolecules between plasma and hepatocytes. To determine whether capillarization contributes to hepatic failure in cirrhosis, we decided to study the plasma clearance (125I) and hepatocyte uptake (electron microscopy) of three tracers in normal and cirrhotic rats. The tracers chosen were a hemeundecapeptide with peroxidatic activity (fluid-phase pinocytosis), asialofetuin (receptor-mediated endocytosis of a medium size protein) and ferritin (receptor-mediated endocytosis of a large size protein). The results demonstrate a decreased hepatocyte uptake of hemeundecapeptide; a significant delay in plasma clearance of asialofetuin; and a minor delay in plasma clearance of ferritin, but a striking trapping of ferritin in the cirrhotic capillary basement membrane. The delayed plasma clearance in cirrhosis cannot be ascribed to a decreased number of surface receptors because, in isolated hepatocytes, the number of molecules bound per cell was equivalent in normal and cirrhotic livers. These findings support the concept of capillarization, with the formation of continuous diffusion and filtration barriers between plasma and hepatocytes, representing a significant hindrance to the bidirectional macromolecular exchange normally taking place between these two compartments. Furthermore, at least in the case of ferritin, the capillary basement membrane of cirrhotic livers seems to be the major filtration barrier. This hindrance to hepatocyte uptake, and presumably also to secretion, may be the cause (or at least a major determinant) of the hepatic failure characteristic of cirrhosis.

Animals↗
PubMed1999

Reversible denaturation of carbonic anhydrase provides a method for its adsorptive immobilization.

Palmityl-substituted sepharose 4B has been used for adsorptive immobilization of heat-denatured carbonic anhydrase. The native form of this enzyme does not show any affinity for binding to this hydrophobic support. However, through the process of denaturation-renaturation performed by heating and subsequent cooling of an enzyme solution in the presence of the matrix, it was possible to obtain a catalytically active immobilized preparation, which was used successfully in continuous catalytic transformations. It is suggested that this simple procedure may provide a convenient method of immobilization for proteins, which are not normally adsorbed on hydrophobic supports.

Adsorption↗
PubMed2005

Sexual dimorphism in the Japanese cranial base: a Fourier-wavelet representation.

An approach, computational shape analysis (CSA), is presented here which utilizes a Fourier-wavelet representation to numerically describe shape features of biological forms. Two elements are involved: 1) elliptical Fourier functions (EFFs), to provide estimates of global aspects, and 2) continuous wavelet transforms (CWTs) to generate an objective estimate of localized features. EFFs are computed, using a set of pseudohomologous points, to create a precise analog of the boundary. This computed contour is then normalized by scaling and rotated in two-dimensional space to insure a representation that is invariant with respect to starting point, size, and orientation. The predicted point coordinates derived from the EFFs are submitted to CWT for further processing. Wavelet coefficients are then computed to identify localized features, localization being a subjective process with EFFs. The advantage of wavelets is that they eliminate the inevitable subjectivity inherent in the choice of measurements. To test the usefulness of CSA, a sample of cranial base (CB) lateral radiographic outlines was available. Five archaeological periods, Yayoi, Kofun, Kamakura, Edo, and Modern, were utilized (n = 297). Statistically significant differences in sex and archaeological age were found. Although archaeological age differences were present, they were small and random in character, suggesting stability in the CB structures. In contrast, sexually dimorphic differences were present in every group from the Yayoi to the Modern period. This presence of sexually dimorphic differences in shape was consistent with earlier studies of M. nemestrina, G. gorilla, and P. troglodytes. Consequently, it is suggested that the pattern of sexual dimorphism documented in the Japanese CB is a primate pattern with an ancient evolutionary history. The results demonstrate, both visually and numerically, that CSA is a powerful approach for describing both global and localized features of craniofacial structures such as the CB.

Anthropology, Physical↗
PubMed1995

Dynamics of follicular growth and atresia of large follicles during the ovarian cycle of the guinea pig: fate of the degenerating follicles, a quantitative study.

