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At least 307 records · Page 17Linked to original sources
PubMed1999

Site-directed mutagenesis analysis of draTG genes and their downstream region from Azospirillum brasilense Yu62.

draT and draG genes are involved in posttranslational regulation of nitrogenase activity of Azospirillum brasilense Yu62. Both genes and their downstream region were mutagenized by Kmr cassette insertions. Analysis of mutations introduced into the draTG region on the A. brasilense Yu62 chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Analysis of mutations introduced into the draTG downstream region (the mutagenized site is about 2 kb downstream from draG) showed that the mutant had higher nitrogenase activity than the wild strain while growing in nitrogen-free medium and medium with 2 mmol/L ammonium. These results reveal that there is no gene required for nitrogen fixation in this mutagenized region, but it is possible that there are genes which play a role in regulating nitrogen fixation. The results of monitoring the expression of transcriptional nifH-lacZ gene fusion in the mutant YZ4 showed that the transcriptional regulation of nif gene in the mutant YZ4 was the same as that in the wild type.

Azospirillum brasilense↗
PubMed2001

Two distal downstream enhancers direct expression of the human apolipoprotein E gene to astrocytes in the brain.

Two distal downstream enhancers controlling astrocyte expression of the human apolipoprotein E (apoE) gene in the brain were identified by analysis of transgenic mice generated with various constructs of the apoE/C-I/C-IV/C-II gene cluster. In wild-type mice, the highest overall levels of apoE mRNA were found in astrocytes in the glomerular layer of olfactory bulbs and in Bergmann glia in the cerebellum. This pattern of expression was reproduced in transgenic mice expressing the entire human apoE gene cluster and also in transgenic mice expressing specific enhancer segments within the cluster. Expression of the human apoE transgene at these sites was specified by two enhancer domains: one enhancer is located 3.3 kb downstream of the apoE gene, and a duplication of this sequence is located 15 kb downstream of the apoE gene. Astrocyte enhancer activity was contained within 620 and 619 bp segments of these domains that show subtle differences in regional expression. In the absence of these distal enhancers, the apoE gene was not expressed in astrocytes. The relatively high levels of apoE expression at specific sites in the olfactory bulb and cerebellum suggest the presence of unique regulatory signals at these locations that may reflect common cellular properties and apoE gene functions. The localization of the two astrocytic enhancers reveals an unexpected complexity in the control of apoE production that is essential to understanding apoE function in the brain.

Animals↗
PubMed2002

[A methodological study of measuring quantitatively turbulent shear stree downstream of mitral stenosis in vivo using Doppler echocardiography].

In order to establish a method for measuring quantitatively turbulent shear stress (TSS) downstream of mitral stenosis in vivo based on Doppler echocardiography and computer-aided image analysis, we used doppler echocardiography to record the spectrum of flow velocity downstream of mitral valve at several locations in normal persons and in patients with mitral stenosis. With the computer-aided analysis of spectrum images, the magnitude of TSS was measured at the locations. The results demonstrate that no matter how severe the mitral stenosis is, the TSS and relative turbulent intensity(Irel) at the central locations of jet are lower than those at the marginal ones. A significant difference in the quantitative items of TSS, Irel and flow field uniformity between normal persons and patients with varying-degree of mitral stenosis was noticed (P < 0.05). There was a significant correlation between these items and effective orifice area (EOA), and we found that the smaller EOA is, the more severe the extent of stenosis is and the greater the magnitude of both TSS and Irel are, and that the highest magnitude of TSS is focused on the marginal area of jet. These results indicate that there is an obvious correlation between TSS(measured by Doppler echocardiography combined with computer-aided image analysis) and flow field uniformity. They can coincidently reveal the hemodynamic changes resulting from mitral stenosis of varied severeness, implying that our method could exactly depict the magnitude of TSS downstream of mitral stenosis in vivo and is non-invasive and good for anti-disturbance. The method can be used to analyze quantitatively TSS in the flow field of heart valve in patients with valvular diseases.

