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At least 991 records · Page 55Linked to original sources
PubMed2006

Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis.

Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments xylose along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting with the native glycolytic Entner Doudoroff (ED) pathway has been hypothesized. We have investigated this putative reaction network by developing a kinetic model incorporating all of the enzymatic reactions of the PP and ED pathways, including those catalyzed by the heterologous enzymes. Starting with the experimental literature on in vitro characterization of each enzymatic reaction, we have developed a kinetic model to enable dynamic simulation of intracellular metabolite concentrations along the network of interacting PP and ED metabolic pathways. This kinetic model is useful for performing in silico simulations to predict how varying the different enzyme concentrations will affect intracellular metabolite concentrations and ethanol production rate during continuous fermentation of glucose and xylose mixtures. Among the five enzymes whose concentrations were varied as inputs to the model, ethanol production in the continuous fermentor was optimized when xylose isomerase (XI) was present at the highest level, followed by transaldolase (TAL). Predictions of the model that the interconnecting enzyme phosphoglucose isomerase (PGI) does not need to be overexpressed were recently confirmed through experimental investigations. Through such systematic analysis, we can develop efficient strategies for maximizing the fermentation of both glucose and xylose, while minimizing the expression of heterologous enzymes.

Fermentation↗
PubMed2005

Systematic review and meta-analysis of technique for closure of the pancreatic remnant after distal pancreatectomy.

BACKGROUND: Appropriate closure of the pancreatic remnant after distal pancreatectomy is still debated. A variety of procedures have been recommended to reduce the frequency of pancreatic fistula. This review quantitatively compares the available techniques. METHODS: Original articles and abstracts published up to the end of June 2004 were searched without language restriction in the Cochrane Controlled Trials Register, Medline and Embase. Three reviewers independently assessed each study's eligibility and quality, and extracted the data. A random effects model was performed using weighted odds ratios. RESULTS: Only ten of 262 articles could be included, two randomized clinical trials and eight observational studies. Reported postoperative morbidity varied from 13.3 to 64 per cent. The primary outcome measure, pancreatic fistula rate, occurred within the range 0-60.9 per cent. Meta-analysis of the six studies comparing stapler versus hand-sutured closure showed a non-significant combined odds ratio for occurrence of a pancreatic fistula of 0.66 (95 per cent confidence interval 0.35 to 1.26, P = 0.21) in favour of stapler closure. CONCLUSIONS: The quality and quantity of information extracted from the available trials are insufficient to enable any firm conclusion to be drawn on the optimal surgical technique of pancreatic stump closure; there is a trend in favour of the stapling technique.

Epidemiologic Methods↗
PubMed1984

Gas chromatography mass spectrometry of tert-butyldimethylsilyl ethers of phthiocerols and mycocerosic alcohols from Mycobacterium tuberculosis.

Mycobacteria synthesize a variety of unusual long-chain fatty acid esters and the systematic analysis of these is of great potential in the classification and identification of these bacteria. This paper describes the extraction, reductive fission and derivatization to tert-butyldimethylsilyl ethers of beta-diesters, the phthiocerol dimycocerosates. The mass spectra of these ethers are characteristic of the parent alcohols and selected ion monitoring techniques have been applied to mixtures extracted from strains of Mycobacterium tuberculosis.

Chemical Phenomena↗
PubMed2001

Connections of the nucleus incertus.

The nucleus incertus (NI) is a distinct cell group in caudoventral regions of the pontine periventricular gray, adjacent to the ventromedial border of the caudal dorsal tegmental nucleus. Recent interest in the NI stems from evidence that it represents one of the periventricular sites with the highest expression levels of mRNA encoding the type 1 corticotropin-releasing hormone (CRH) receptor, which has a high affinity for naturally occurring CRH, perhaps accounting for some of the extrapituitary actions of the peptide on autonomic and behavioral components of the stress response. However, almost nothing is known about NI function and hodological relationships. In this paper, we present the results of a systematic analysis of NI inputs and outputs using cholera toxin B subunit as a retrograde tracer and Phaseolus vulgaris-leucoagglutinin as an anterograde tracer. Our retrograde tracer experiments indicate that the NI is in a strategic position to integrate information related to behavioral planning (from the prefrontal cortex), lateral habenular processing, hippocampal function, and oculomotor control. Based on its efferent connections, the NI is in a position to exert significant modulating influences on prefrontal and hippocampal cortical activity, and the nucleus is also in a position to influence brain sites known to control locomotor behavior, attentive states, and learning processes. Overall, the present results support the idea that the NI is a distinct region of the pontine periventricular gray, and together with the superior central (median raphé) and interpeduncular nuclei the NI appears to form a midline behavior control network of the brainstem.