BACKGROUND: A quantitative integrated study of healthy ovarian follicles of different sizes and their mitotic activity and of clearly defined atretic stages of involuting large growing follicles at different stages of the guinea pig ovarian cycle is not available in the literature. We considered that such a study would reveal new aspects of ovarian tissue dynamics and provide new information in an organ with a continuous phenotypic transformation of its cellular components. METHODS: Ovaries from guinea pigs were removed on days 1 (opening of the vagina), 3, 6, 9, 13, and 16 of the cycle, and the following were measured in serial sections: (1) total number of healthy follicles falling into categories based on the volume occupied by granulosa cells, (2) total number of atretic follicles falling into clearly defined morphological stages of the degenerative and involutionary process affecting medium to large follicles, and (3) proportion of metaphase-arrested granulosa cells, after colcemid injection, in healthy follicles of different size categories. RESULTS: Dynamic patterns of follicular growth and degeneration were revealed that permitted the following main conclusions and observations: (1) small to middle-size follicles can reach the maximal category mass of granulosa mass within 6-7 days, and the number of granulosa cells can increase 6-7-fold during this interval, (2) the cohort that gives rise to 2-6 preovulatory follicles and to the large follicles that will undergo atresia during each cycle varied from 68 to 108 follicles, (3) cell death starts in the granulosa cell layers of large follicles even when neighbouring cells maintain a high mitotic activity and it spreads rapidly; dead granulosa cells are cleare by nucleolysis and cytolysis in the absence of blood leucocytes or neovascularization, (4) foci of atresia are observed also in a few preovulatory follicles, (5) antral cavities of follicles with dead granulosa cells in the process of being lysed shrink and are filled within 2-3 days with large fibroblast-like cells arising from phenotypic transformation of inner layers of theca interna, with no evidence of mitotic activity or angiogenesis; the outer layers of theca interna involute, and by progressive atrophy and a process of cell death, minute nodular structures arise with remnants of the ovum and zona pellucida, and (6) a transient wave of degeneration affects a proportion of small and middle-size follicles during the metestrous period. This process does not resemble the morphological phenomenology of follicular involution, which affects only large follicles. CONCLUSIONS: This study contributes to a fuller understanding of the dynamics and time relationships of follicular growth and loss in the guinea pig ovary and provides new morphogenetic information on the atretic process. It would be valuable for the design of experiments on endocrine and paracrine interactions involved in follicular growth and atresia.

Animals↗
PubMed1994

Topological analysis of the brainstem of the bowfin, Amia calva.

This paper presents a survey of the cell masses in the brainstem of the generalized actinopterygian fish Amia calva, based on transversely cut Nissl-, Klüver-Barrera-, and Bodian-stained serial sections. This study is intended to serve a double purpose. First it forms part of a now almost complete series of publications on the structure of the brainstem in representative species of all groups of vertebrates. Within the framework of this comparative program the cell masses in the brainstem and their positional relations are analyzed in the light of the Herrick-Johnston concept; according to this the brainstem nuclei are arranged in four longitudinal, functional zones or columns, the boundaries of which are marked by ventricular sulci. The procedure employed in this analysis essentially involves two steps: first, the cell masses and large individual cells are projected upon the ventricular surface, and next, the ventricular surface is flattened out, that is, subjected to a one-to-one continuous topological transformation (Nieuwenhuys [1974] J. Comp. Neurol. 156:255-267). The second purpose of the present paper is to provide a cytoarchitectonic basis for experimental analysis of the fiber connectivity in the brainstem of Amia. Five longitudinal sulci--the sulcus medianus inferior, the sulcus intermedius ventralis, the sulcus limitans, the sulcus intermedius dorsalis, and the sulcus lateralis mesencephali--could be distinguished. Some shorter grooves, present in the isthmal region, clearly deviate from the overall longitudinal pattern of the other sulci. Although in Amia most neuronal perikarya are contained within a diffuse periventricular gray, 40 cell masses could be delineated: Eight of these are primary efferent or motor nuclei, 10 are primary afferent or sensory centers, seven are considered to be components of the reticular formation, and the remaining 15 may be interpreted as "relay" nuclei. The topological analysis yielded the following results. In the rhombencephalon the gray matter is arranged in four longitudinal columns or areas, termed area ventralis, area intermedioventralis, area intermediodorsalis, and area dorsalis. The sulcus intermedius ventralis, the sulcus limitans, and the sulcus intermedius dorsalis mark the boundaries between these morphological entities. These longitudinal areas coincide largely, but not entirely, with the functional columns of Herrick and Johnston. The most obvious incongruity is that the area intermediodorsalis contains, in addition to the viscerosensory nucleus of the solitary tract, several general somatosensory and special somatosensory nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Lactate doublet quantification and lipid signal suppression using a new biexponential decay filter: application to simulated and 1H MRS brain tumor time-domain data.