Adult↗
PubMed2002

[Mutations in beta'-subunit of the Escherichia coli RNA-polymerase influence interaction with downstream duplex DNA in the elongation complex].

RNA polymerase (RNAP) exhibits absolute processivity being capable of synthesizing RNA 10(3)-10(5) nucleotides in length without breaking contact with the DNA template. Stability of the elongation complex is thought to depend, in particular, on the RNAP-DNA interactions downstream along the run of transcription. We studied the effects of several deletions and insertions in the RNAP beta'-subunit N-terminal region, which presumably interacts with the downstream duplex DNA in the elongation complex. Most of the mutations obtained led to gross defects in RNAP assembly and disturbed catalytic activity of the enzyme. The mutations reduced stability of both promoter and elongation complexes, probably because they altered the contacts between RNAP and the downstream duplex DNA.

Amino Acid Sequence↗
PubMed2003

Immunohistochemical expression of cyclooxygenase isoenzymes and downstream enzymes in human lung tumors.

PURPOSE: Prostanoids are important mediators of pulmonary vaso- and bronchotone regulation and strongly influence inflammatory reactivity. The product of cyclooxygenase (Cox), prostaglandin H(2), is further metabolized via downstream enzymes into the different effective metabolites. The specific cellular equipment with certain downstream enzymes crucially determines the cellular reactivity by generation of functionally different prostanoid metabolites. EXPERIMENTAL DESIGN: To elucidate the role of arachidonic acid metabolism via the cyclooxygenase pathway in different human lung tumors, expression of cyclooxygenase isoenzymes (Cox-1 and Cox-2) and downstream enzymes of prostanoid metabolism was investigated in human non-small cell lung cancer and normal human lung tissue by immunohistochemical techniques. RESULTS: In comparison to strong Cox-1 reactivity in airways and endothelial cells of normal lung specimens, only 4 of 15 adenocarcinomas showed infrequent Cox-1 expression. All lung cancer specimens displayed an increased Cox-2 immunostaining pattern with strong reactivity in adenocarcinomas and lower reactivity in squamous cell carcinomas. Adenocarcinomas and squamous cell carcinomas were also positive for thromboxane A(2) synthase, prostaglandin D(2) synthase, and prostaglandin E(2) synthase, but not for prostacyclin synthase. Endothelial cells of vessels found within or near the tumor show extensive immunostaining of Cox-2 and thromboxane A(2) synthase, whereas endothelial cells of normal lung specimens, in contrast, expressed Cox-1 and prostacyclin synthase. CONCLUSIONS: We conclude that non-small cell lung cancer shows a specific Cox-/downstream-enzyme expression pattern, which is specifically altered in lung tumor cells and tumor supplying vessels in contrast to normal lung tissue. This may have major impact on tumor progression and tumor-associated inflammation via an altered prostanoid metabolism with consecutive tumor-associated blood flow distribution.

Adenocarcinoma↗
PubMed2005

[Design and adjustment of a hydrodynamic model of turbulent flow separation area for in vitro experiment on the downstream of tubal stenosis].

This paper reports the establishment of a model of turbulent flow separation area for experiment on the downstream of tubal stenosis, and adjust it to cooperate with the velocity and turbulent shear stress (TSS) detection by means of the particle image velocimetry (PIV), and with the pressure detection of pressure sensor in vitro. The velocity, TSS and wall pressure characteristics of the downstream of tubal stenosis were quantitatively detected and analyzed via the PIV and pressure sensor. And the hydrodynamic characteristics of the velocity, TSS and wall pressure in the flow separation area were primarily understood. The model can cooperate smoothly with the PIV and pressure sensor to detect the velocity, TSS and wall pressure; there exist low velocity, low TSS and low pressure in the flow separation area downstream of tubal stenosis.

Blood Flow Velocity↗
PubMed2006

[Model experiment and numerical simulation of marginal pressure in the flow field downstream of the stenostic vessel in vitro].

