Biomedical subjects
Publications and source records attributed to R Sousa.
Retrospective studies, based on autopsy findings, indicate that infiltration of the gastrointestinal tract complicates leukemia of all types and may be present from the esophagus to the rectum. This report presents what is believed to be the first reported case of a patient with chronic lymphocytic leukemia with evidence of esophageal, gastric, duodenal bulb, and colonic involvement confirmed by mucosal biopsy at endoscopy.
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BACKGROUND: Homologous blood transfusion is associated with immunosuppressive consequences. Some clinical and experimental studies have suggested an immunostimulating action of autologous blood transfusion. The aim of this paper is to ascertain the effects of either homologous blood transfusion or autologous blood transfusion on the lymphocyte subsets and cytokines in a model of intra-abdominal sepsis. MATERIALS AND METHODS: There were three study groups. Group A: 10 Wistar-Furth (WF) rats underwent cecal ligation and puncture (CLP) aimed at causing an intra-abdomial sepsis; Group B: 10 WF rats underwent CLP plus 1 ml homologous blood perioperative transfusion obtained from Fisher-344 rat while Group C: 10 WF rats underwent CLP plus 1 ml autologous blood perioperative transfusion. Changes of peripheral lymphocyte subsets, percentages of total T-lymphocytes (CD3), Helper T-lymphocytes (CD4), supressor/cytotoxic T-lymphocytes (CD8), CD4/CD8 ratio, Interleukin-2 receptor expression (IL-2R) and cytokines IL-1 and TNF-alpha were measured in peripheral blood on the preoperative, 1st, 3rd and 7th postsepsis (PO) days. RESULTS: Rats in homologous transfused group showed a decrease of %CD4 on the 3rd PO (from preoperative to 3rd PO;p < 0.01; and from 1st to 3rd PO; p < 0.05) and on the 7th PO (from preoperative to 7th PO; p < 0.05); %CD8 increased from preoperative to 3rd PO (p < 0.05), from 1st to 3rd PO (p < 0.01) and from 1st to 7th PO (p < 0.05). An initial decrease on day 1 (p < 0.01) followed by an increase on the 3rd PO (p < 0.01) with regard to IL-2R and a significant increase of IL-1 levels within the first 24h (p < 0.01). Rats in autologous transfused group showed an increase of %CD3 from preoperative to 7th PO (p < 0.05), and from 3rd to 7th PO (p < 0.01). CONCLUSIONS: We observed that homologous blood transfusions induce a greater alteration in the cellular immune response and of the cascade of cytokines than autologous transfusions. This modulates the variations of the immune response induced by sepsis.
BACKGROUND: S-adenosylmethionine (SAMe) is a forerunner of glutathione. AIMS: The aim of the present study is to ascertain this drug effect on T-lymphocytes and cytokines in an experimental model of surgical sepsis. METHODS: Rats were allotted in two groups. In the control group, rats underwent anaesthesia and laparatomy with cecal ligation and puncture (CLP). In the second group, rats underwent the same CLP and received SAMe (14 mg/kg) i.m., on the 1st (1PO) and 2nd (2PO) postoperative days. A week before surgery (PRE), on the 1PO and on the 3PO: IL-1, IL-2, IL-4, IL-6, IL-10 and TNF levels (ELISA & MoAb), and CD3, CD4, CD8 cell and IL-2R percentages (%) (flow cytometry & MoAb) were determined in peripheral blood. RESULTS: Rats receiving SAMe do not show changes of CD3%, CD4% and IL-1 levels but show a significant increase of CD8% on the 3PO, showing a significant difference with regard to controls (p < 0.01). Both groups show a similar IL-2R variation pattern: increasing on 1PO (p < 0.05) and decreasing on 3PO (p < 0.05). CONCLUSION: In sepsis: SAMe inhibits the decrease of circulating immune-active cells and the IL-1 increase. This drug seems to have effects useful in avoiding immunological alterations in sepsis, that need to be tested in humans.
