Identification of an N-terminal region of sigma 54 required for enhancer responsiveness

Sigma 54 associates with bacterial core RNA polymerase and converts it into an enhancer-responsive enzyme. Deletion of the N-terminal 40 amino acids is known to result in loss of the ability to respond to enhancer binding proteins. In this work PCR mutagenesis and genetic screens were used to identify a small patch, from amino acids 33 to 37, that is required for proper response to activator in vivo. Site-directed single point mutants within this segment were constructed and studied. Two of these were defective in responding to the enhancer binding protein in vitro. The mutants could still direct the polymerase to bind to DNA and initiate transient melting. However, they failed in directing activator-dependent formation of a heparin-stable open complex. Thus, amino acid region 33 to 37 includes critical activation response determinants. This region overlaps the larger leucine patch negative-control region, suggesting that anti-inhibition and positive activation are closely coupled events.     

Comparison of the effects of ischemic preconditioning and surgical delay on pedicled musculocutaneous flap survival in a rat model

Both surgical delay (SD) and ischemic preconditioning (IP) have been shown to be effective in improving the survival of pedicled musculocutaneous flaps. The goal of our study was to determine the effects of IP and SD, separately and together, on the survival of pedicled transverse rectus abdominis musculocutaneous (TRAM) flaps in a rat model. Thirty-two male Sprague-Dawley rats were divided into four groups of 8 rats each: (1) control, (2) 2-week SD, (3) IP, and (4) SD plus IP. A TRAM flap was elevated in each rat. Flap viability was assessed on the fifth postoperative day by computerized video planimetry. Mean area of flap survival was compared between the control, IP, SD, and SD plus IP groups using analysis of variance and Student's t-test. Improvement in surface area survival was seen in musculocutaneous flaps subjected to IP, SD, and SD plus IP compared with the control. IP and SD improved survival 1.3 and 1.4 times the control area respectively. Differences between treatment and control flaps were statistically significant (p < 0.04). In addition, the combination of SD plus IP improved survival by 1.8 times, which is statistically different from controls and from either technique individually (p < 0.002). IP and SD have similar efficacy in improving survival in this musculocutaneous flap model. The effects of IP and SD appear to be additive. The advantage of IP over SD is that IP can be performed during the same operative session as the flap elevation and only adds 1 hour to the surgical procedure.     

Rat pachytene spermatocytes down-regulate a polo-like kinase and up-regulate a thiol-specific antioxidant protein, whereas sertoli cells down-regulate a phosphodiesterase and up-regulate an oxidative stress protein after exposure to methoxyethanol and methoxyacetic acid

2-Methoxyethanol (ME) and its metabolite, methoxyacetic acid (MAA), produce testicular lesions characterized by pachytene spermatocyte degeneration. To understand the molecular basis of this action on meiotic prophase cells, mRNA differential display was used to identify gene expression changes in control and treated cells. When pachytene spermatocytes were cultured with 5 mM ME or 5 mM MAA for 24 h, two complementary DNAs (cDNAs), of 557 nucleotides (clone 5) and 388 nucleotides (clone 6), were up-regulated; and a cDNA of 648 nucleotides (clone 1) was down-regulated. The altered expression pattern shown by differential display was confirmed by Northern blotting. Sequence analyses indicate that clones 1 and 6 have 83% and 79% homology at the nucleotide level to a polo-like kinase and a thiol-specific antioxidant, respectively. Clone 5 shows no homology to any known gene in the database. Messenger RNAs (mRNAs) encoding the thiol-specific antioxidant and clone 5 are up-regulated within 30 min of the addition of MAA, whereas the polo-like kinase mRNA decreased to undetectable levels after 6 h. Changes in Sertoli cell gene expression were also detected when Sertoli cells were cultured with 5 mM ME or MAA for 24 h. Two cDNAs, of 367 nucleotides (clone 2) and 676 nucleotides (clone 3), were up-regulated; and a cDNA of 538 nucleotides (clone 4) was down-regulated. Homology searches revealed that clones 3 and 4 have 90 and 91% homology at the nucleotide level to an oxidative stress protein and a phosphodiesterase (PDE), respectively. Northern blotting confirmed the differential display expression pattern for the PDE and oxidative stress protein. mRNAs for the latter were induced within 30 min, and PDE mRNAs were down-regulated within one h, after the addition of MAA. To determine whether the changes in gene expression seen with cells in culture also occur in vivo, rats were given a single oral dose of 250 mg/kg ME or MAA. After 24 h, total testis RNAs from control and treated rats were purified and hybridized. The expression patterns seen in vivo for the differentially expressed cDNAs were identical to those seen in vitro. We conclude that, although pachytene spermatocytes seem to be selectively affected by ME and MAA, changes in gene expression are also detected in Sertoli cells, suggesting that the action(s) of ME or MAA on pachytene spermatocytes could be mediated through Sertoli cells.     