A new postprocessing filter based on the continuous wavelet transform (CWT) method modeled as a biexponential decay function to isolate the lactate doublet from overlapping lipid resonance(s) and estimate its magnetic resonance spectroscopy (MRS) parameters (signal amplitude, resonance frequencies, and apparent relaxation time (T(*) (2))) is proposed. The new filter employs the same iterative process used in the previously single exponential decay filter. A comparison of the results obtained from application of both filters to simulated data and real (1)H MRS data collected from human blood plasma and brain tumors demonstrates that the new filter provides a better estimate of MRS parameters of lactate, with less computation time. Furthermore, the results show that the new filter is less sensitive to noise and provides a direct estimate of J-coupling value of the lactate doublet.

Adipose Tissue↗
PubMed2003

Stability of multivariable fractional polynomial models with selection of variables and transformations: a bootstrap investigation.

Sauerbrei and Royston have recently described an algorithm, based on fractional polynomials, for the simultaneous selection of variables and of suitable transformations for continuous predictors in a multivariable regression setting. They illustrated the approach by analyses of two breast cancer data sets. Here we extend their work by considering how to assess possible instability in such multivariable fractional polynomial models. We first apply the algorithm repeatedly in many bootstrap replicates. We then use log-linear models to investigate dependencies among the inclusion fractions for each predictor and among the simplified classes of fractional polynomial function chosen in the bootstrap samples. To further evaluate the results, we define measures of instability based on a decomposition of the variability of the bootstrap-selected functions in relation to a reference function from the original model. For each data set we are able to identify large, reasonably stable subsets of the bootstrap replications in which the functional forms of the predictors appear fairly stable. Despite the considerable flexibility of the family of fractional polynomials and the consequent risk of overfitting when several variables are considered, we conclude that the multivariable selection algorithm can find stable models.

Algorithms↗
PubMed1992

Dynamic changes in cytokine secretion by stromal cells during prolonged maintenance under protein-free conditions.

Stromal cells of bone marrow origin produce a variety of known cytokines and some factors exhibiting apparently new biological activities. Several of these were identified by the study of cell to cell interactions and were not found in detectable amounts in media conditioned by the cells. We describe here a culture system that enables the release of stromal cytokines into medium free of any added proteins and supplemented with peptides from casein hydrolysate (0.1%). The absence of serum proteins allows extensive concentration and monitoring of activities that are otherwise undetectable. Stromal cells of the MBA-2.1 clonal cell line were seeded in a stationary bed reactor packed with a carrier of non-woven fabric matrix. After a proliferation phase with serum containing medium, the cells were maintained for over 10 months in protein-free medium. Throughout this extended incubation in the absence of serum or serum replacing proteins, stromal cells retained their viability and continuously released transforming growth factor-beta (TGF-beta), macrophage-colony stimulating factor (M-CSF) and restrictin-P, a cytotoxic factor that specifically arrested the growth of plasmacytoma cells. In addition, interleukin-6 (IL-6) was first undetectable, and later in culture its titer reached a maximum of 180,000 international units (IU)/ml. Concomitantly, the production of restrictin-P diminished and reached its lowest levels at the end of 10 months. The results may imply a possible causal relationship between the expression of IL-6 and restrictin-P, since no similarly significant changes were observed in the titers of M-CSF and TGF-beta. This novel bioreactor system may be adaptable for efficient production of different cytokines under absolute serum-free conditions.