For the defects of interpreting the relationship between the distribution and severity of vascular stenostic disease and the low shear stress, the study focus on and explore the rules of the pressure distribution and variation downstream of the stenostic vessel with the numerical simulation and the hydrodynamic model of turbulent flow separation area for in vitro experiment. The results reveal that there is a good compatibility between the model experiment and the numerical simulation; there exists a marked pressure decrease downstream of the stenosis. And the distribution area of low pressure is similar to the characteristic distribution of the stenostic vessel diseases. The study suggested that the numerical simulation can be applied in the study of micro-flow field; the presure distribution and variatioin downstream of the stenostic vessel maybe another factor to trigger the pathogenesis of vessel stenostic diseases.

Blood Flow Velocity↗
PubMed1993

A far-downstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice.

The human apolipoprotein (apo) E and apoC-I genes are located 5 kilobases apart in the same transcriptional orientation on chromosome 19, and they are expressed at high levels in the liver with lower levels of expression in selected other tissues. Analysis of a series of overlapping human apoE and apoC-I genomic fragments in transgenic mice revealed that the expression of these transgenes in the liver requires a common cis-acting regulatory domain. This hepatic control region (HCR) was localized to a 764-base pair region that is located about 18 kilobases downstream of the apoE promoter and about 9 kilobases downstream of the apoC-I promoter. All the transgenic animals that had been prepared with a construct that contained this region had relatively high levels of transgene expression in the liver, whereas constructs that lacked this region showed no expression in the liver. In situ hybridization studies showed that the HCR directed apoE and apoC-I transgene expression in hepatocytes. When the HCR from the apoE/C-I gene locus was ligated proximal to a human apoA-IV gene fragment, which is not normally expressed in the liver, the resulting apoA-IV/HCR fusion construct was expressed at high levels in the liver, indicating that the HCR could direct high level liver expression of a heterologous promoter/gene construct. Expression of the apoE transgene in the liver and kidney, and perhaps other tissues, required the presence of a nonspecific proximal enhancer element in the apolipoprotein E gene promoter, located between 161 and 141 bp relative to the transcription initiation site. However, the proximal apoE gene promoter, including this enhancer element, contained no sequences capable of directing hepatocyte expression in the absence of the HCR. Thus, the far-downstream HCR appears to contain all of the sequences necessary for determining high level liver-specific gene expression.

Animals↗
PubMed1995

Cell type-specific activity of the N-myc promoter in human neuroblastoma cells is mediated by a downstream silencer.

The N-myc oncogene is actively transcribed in many neuroblastoma tumors, but is not expressed in mature, normal tissue of any type. Chloramphenicol acetyl transferase (CAT) assays of constructs containing N-myc sequence transfected into N-myc expressing LA-N-5 neuroblastoma cells or non-expressing HeLa carcinoma cells have revealed a 201 base pair (bp) regulatory region mediating the cell type-specific activity of the promoter. While located downstream from 5' mRNA cap sites, the region appears to function by preventing transcriptional initiation. This downstream region is capable of suppressing promoter activity independently of position, and contains an element having 100% homology with the 9 bp consensus sequence of a transcriptional silencer found in the upstream region of the lysozyme gene. DNA gel retardation assays have shown that this sequence is involved in a specific DNA-protein interaction with nuclear extract from HeLa cells that is distinct from that occurring with extract from LA-N-5 cells. These results suggest that the N-myc promoter's cell type-specific activity is regulated by a downstream silencer, and that differential binding of regulatory protein from that present in non-expressing cells may result in the constitutive N-myc expression seen in neuroblastoma.

Base Sequence↗
PubMed1994

Protein-DNA interactions within DNase I-hypersensitive sites located downstream of the HIV-1 promoter.