The effect of prolonged clamping and vascular stasis on the patency of arterial and venous microanastomoses

This study was designed to investigate the influence of the volume irradiated on the probability of ureteral complications and to provide data for volume modeling. One hundred thirty-four purpose-bred beagle dogs received single intraoperative doses of 6 MeV electrons ranging from 12 to 54 Gy to three lengths of ureter: 2, 4 or 8 cm. The response was evaluated by excretory urography. The ED50 was 21.9 Gy (95% CI 13.3-30 Gy) for 8 cm 3 years after treatment. The estimated ED50's were greater than 43 Gy for 4 cm and 85 Gy for 2 cm. Reducing the length of ureter irradiated from 8 cm to 4 cm increased the ED50 for ureteral dilation by at least a factor of 2, while reduction from 8 cm to 2 cm increased the ED50 by at least a factor of 4. The ED50 for renal injury secondary to stenosis was 30.5 Gy (95% CI 17.2-232 Gy) when an 8-cm field was irradiated. There was a significant effect of volume irradiated on the frequency of ureteral stenosis. Reducing the length of ureter included in the treatment field should allow delivery of higher doses to tumors without increased complications.     

Effect of subcutaneous injection of granulocyte-macrophage colony stimulating factor (GM-CSF) on healing of chronic refractory wounds

OBJECTIVE: To assess the effect of granulocyte macrophage colony-stimulating factor (GM-CSF) on healing of chronic wounds. DESIGN: Prospective open study. SETTING: 1 cancer hospital and 2 University hospitals, Pakistan. SUBJECTS: 35 patients with chronic wounds (duration 3 months or longer) that had not responded to standard treatment. Patients with malignant ulcers were excluded. INTERVENTION: GM-CSF 10 microg/cm2 was injected subcutaneously along the edges and base of the wound. The treatment was given only once and patients were followed weekly for a minimum of six weeks. MAIN OUTCOME MEASURES: Changes in size, shape, depth, edges and base of the wound, amount and quality of the granulation tissue, and type of discharge. RESULTS: Six patients were excluded from the analysis, 4 who died early of underlying disease and were not evaluable, and 2 who were excluded when histological examination of the wound showed that it was malignant. Although various types of wounds were studied, most (n = 10, 34%) were postoperative. 23 of the wounds were over 2 cm in diameter (mean 4.8 x 4.6 cm) with thin granulation tissue, and almost half were infected. Nine of the 29 wounds healed completely within six weeks while another 11 decreased in size by more than 50%; 7 patients responded slightly. Only two wounds showed no evidence of healing during the observation period. More than half of the 20 wounds that responded had healed by three weeks. Response did not correlate with any clinical variable including the presence of infection. Toxicity was negligible. CONCLUSIONS: We conclude that subcutaneous injection of a single dose of GM-CSF may induce healing in refractory chronic wounds. Trials are necessary to validate these initial observations and to decide the optimal dose and route, and whether any additional benefit may be derived from repeated injections.     