Animals↗
PubMed2000

Water peak suppression: time-frequency vs time-scale approach.

Wavelets are the most popular time-scale analysis tool. A well-known application of wavelets in nuclear magnetic resonance spectroscopy is water peak extraction/suppression. However, spectroscopists are more familiar with frequency than scale. So, from a spectroscopist point of view, a time-scale analysis tool (i.e., wavelets) is not natural and a time-frequency approach would be much more satisfactory. We explain a time-frequency solution to this problem based on Gabor analysis. As the two formalisms are closely linked together we continuously emphasize their similarities and differences. In particular we show that, here, the Gabor method is as efficient as the wavelet approach, and we give some examples. Those remarks also apply to other NMR problems solved previously with the continuous wavelet transform, such as quantification or dynamical phase correction.

Algorithms↗
PubMed2001

Water modeled signal removal and data quantification in localized MR spectroscopy using a time-scale postacquistion method.

We have previously shown the continuous wavelet transform (CWT), a signal-processing tool, which is based upon an iterative algorithm using a lorentzian signal model, to be useful as a postacquisition water suppression technique. To further exploit this tool we show its usefulness in accurately quantifying the signal metabolites after water removal. However, due to the static field inhomogeneities, eddy currents, and "radiation damping," the water signal and the metabolites may no longer have a lorentzian lineshape. Therefore, another signal model must be used. As the CWT is a flexible method, we have developed a new algorithm using a gaussian model and found that it fits the signal components, especially the water resonance, better than the lorentzian model in most cases. A new framework, which uses the two models, is proposed. The framework iteratively extracts each resonance, starting by the water peak, from the raw signal and adjusts its envelope to both the lorentzian and the gaussian models. The model giving the best fit is selected. As a consequence, the small signals originating from metabolites when selecting, removing, and quantifying the dominant water resonance from the raw time domain signal are preserved and an accurate estimation of their concentrations is obtained. This is demonstrated by analyzing (1H) magnetic resonance spectroscopy unsuppressed water data collected from a phantom with known concentrations at two different field strengths and data collected from normal volunteers using two different localization methods.

Artifacts↗
PubMed1998

Shaping space: the possible and the attainable in RNA genotype-phenotype mapping.

Understanding which phenotypes are accessible from which genotypes is fundamental for understanding the evolutionary process. This notion of accessibility can be used to define a relation of nearness among phenotypes, independently of their similarity. Because of neutrality, phenotypes denote equivalence classes of genotypes. The definition of neighborhood relations among phenotypes relies, therefore, on the statistics of neighborhood relations among equivalence classes of genotypes in genotype space. The folding of RNA sequence (genotypes) into secondary structures (phenotypes) is an ideal case to implement these concepts. We study the extent to which the folding of RNA sequence induces a "statistical topology" on the set of minimum free energy secondary structures. The resulting nearness relation suggests a notion of "continuous" structure transformation. We can, then rationalize major transitions in evolutionary trajectories at the level of RNA structures by identifying those transformations which are irreducibly discontinuous. This is shown by means of computer simulations. The statistical topology organizing the set of RNA shapes explains why neutral drift in sequence space plays a key role in evolutionary optimization.

Animals↗
PubMed1993

Circadian and light-regulated expression of nitrate reductase in Arabidopsis.