We have examined by in vitro footprinting a region located downstream of the human immunodeficiency virus, type 1 (HIV-1) promoter found to be hypersensitive to DNase I digestion in vivo. Recognition sites for several constitutive or inducible DNA binding factors were identified. Three AP-1 binding sites and an AP-3-like motif were situated within the R-U5 region of the long terminal repeat. A novel purine-rich motif (5'-GAAAGC-GAAAGDD-3' (D represents G, A, or T residues)), which interacts with a nuclear factor designated downstream binding factor 1 (DBF1), and two juxtaposed Sp-1 binding sites were located in the untranslated sequence immediately downstream of the 5'-long terminal repeat. Genomic footprinting of these sequence elements in the HIV-1 chronically infected cell lines revealed that the DBF1 and Sp-1 sites are occupied in vivo. Furthermore, transient transfection assays showed that point mutations in the DBF1 binding site decreased significantly the HIV-1 basal promoter activity. Taken together, these results suggest that the DBF1 play a role in the HIV-1 transcription regulation.

Base Sequence↗
PubMed1994

Turbulent stresses downstream of three mechanical aortic valve prostheses in human beings.

High levels of turbulent stresses resulting from disturbed blood flow may cause damage to red blood cells and platelets. The purpose of this study was to evaluate the spatial distribution and temporal development of turbulent stresses downstream of three mechanical aortic valve prostheses in human subjects: the St. Jude Medical, the CarboMedics, and the Starr-Edwards silicone rubber ball. Blood velocity measurements were taken at 17 measuring points in the cross-sectional area of the ascending aorta 5 to 6 cm downstream of the aortic anulus with the use of a perivascular pulsed Doppler ultrasound system. Turbulence analysis was done for each of the 17 measuring points by calculating the radial Reynolds normal stresses within 50 msec overlapping time windows during systole. By coordinating the calculated Reynolds normal stress values for each time window and for all measuring points, computerized two-dimensional color-coded mapping of the turbulent stress distribution during systole was done. For the St. Jude Medical valves the highest Reynolds normal stress (27 to 63 N/m2) were found along the central slit near the vessel walls. The temporal development and spatial distribution of Reynolds normal stresses for the CarboMedics valves were quite similar to those of the St. Jude Medical valves with maximum Reynolds normal stress values ranging from 19 to 72 N/m2. The typical Reynolds normal stress distribution for the Starr-Edwards silicone rubber ball valves was asymmetric, revealing the highest Reynolds normal stresses (11 to 56 N/m2) at various locations in the annular region between the ball and the vessel wall. The spatial distribution and temporal development of turbulent stresses downstream of the three investigated mechanical aortic valve prostheses correlated well with the superstructure of the valves. The maximum Reynolds normal stresses for the three valve types were in the same order of magnitude with exposure times sufficient to cause sublethal damage to red blood cells and platelets.

Adult↗
PubMed1996

Shortening and superlon-wrapping the downstream tube can increase the useful range of the blood warmer.

BACKGROUND: Hypothermia occurs frequently in clinical anesthesia. Many apparatus were introduced to keep intravenous fluid warm. But at low to medium flow rates little effects have been achieved because of heat lost along the tube. Superlon, an industrial heat-preserving material widely used in air conditioning to prevent temperature change, has never been used in the blood warmer. METHODS: The study was performed in three groups group I using standard (120 cm) unwrapped tube (Animac AM-4 set), group II using a shortened downstream tube (60 cm), and group III using the downstream tube which was further wrapped with superlon (Super LTD). The blood warmer used was identical in all 3 groups. We recorded the fluid temperatures at different points downstream at various flow rates. The values were then compared among groups. RESULTS: The fluid temperatures before entering the warmer were around 20 degrees C and 37 degrees C at the 2 cm post-warmer points at various flow rates. The end point temperatures rose with flow rates and were the highest in group III and lowest in group I at each flow rate (p < 0.01). In group III the temperature could be kept above 32 degrees C at a very low flow rate (150 ml/h) and above 35 degrees C at 300 ml/h. In group I, 1,000 to 2,000 ml/h were needed to keep the same temperature. CONCLUSIONS: These refinements increase the useful range of the Animac AM-4 warmer and make it useful in pediatric rapid fluid resuscitation and current fluid management. Moreover, they are easy to set up, inexpensive, and can be applied to any kind of blood warmers.

Anesthesia↗
PubMed1997

Virtually identical enhancers containing a segment of homology to murine 3'IgH-E(hs1,2) lie downstream of human Ig C alpha 1 and C alpha 2 genes.