Bacteriophage T7 lysozyme binds to T7 RNA polymerase (RNAP) and regulates its transcription by differentially repressing initiation from different T7 promoters. This selective repression is due in part to a lysozyme-induced increase in the KNTP of the initiation complex (IC) and to intrinsically different NTP concentration requirements for efficient initiation from different T7 promoters. While lysozyme represses initiation, once the enzyme has left the promoter and formed an elongation complex (EC) it is generally resistant to the effects of lysozyme. The mechanism by which the inhibitory effects of lysozyme are largely restricted to the initiation phase of transcription is not well understood. We find that T7 lysozyme destabilizes initial transcription complexes (ITCs) and increases the rate of release of transcripts from these complexes but does not destabilize ECs. However, if the RNA:RNAP interaction proposed to be important for EC stability is disrupted by proteolysis of the RNA-binding domain or use of templates which interfere with establishment of this RNA:RNAP interaction, the EC becomes sensitive to lysozyme. Comparison of the X-ray structures of T7RNAP and of a T7RNAP:T7 lysozyme complex reveals that lysozyme causes the C terminus of the polymerase to flip out of the active site. Experiments in which carboxypeptidase A is used to probe the lysozyme-induced exposure of the C terminus reveal a large decrease in carboxypeptidase sensitivity following transcription initiation, suggesting that interactions with the 3'-end of the RNA help stabilize the active site in a functional (carboxypeptidase protected) conformation. Thus, the resistance of the EC to lysozyme appears to be due to the consecutive establishment of two sets of RNA:RNAP interactions. The first is made with the 3'-end of the RNA and helps stabilize a functional conformation of the active site, thereby suppressing the effects of lysozyme on KNTP. The second is made with a more upstream element of the RNA and keeps the EC from being destabilized by lysozyme binding.
We have characterized the roles of the phage T7 RNA polymerase (RNAP) thumb subdomain and the RNA binding activity of the N-terminal domain in elongation complex (EC) stability by evaluating how disrupting these structures affects the dissociation rates of halted ECs. Our results reveal distinct roles for these elements in EC stabilization. On supercoiled or partially single-stranded templates the enzyme with a deletion of the thumb subdomain is exceptionally unstable. However, on linear duplex templates the polymerase which has been proteolytically cleaved within the N-terminal domain is the most unstable. The differences in the effects of these RNAP modifications on the stability of ECs on the different templates appear to be due to differences in EC structure: on the linear duplex templates the RNA is properly displaced from the DNA, but on the supercoiled or partially single-stranded templates an extended RNA:DNA hybrid makes a larger contribution to the conformational state of the EC. The halted EC can therefore exist either in a conformation in which the RNA is displaced from the DNA and forms an interaction with the RNAP, or in a conformation in which a more extended RNA:DNA hybrid is present and the RNA:RNAP interaction is less extensive. The partitioning between these competing conformations appears to be a function of the energetics of template reannealing and the relative strengths of the RNA:RNAP interaction and the RNA:DNA hybrid.
We have characterized the blocks to progression of T7 and T3 RNA polymerase transcription complexes created when a Tus protein is bound to the template. The encounter with Tus impedes the progress of the transcription complexes of either enzyme. The duration of the block depends on which polymerase is used and the orientation of Tus on the DNA. Both genuine termination (dissociation of the transcription complex) and halting followed by continued progression after the block is abrogated are observed. The fraction of complexes that terminates depends on which polymerase is used and on the orientation of the Tus molecule. The efficiency of the block to transcription increases as the Tus concentration is increased, even if the concentration of Tus is already many times in excess of what is required to saturate its binding sites on the template in the absence of transcription. The block to transcription is rapidly abrogated if an excess of a DNA containing a binding site for Tus is added to a transcription reaction in which Tus and template have been preincubated. Finally, we find that transcription will rapidly displace Tus from a template under conditions that generate persistent blocks to transcription. These observations reveal that during the encounter with the transcription complex Tus rapidly dissociates from the template but that at sufficiently high concentrations Tus usually rebinds before the transcription complex can move forward. The advantage of a mechanism which can create a persistent block to transcription or replication complex progression, which can nevertheless be rapidly abrogated in response to down regulation of the blocking protein, is suggested.
We have cloned cDNAs encoding the last iron-sulphur protein of complex I from Neurospora crassa. The cDNA sequence contains an open reading frame that codes for a precursor polypeptide of 226 amino acid residues with a molecular mass of 24972 Da. Our results indicate that the mature protein belongs probably to the peripheral arm of complex I and is rather unstable when not assembled into the enzyme. The protein is highly homologous to the PSST subunit of bovine complex I, the most likely candidate to bind iron-sulphur cluster N-2. All the amino acid residues proposed to bind such a cluster are conserved in the fungal protein.