The expression of a number of plant genes is regulated by an endogenous circadian clock. We report that the Arabidopsis NIA2 (nitrate reductase) gene shows robust circadian oscillations in mRNA accumulation which persist for at least 5 days in plants that have been grown in a light-dark (LD) cycle and then transformed to continuous light (LL). We further show that NIA2 mRNA accumulation oscillates in a circadian fashion in plants that have been grown in LD and then transferred to continuous darkness (DD). Results from nuclear run-on transcriptional analysis suggest that the oscillations in steady-state levels of NIA2 mRNA abundance are not primarily due to changes in transcription but, instead, reflect post-transcriptional regulation. The circadian oscillations in NIA2 mRNA abundance are paralleled by circadian oscillations in nitrate reductase enzyme activity (NR activity) in Arabidopsis plants that have been grown in LD and then transferred either to DD or to LL. Etiolated Arabidopsis seedlings express neither NIA2 mRNA nor NR activity. However, both NIA2 mRNA accumulation and NR activity are induced by exposure to white light. The inductive effects of light on NIA2 mRNA accumulation are due, at least in part, to a very low fluence phytochrome-mediated response. However, the persistence of circadian oscillations in NIA2 mRNA abundance for at least 5 days in LL demonstrates that the circadian clock is capable of overriding or gating the inductive effects of light on NIA2 mRNA accumulation in Arabidopsis for an extended, continuous period of time.

Arabidopsis↗
PubMed1986

Spontaneous amplification of yeast CEN ARS plasmids.

Transformation of Saccharomyces cerevisiae with several yeast CEN4 ARS1 plasmids containing the his3-delta 4 allele (as well as the URA3 and TRP1 markers) yielded His+ transformants at 0.1%-50% the frequency of Ura+ Trp+ transformants. Additional His+ derivatives arose on continuous growth of transformants originally scored as His- Ura+ Trp+. In all cases, the His+ phenotype was not due to plasmid or host mutations but invariably correlated with an up to 12-fold increase in plasmid copy number. On removal of selective pressure, the His+ phenotype was lost more readily than the Ura+ Trp+ markers, with a corresponding decrease in plasmid copy number. Also, the amplification did not decrease the mitotic loss rate of the Ura+ Trp+ markers. These results indicate that CEN ARS plasmids can be spontaneously amplified to higher levels than previously observed. However, when amplified, apparently not all copies exhibit the characteristic stability of CEN ARS plasmids.

Alleles↗
PubMed1995

Evolution and ultrastructure of the bovine spermatogonia precursor cell line.

The spermatogonial stem cell line in prepubertal and adult bovine testis was studied by electron microscopy and protein gene product 9.5 immunohistochemistry. Three successive spermatogonia precursor cell configurations were observed. Small basal stem cells were found to possess a spherical shape and nuclei with two to three nucleoli. They were observed in prepubertal testes (25 and 30 weeks) and in low numbers during all the stages of the seminiferous epithelial cycle in the adult. Aggregated spermatogonia precursor cells are the dominating germ cell type in the 25-week-old and 30-week-old calf. In the adult seminiferous epithelium, they cause expansion of the basal tubular compartment as they form dense groups containing up to 15 cells. These groups are observed concomitantly with cycling A-spermatogonia and preleptotenes at the beginning of spermatocytogenesis. At the end of A-spermatogonia propagation, the aggregated spermatogonia precursor cells separate and intermingle with cycling A-spermatogonia. The spermatogonia precursor cells can later be found together with I-spermatogonia as members of an interconnected cellular network of medium-sized cells. When the I-spermatogonia divide to form the smaller B-spermatogonia, the precursor cells, which stay connected with the cycling spermatogonial population, pass through a growth phase. They can now be considered as committed spermatogonia precursor cells and are continuously being transformed into A1-spermatogonia to start a new round of spermatocytogenesis. Ultrastructurally, all members of the precursor cell line are similar. However, a number of features have been found to show a quantitative increase (endoplasmic reticulum, mitochondria) or to exhibit a rising degree of complexity (nucleolus) during the progression from basal stem cells to committed spermatogonia precursor cells.

Animals↗