We have isolated sequences downstream of human Ig C alpha 1 and C alpha 2 genes and have identified two enhancers in these regions. One enhancer is located approximately 9 kb downstream of C alpha 1, and the second enhancer is located approximately 11 kb downstream of C alpha 2. These approximately 1.6-kb enhancers are virtually identical to each other except for varying numbers of a approximately 53-bp motif. The C alpha 2-associated enhancer contains four copies of this motif in tandem, whereas the C alpha 1-associated enhancer has only a single copy. Within the human enhancers is a 177-bp segment that is homologous to a 191-bp segment of one of four enhancers from the 3' regulatory region of murine (and rat) DNA, namely 3'IgH-E(hs1,2). Like the murine and rat enhancers, both human enhancers are flanked by inverted repeats; furthermore, the human enhancers generally appear to be inverted with respect to each other. The evolutionarily conserved region of homology has substantial core enhancer activity. Contained within this region are the single octamer and one copy of the approximately 53-bp motif, both of which contribute to the activity of the full-length enhancer. A comparison of the DNA sequences and the results of transient transfection assays imply that the human C alpha-associated enhancers may be regulated (in part) differently than the murine enhancer 3'IgH-E(hs1,2).

Animals↗
PubMed1997

Cloning and sequencing of draTG genes and their downstream region of Azospirillum brasilense Yu62.

An 8-kb fragment was cloned by probing the gene library of Azospirillum brasilense Yu62 with the 4.0-kb draTG fragment of A. brasilense Sp7. DNA hybridization of this fragment demonstrated that draTG genes were located in a 3.0-kb EcoR I-Kpn I fragment, and were contiguous to the nifH gene. This 3.0-kb fragment was completely sequenced on both strands. Sequence analysis of the fragment revealed that it included the full-length draTG genes and two ORFs downstream of draG (ORF3 and incomplete ORF4). The draTG and downstream ORF3 are presumed to be cotranscribed as a single operon. Promoter element analysis of the sequenced region showed that there were some elements of the sigma 54-dependent promoter (DPEs and UASs) in the upstream region of draG and ORF3. This suggests that the draG and ORF3 might be transcribed independently in addition to being cotranscribed with draT. Both the DNA and amino acid sequences of the draTG genes from A. brasilense Yu62 were compared with those of other nitrogen-fixing bacteria. The results showed the draTG genes were highly conservative. There were only a few changes among strains and/or species. Homology analysis of ORF revealed that the ORF3 immediately downstream of draG was homologous not only with the ORF at the same position in Rhodospirillum rubrum and A. lipoferum, but also with the ORF14 of Azotobacter vinelandii and the ORF4 showed extensive similarity to yafJ of Escherichia coli.

Amino Acid Sequence↗
PubMed1998

Medtronic Hall versus St. Jude Medical mechanical aortic valve: downstream turbulences with respect to rotation in pigs.

BACKGROUND AND AIMS OF THE STUDY: Turbulences downstream of mechanical aortic valves are known to contribute to most valve-related complications such as thrombosis, embolization or damage to blood components. In vitro studies have demonstrated the impact of the orientation of prostheses on transvalvular energy loss. This study evaluates the influence of valve orientation on turbulences in the supravalvular aorta in pigs. METHODS: A rotation device which could carry a Medtronic Hall (MH) or St. Jude Medical (SJM) aortic valve prosthesis (23 mm) was constructed and implanted into four healthy pigs. Turbulence measurements using pulsed Doppler ultrasonography were carried out 3 cm downstream of the valve, while the prostheses were rotated in 45 degrees steps. Reynold's normal stress values (RNS) were calculated as key markers for turbulent stresses. RESULTS: Turbulences downstream of MH and SJM valves demonstrated a significant change with rotation. The MH valve showed minimum RNSmean values with orientation of the large orifice to the right posterior aortic wall, which is the area of highest velocities during ejection. With this orientation, aortic flow almost complied with physiologic conditions. Increase of turbulence was observed with any other position. The SJM valve revealed significant turbulent flow at any orientation. Minimum RNSmean values were also measured with one orifice facing the right posterior wall of the aorta. CONCLUSION: With optimum orientation (major orifice facing the right posterior aortic wall) the MH valve matches the aortic flow pattern to near-normal physiology. The flow patterns of the SJM valve are less susceptible to rotation, but cannot attain the optimum RNS values of the MH prosthesis.