Afferent Pathways↗
PubMed2004

Distinct migratory behavior of early- and late-born neurons derived from the cortical ventricular zone.

Time-lapse studies indicate that ventricular zone (VZ)-derived cells show two migratory modes in the cerebral cortex at different stages of mammalian embryogenesis: somal translocation and locomotion. We carried out a systematic analysis to examine whether the migratory behavior of cortical neurons derived from the cortical VZ is stage-dependent. We labeled VZ cells of mouse embryos with green fluorescent protein (gfp) -encoding plasmids by in utero electroporation and evaluated the labeled cells after appropriate survival periods. After electroporation at either embryonic day (E) 12.5 or E15.5, GFP+ VZ cells were initially spindle-shaped and radially oriented. After leaving the VZ, they transformed into round or horizontally oriented fusiform neurons with many short processes. They then seemed to gradually change into radially oriented bipolar cells as they moved upward. Whereas the earliest emigrants from the VZ labeled at E12.5 (early-born neurons) reached the top of the cortical plate (CP) after these changes, VZ cells labeled at E15.5 (late-born neurons) further migrated along the length of radial fibers to reach the top of the CP. A dominant negative form of the gene for cyclin-dependent kinase 5 (Cdk5DN) was then introduced into VZ cells. Transfection of E12.5 VZ with cdk5dn did not disrupt the migration of the early-born neurons. However, this caused a failure in migration of the late-born neurons, although they transformed into bipolar shapes in the intermediate zone. Thus, there appear to be at least two distinct migratory phases of cortical neurons: one common to the early- and late-born neurons, and the other specific to late-born neurons and Cdk5-dependent.

Animals↗
PubMed1987

Distribution of neuropeptide Y-like immunoreactivity in the hypothalamus of the adult golden hamster.

The distribution of neuropeptide Y (NPY)-like immunoreactivity within the hypothalamus of the adult golden hamster was investigated with conventional immunohistochemical techniques. Neuropeptide Y immunoreactive cell bodies were found in greatest numbers in the arcuate nucleus while a few stained perikarya were seen in the internal and subependymal zones of the median eminence. Isolated perikarya were observed in the anterior commissure and supracommissural portion of the interstitial nucleus of the stria terminalis. Immunoreactive axons were located throughout the hypothalamus with the highest concentrations in the subependymal and internal zones of the median eminence, the interstitial nucleus of the stria terminalis, the medial preoptic area, and in the following nuclei: periventricular, suprachiasmatic, paraventricular, perifornical, median preoptic, and arcuate. Moderate to dense plexuses of immunoreactive fibers were observed in the anterior, lateral, and posterior hypothalamic areas and in the infundibular stalk. The supraoptic nucleus and lateral preoptic area displayed a small number of labeled axons whereas the ventromedial nucleus contained only a few fibers. NPY immunoreactive fibers were present in the optic tract and in the dorsomedial aspect of the optic chiasm. Labeled fibers penetrated the ependymal lining of the third ventricle throughout the ventral aspect of the periventricular zone. Additional fibers were observed in the pia lining the ventral aspect of the hypothalamus. This systematic analysis of hypothalamic NPY immunoreactivity in the adult golden hamster suggests that a portion of the labeled fibers display a distribution that is similar to previously described noradrenergic fibers in the hypothalamus.

Animals↗
PubMed1987

The entorhinal cortex of the monkey: II. Cortical afferents.

The entorhinal cortex of the monkey is commonly viewed as the major link between the cerebral cortex and the other fields of the hippocampal formation. Until recently, however, little was known about the origins of the cortical projections to the entorhinal cortex, and most of the available information is still based on degeneration studies. We have carried out a systematic analysis of these connections by placing small injections of the retrograde tracer wheat germ agglutinin conjugated to horseradish peroxidase into each of the fields of the entorhinal cortex of the Macaca fascicularis monkey. Retrogradely labeled cells were observed in several areas of the frontal and temporal lobes, the insula, and the cingulate cortex. In the frontal lobe, the greatest number of labeled cells were observed in the orbital region and specifically in areas 13 and 13a: labeled cells were also seen in areas 14, 11, and 12. In the dorsolateral frontal cortex, labeled cells were observed mainly in the rostral half of area 46; occasionally cells were also seen in areas 9, 8, and 6. In the cingulate cortex, labeled cells were observed in area 25, area 32, and rostral levels of area 24; fewer cells were observed at caudal levels of area 24 or in area 23. The retrosplenial region (areas 30 and 29), including its caudal extension along the rostral calcarine sulcus and its ventral extension into the temporal lobe, contained numerous labeled cells. In the temporal lobe, retrogradely labeled cells were arranged in two rostrocaudally oriented bands. Rostral to the hippocampal formation, the first band encompassed the piriform and periamygdaloid cortices and areas 35 and 36; the labeling in area 36 was continuous to the temporal pole. At more caudal levels this band was located immediately lateral to the hippocampal formation and included areas 35 and 36 rostrally and areas TH and TF caudally. The second band was situated in the superior temporal gyrus where labeled cells were observed in several distinct cytoarchitectonic fields, including the parainsular cortex in the fundus of the inferior limiting sulcus. In the insula proper, retrogradely labeled cells were seen mainly in the rostral or agranular division; far fewer were observed in the dysgranular and granular insula. Whereas there is little available physiological information concerning many of the cortical regions that project to the entorhinal cortex, on anatomical grounds they may be generally characterized as polysensory associational regions.