Reversal of vinblastine transport by chlorpromazine in membrane vesicles from multidrug-resistant human CCRF-CEM leukaemia cells

In this study, anti-basic fibroblast growth factor (anti-bFGF) antibody was used to determine whether the mitogenic effect of angiotensin II in vivo could be blocked by neutralizing bFGF in the vessel wall. Animals, divided into six experimental groups, were given (1) angiotensin II, (2) angiotensin II + anti-bFGF antibody, (3) angiotensin II + normal goat IgG (ngIgG), (4) anti-bFGF antibody, (5) ngIgG, and (6) Ringer's solution. Angiotensin II at 435 ng x kg(-1) x min(-1) was infused into rats continuously for 1 week to induce smooth muscle cell replication, and anti-bFGF antibody or ngIgG was injected intravenously 4 times over the 1-week period at a dose of 60 mg/injection. Bromodeoxyuridine (30 mg/mL) was also continuously infused during the 1-week period. The left carotid artery of all animals was balloon-injured on day 4 of the treatment, and all groups were killed for study on day 7. The results showed that angiotensin II significantly stimulated smooth muscle replication in the balloon-injured carotid artery, intact carotid artery, and three branch levels of the mesenteric vascular tree. Anti-bFGF was able to block the mitogenic effect of angiotensin II in larger vessels but not the smallest (type I) microvessels of the mesenteric arterial tree. This differential response may be attributable to the nature of the lesions in type I vessels versus larger vessels: the type I vascular lesion has a large component of proliferating macrophages, whereas the larger vessels show less injury, few macrophages, and varying levels of smooth muscle replication. Our data suggest that the vessel wall remodeling in the angiotensin II-treated larger vessels involves DNA replication that is dependent on the presence of bFGF.     

Application of exergy analysis to various psychrometric processes

The relation between work and changes in entropy generation arises from the simultaneous treatment of the first and second laws referred to as exergy (or available energy) analysis. In this paper, we discuss thermodynamic analysis of various psychrometric processes using the concept of exergy. A parametric study of each of the processes is carried out to determine the variation of second-law efficiency as a function of mass flow rate, relative humidity and temperature. Other trends such as variation of temperature with relative humidity are also shown where applicable. Irreversible losses are calculated by applying an exergy balance on each system. In this regard, an engineering equation solver (EES) programme is used, which is unique because it has built-in functions for most thermodynamic and transport properties; removing the need for approximate equations. The concept of total exergy as the sum of thermomechanical and chemical parts is employed in calculating the flow exergies for air and water vapor mixtures. It is shown for some processes investigated that an increase in the relative humidity of the incoming air stream increases second-law efficiency. We notice that a decrease in mass flow rate of fresh air (second incoming stream) in the case of adiabatic mixing decreases the second-law efficiency of the process. Also, it is shown that the mass flow rate (of both water and steam) has almost a linear relationship with relative humidity in the range investigated. Copyright © 2003 John Wiley & Sons, Ltd.     

Electroconductive performance of polypyrrole/graphene nanocomposites synthesized through in situ emulsion polymerization

The present study demonstrates a modified in situ emulsion polymerization (EP) approach convenient for the formation of polypyrrole/graphene (PPy/GN) nanocomposites with harnessed conductivities. A series of PPy/GN nanocomposites were prepared by loading different weight percent (wt %) of GN during in situ EP of pyrrole monomer. The polymerization was carried out in the presence of dodecyl benzene sulfonic acid, which acts as an emulsifier and protonating agent. The microstructures of the nanocomposites were studied by scanning electron microscopy, transmission electron microscopy, X‐ray diffraction, Fourier transform infrared, X‐ray photoelectron spectroscopy, UV–vis spectroscopy, Raman spectroscopy, photoluminescence spectroscopy and thermogravimetric analyses. The electrical conductivities of the nanocomposite pellets pressed at different applied pressures were determined using four probe analyzer. The electrical conductivities of the nanocomposites were considerably enhanced as compared to those of the individual PPy samples pressed at the same pressures. An enhanced conductivity of 717.06 S m^?1 was observed in the sample with 5 wt % GN loading and applied pressure of 8 tons. The results of the present study signify that the addition of GN in the PPy polymer harnesses both electrical and thermal properties of the polymer. Thus, PPy/GN nanocomposites with superior properties for various semiconductor applications can be obtained through direct loading of GN during the polymerization process. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41800.