Animals↗
PubMed1999

Stabilization of a proteolytically sensitive cytoplasmic recombinant protein during transition to downstream processing.

The influence of aeration and glucose feeding on the stability of recombinant protein A in Escherichia coli during the transition period from a fed-batch cultivation to downstream processing was studied. Neither interruption of the feeding under aerobic conditions nor anaerobic conditions in presence of glucose could stabilize protein A completely and the intracellular ATP pool did not decrease to less than 0.75-1 mM by this treatment. On the other hand, the absence of both oxygen and glucose resulted in a decrease of the ATP pool to less than 0.5 mM and almost complete stabilization of protein A. The decrease of ATP was more severe when sulfite was used instead of nitrogen gas to create anaerobic conditions in presence of glucose. This also resulted in nearly complete stabilization of protein A, which might be explained by an inhibiting effect of sodium sulfite on fermentation. Therefore, protein stabilization and decrease of the ATP pool were correlated in experiments in vivo. The concentrations of ADP and AMP increased during starvation and may also play a role in stabilization of the protein in vivo. ATP may be a limiting factor of proteolysis also during further steps of downstream processing. Its concentration decreases by 80-90% during harvesting and centrifugation of biomass and even further during disruption of cells. However, neither addition nor regeneration of ATP in cell disintegrate was enough to restore degradation of protein A, indicating that an additional factor limits proteolysis in vitro.

Adenosine Triphosphate↗
PubMed1998

Platelet-activating factor is a downstream messenger of kainate-induced activation of mitogen-activated protein kinases in primary hippocampal neurons.

Excitatory amino acids transduce physiological and pathological signals to neurons. Similarly, the neuroactive lipid platelet-activating factor (PAF) has been implicated in modulating long-term potentiation and neuronal survival. Excitatory amino acids and PAF have been shown to increase mitogen-activated protein (MAP) kinases in different cell types. Here, we have investigated the similarities and differences between PAF and kainate in activating MAP kinases in primary hippocampal neurons in vitro. Extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 kinases were activated by kainate or PAF in hippocampal neurons. This activation was blocked by the receptor antagonists CNQX and BN 50730 for kainate and PAF, respectively. The PAF receptor antagonist BN 50730 also blocked kainate activation. CNQX had no effect on PAF activation of the kinases, indicating that PAF is downstream of kainate activation. Coapplication of submaximal concentrations of PAF and kainate resulted in a less than additive activation, suggesting similar routes of activation by the two agonists. Both CNQX and BN 50730 blocked kainate-induced neurotoxicity. These results indicate that PAF and kainate activate similar kinase pathways. Therefore, PAF acts downstream of the kainate subtype of glutamate receptors, and when excessive receptor activation takes place, this bioactive lipid may contribute to neuronal cell death.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2006

Identification of Flt3 internal tandem duplications downstream targets by high-throughput immunoblotting protein array system.

The receptor tyrosine kinase Flt3 plays an important role in proliferation and survival of hematopoietic cells. Flt3 is the most frequently mutated gene (20-30%) in cases of acutemyeloid leukemia (AML). The majority of Flt3 mutations are internal tandem duplications (ITD) in the juxtamembrane domain of Flt3 receptor. This mutation results in the constitutive activation of STAT5 and Ras/mitogen-activated protein kinase pathways, leading to the aberrant growth of AML cells. In this study, to better understand the mechanisms of Flt3-ITD to the downstream pathways, a high-throughput immunoblotting protein array system was employed. As a result, c-Jun and c-Raf were markedly induced, suggesting that these factors are functional downstream targets of Flt3-ITD.

Animals↗