Afferent Pathways↗
PubMed1993

Synaptophysin immunohistochemistry reveals inside-out pattern of early synaptogenesis in ferret cerebral cortex.

Synaptogenesis in the ferret cerebral cortex was examined from the day of birth to adulthood with an antibody against synaptophysin at the light and electron microscopic levels. Due to the premature birth of ferrets, the generation of cells destined to the upper cortical layers and their subsequent migration to their final positions in the cortical plate are largely postnatal events. Throughout the newborn ferret cerebral cortex, a high amount of synaptophysin immunoreactivity was present within the marginal zone and subplate region. Staining was also conspicuous within the forming cortical plate. The typical layering pattern of synaptophysin immunoreactivity in the developing cortical plate correlated with the migration pattern of cortical neurons. The synaptic density was lowest directly below the marginal zone, where the youngest neurons just stopped their migration. Below this zone, the density of the synaptic staining increased gradually toward lower (and older) cortical plate layers. As the cortex expanded, the synaptophysin immunoreactivity pattern closely followed the expansion, suggesting that synapses were formed in a given layer shortly after the cells migrating to this layer reached their final position. As soon as cell migration had finished, the entire cortical plate contained dense synaptophysin immunoreactivity, in a pattern similar to that observed in the adult animal. During cortical development, a rostrocaudal and a laterodorsal gradient of synaptogenesis was observed. At any given time, rostral and lateral regions of the cerebral cortex were more advanced in their development than caudal and dorsal regions. Electron microscopic examination of synaptophysin immunoreactivity in the developing cerebral cortex of ferrets confirmed that labeling was solely associated with synaptic vesicles. These vesicles were typically, but not exclusively, confined to synaptic boutons. Especially around the end of the first postnatal week, long fiber profiles loaded with synaptic vesicles were occasionally detected. As some of these fibers also showed en passant synapses along their course, we concluded that synaptic vesicle labeling may be reliably used to study synaptogenesis at the light microscopic level. A systematic analysis of samples from postnatal days 0 and 7 corroborated this conclusion, showing that synaptic profile distribution completely matched the distribution of synaptophysin immunoreactivity seen in the light microscope. In conclusion, synaptogenesis begins as soon as migratory cells reach their final position in the cortical plate. As long as cell migration continues, synaptogenesis is under the constraints of neurogenesis, following its gradients.

Aging↗
PubMed1995

Cardiovascular responses to electrical stimulation of the bed nucleus of the stria terminalis.

To determine whether the influence of the bed nucleus of the stria terminalis (BST) on cardiovascular function can be localized to specific cytoarchitectural areas within the BST, urethane (1.3 g/kg)-anesthetized male Sprague-Dawley rats were probed for cardiovascular reactive sites. Electrical stimuli (50 microA, 50 Hz, and a 0.5 ms pulse duration), delivered through stereotaxically placed glass semimicroelectrodes, were localized to the BST. Sham-stimulated animals served as controls. Stimulation sites were correlated with cytoarchitecturally distinct areas within the BST, and changes in mean arterial pressure (MAP) were subjected to statistical analysis. Systematically probing the BST for cardiovascular reactive sites showed a correlation between evoked responses and distinct cytoarchitectural areas. Stimulation of the medial BST produced increases in MAP; stimulation of the lateral aspect of the BST produced decreases in MAP. Both pressor and depressor responses were evoked from the area ventral to the anterior commissure. Pressor responses were elicited from the area immediately ventral to the anterior commissure, and depressor responses followed stimulation of an area more ventral. All subnuclei showed corroborating cardiovascular responses to 20-30 nl microinjection of sodium glutamate. Taken together, these data provide substantial evidence to indicate that the BST, particularly at more rostral areas, consists of a medial pressor area, a lateral depressor area, and a ventral area with both pressor and depressor zones.

Animals↗
PubMed2006

Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling.

A growing body of evidence implicates Wnt signaling in the control of angiogenesis. To better understand the role of the Wnt/beta-catenin pathway in endothelial cells (EC), we examined endogenous signaling activity and signaling component expression in vascular cells. We observed stabilization of cytosolic beta-catenin and activation of a T-cell factor (TCF) -luciferase promoter, hallmarks of canonical Wnt signaling activity, in cultured EC. This activity was increased in subconfluent EC, which are known to display characteristics of angiogenic EC, compared with confluent EC, which have a more differentiated phenotype. Endogenous TCF activity was inhibited by transfection with a secreted inhibitor of canonical Wnt signaling. A systematic analysis of Wnt, Fzd, SFRP, and Dkk gene expression in human EC (cultured and freshly isolated), smooth muscle cells (cultured), and aorta demonstrated that numerous Wnt signaling components are expressed by vascular cells. We conclude that Wnt signaling components are expressed and active in cultured EC.

Cell Movement↗
PubMed1991

Lethal(1) aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster.

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tuml and tu(1)Szts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from "autoimmune responses" or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.

Animals↗
PubMed1993

Peptides of 23 residues or greater are required to stimulate a high affinity class II-restricted T cell response.

Helper T cells recognize fragments of antigen bound to the class II molecules on the surface of antigen-presenting cells. Naturally processed antigenic fragments have been isolated from the class II molecules and shown to be heterogeneous in length, ranging from 13 to 25 residues, and to vary at both the N and C termini. A 15-residue peptide in an extended conformation is predicted to fit in an open peptide-binding cleft of the class II molecules. Thus, the longer peptides observed bound to class II presumably have regions which reside outside the cleft. It is not known if the additional length contributes significantly to T cell activation. We have carried out a systematic analysis of the antigenicity of peptides of increasing length beyond the minimally defined T cell antigenic peptide. Here we show that the full functional activities of peptides representing the major antigenic determinant of the protein antigen, cytochrome c, minimally require that the peptides be 23 amino acids long. The long peptides do not require processing and are presented by purified class II molecules incorporated into synthetic membranes, indicating that such peptides associate directly with class II and require no additional cellular machinery for presentation. We also show that a hybrid peptide, 51 residues in length, containing a 29-residue cytochrome c peptide and a "promiscuous" peptide of tetanus toxoid, is more antigenic than the 23-residue peptide alone and significantly, does not require processing. Thus, the additional peptide length, although not predicted to bind in the peptide-binding groove of the MHC class II molecule, has a significant impact on the ability of the peptides to stimulate T cell responses maximally.

Amino Acid Sequence↗
PubMed1997

Comparison of cytotoxic T lymphocyte responses induced by peptide or DNA immunization: implications on immunogenicity and immunodominance.

To study the mechanisms that influence the immunogenicity and immunodominance of potential cytotoxic T lymphocyte (CTL) epitopes, we conducted a systematic analysis of the CTL response raised in HLA-A*0201/Kb (A2/Kb) transgenic mice against the viral antigen, hepatitis B virus polymerase (HBV pol). From a pool of 26 nonamer peptides containing the HLA-A*0201-binding motif, we selected A2-binding peptides, immunized A2/Kb animals, and tested the CTL raised against the peptide for recognition of HBV pol transfectants. Of nine immunogenic CTL epitopes, only four were recognized on HBV pol transfectants, whereas the other five were cryptic. Characterization of the peptide-specific CTL lines indicated that crypticity may result from either poor processing or low T cell receptor (TCR) avidity. To identify the immunodominant epitopes, we determined the CTL specificities induced in A2/Kb animals in response to priming with HBV pol cDNA. We obtained a response against three epitopes that were contained with the set of four epitopes recognized by peptide-specific CTL on HBV pol transfectants. Comparative analysis of cDNA priming and peptide priming revealed, therefore, the presence of a subdominant epitope. We conclude that for the HBV pol antigen, the repertoire of CTL specificities is shaped by major histocompatibility complex class I peptide binding capacity, antigen processing, and TCR availability.

Amino Acid Sequence↗
PubMed1995

Temperature-gradient gel electrophoresis as a screening tool for polymorphisms in multigene families.

Information about sequence variability between different copies of a multigene family is indispensable for understanding the evolutionary mechanisms acting on multigene families. However, their high copy number has been a major obstacle to systematic analysis. Exemplified by the internal transcribed spacer 1 (ITS1) of the rDNA in Drosophila melanogaster, it is shown how temperature gradient gel electrophoresis (TGGE) can be used to study sequence polymorphisms in a multigene family. Experimental conditions influencing the melting behavior of the ITS1 fragment are discussed as well as discrepancies between observed and calculated melting patterns.

Animals↗
PubMed2005

Structural factors determining DNA length limitations in conformation-sensitive mutation detection methods.

Numerous mutations and polymorphisms in human genes remain to be identified using reliable methods. Of the available mutation scanning methods those dependent on structural change-induced mobility shifts are highly effective. Their efficiency is, however, DNA length-sensitive and the reasons for that are poorly understood. In this study, we explain why scanning genes for mutations is less effective in longer DNA fragments, and reveal the factors which are behind this effect. We have performed a systematic analysis of the same sequence variants of exon 11 of the BRCA1 gene in DNA fragments of three different lengths using the combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis (DA) by capillary electrophoresis (CE). There are two major structural factors responsible for the reduced mutation detection rate in long amplicons. The first is increased contribution from other secondary structure modules and domains in longer fragments, which mask the structural change induced by the mutation. The second is higher frequency of single-nucleotide polymorphisms (SNPs) including common polymorphisms in longer fragments. This makes it necessary to distinguish the structural effect of the mutation from that of each polymorphic variant, which is often difficult to achieve. Taking these factors into account, an efficient scanning of genes for sequence variants by conformation-sensitive methods may be performed.

DNA↗
PubMed1996

Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo.

For the systematic analysis of various clinical and molecular aspects of hepatitis B virus (HBV) infection, an experimental small animal system of HBV infection would be a great advance. The susceptibility to HBV infection, therefore, of hepatocytes from the tree shrew species tupaia belangeri was studied in vitro and in vivo. Primary hepatocytes isolated from livers of tupaias can be reproducibly infected with HBV. In vitro infection results in viral DNA and RNA synthesis in hepatocytes and secretion hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) into culture medium. Tupaias can also be infected with HBV in vivo, resulting in viral DNA replication and gene expression in tupaia livers. Similar to acute, self-limited hepatitis B in humans HBsAg is rapidly cleared from serum, followed by seroconversion to anti-HBe and anti-HBs. These data clearly tht HBV is infectious to tupaia hepatocytes in vitro and transiently in vivo. Tupaias, therefore, may become a useful model for the experimental analysis of various molecular and clinical aspects of HBV infection, including the significance of HBV quasispecies, the steps involved in hepatocarcinogenesis as well as the evaluation of various antiviral strategies.

Animals↗
PubMedDate not supplied

Untangling Gordian knots: improving tuberculosis control through the development of 'programme theories'.

We argue that if the lessons from tuberculosis control programmes are to be drawn effectively then a more nuanced understanding is needed that takes account of the complex health system environment within which they sit. We suggest that a conceptual framework that draws upon the World Health Organization's DOTS strategy can be harnessed to assist the systematic analysis of programmes in a way that links this vertical, disease specific strategy to horizontal health system factors so that comparisons can be made. This multi-disciplinary, multi-method approach to the evaluation builds upon the work of others including Pawson and Tilley and their 'programmes theories'. This work has informed the application of an evaluation toolkit which has been successfully applied in a number of settings and assisted in the sustainable implementation of a DOTS strategy in Russia.

Directly Observed Therapy↗
PubMed2006

BMPR2 gene rearrangements account for a significant proportion of mutations in familial and idiopathic pulmonary arterial hypertension.

Mutations of the BMPR2 gene predispose to pulmonary arterial hypertension (PAH), a serious, progressive disease of the pulmonary vascular system. However, despite the fact that most PAH families are consistent with linkage to the BMPR2 locus, sequencing only identifies mutations in some 55% of familial cases and between 10% and 40% of cases without a family history (idiopathic or IPAH). We therefore conducted a systematic analysis for larger gene rearrangements in panels of both familial and idiopathic PAH cases that were negative on sequencing of coding regions. Analysis of exon dosage across the entire gene using Multiplex Ligation-dependent Probe Amplification identified nine novel rearrangements and enabled full characterization at the exon level of previously reported deletions. Overall, BMPR2 rearrangements were identified in 7 of 58 families and 6 of 126 IPAH cases, suggesting that gross rearrangements underlie around 12% of all FPAH cases and 5% of IPAH. Importantly, two deletions encompassed all functional protein domains and are predicted to result in null mutations, providing the strongest support yet that the predominant molecular mechanism for disease predisposition is haploinsufficiency. Dosage analysis should now be considered an integral of part of the molecular work-up of PAH patients.

Bone Morphogenetic Protein Receptors, Type II↗