A T7 RNAP mutant (Y639F) which eliminates discrimination of the chemical character of the NTP ribose 2'-group, facilitates incorporation of non-canonicalsubstrates into nucleic acids. However, transcripts containing a high percentage of non-canonical NMPs are poorly extended due to effects of the 2'-substituents on the transcript:template hybrid conformation. We tested the addition of compounds that stabilize A-type helix geometry to the reaction. High concentrations of polyamines, together with other changes in reaction conditions, greatly increased the synthesis of transcripts heavily substituted with non-canonical ribose 2'-groups. Template structures that facilitate promoter opening increased the efficiency of reactions where non-canonical substrates were incorporated during transcription of +1 to +6.
The hypothesis that active site translocation during initial transcription occurs by a passive sliding mechanism which allows the pre- and post-translocated states to equilibrate on the time scale of bond formation was tested by evaluating the effects of NTP concentration on individual transcript extension steps in the presence of translocation roadblocks created by proteins bound immediately downstream of a T7 promoter, as well as by evaluating the effects of NTP concentration on competing transcript extension pathways (iterative synthesis and "normal" extension). Results are consistent with a passive sliding mechanism for translocation which is driven by NTP binding, and are inconsistent with mechanisms in which the pre- and post-translocated states fail to equilibrate with each other on the time scale of bond formation or in which translocation is driven by NTP hydrolysis. We also find, in agreement with many previous studies, that divergence from consensus in the ITS (initially transcribed sequence) of the T7 promoter decreases productive initiation. However, this appears to be largely due to increases in the NTP concentration requirements for efficient transcription on the divergent ITSs.
The binding of T7 lysozyme to T7 RNAP increases the apparent Km for NTP during initiation (formation of the first phosphodiester bond). It also increases the dissociation constant and dissociation rate of product dinucleotide from the polymerase. Higher NTP concentrations are required for maximal rates of productive initiation from T7 class II versus class III promoters, though individual promoters display distinct responses to changes in NTP concentrations. The greater degree of repression of class II versus class III promoters by T7 lysozyme, which appears to be important for the switch to class III gene expression during the phage life cycle, might therefore be a consequence of: (1) T7 lysozyme generally reducing the affinity of the polymerase for NTPs and increasing the rate of release of transcripts, and (2), intrinsically higher NTP concentration requirements for productive initiation from class II promoters. T7 lysozyme is also found to inhibit the addition of untemplated bases to the transcript which can occur when the elongation complex reaches the end of a template, and its effects are qualitatively similar to those reported for mutations in the extreme C terminus of T7 RNAP. Together with the locations of polymerase mutations which cause resistance or hypersensitivity to T7 lysozyme, these observations suggest that the structural mechanism of lysozyme action might include conformational changes in the C-terminal loop (aa. approximately 820-883) of T7 RNAP.
We study here the effect on phage T7 RNA polymerase activity of lac repressor bound downstream of the T7 promoter. When repressor binds in vitro at an operator centered at +13 or +15 with respect to transcription start, it does not prevent initiation, though the transcript yield is reduced. However, the processivity of the polymerase is depressed and transcript extension is blocked at positions +4 and +6, respectively. These results indicate that repressor and polymerase do not simply exclude each other from the promoter. Rather, they would come into steric conflict and compete for establishment or retention of interactions with the same segment of DNA, without this leading to the immediate displacement of either polymerase or repressor. The resulting destabilization of the transcription complex would depress both initiation rate and enzyme processivity. In contrast to the above results, little reduction in runoff transcription is observed when operator is centered at +47. The decreased sensitivity of polymerase to repressor bound at +47 versus +13 or +15 is likely to be due to the higher stability of the elongation complex during the transcription of downstream regions in comparison with the first transcribed nucleotides. We also show that under conditions of leaky repression and with operator centered at +13, a mutant T7 RNA polymerase showing normal promoter affinity but a slower elongation rate is more sensitive to repression than the wild-type enzyme, both in vitro and in vivo. In vitro, this higher sensitivity is largely due to a reduced ability of the mutant to overcome the elongation block at position +4. The parallel between the in vitro and in vivo data suggests that in vivo the repressor also does not prevent polymerase from binding to promoter, but interferes with subsequent steps in initiation and transcript extension, in this case presumably largely extension beyond +4.
BACKGROUND: Intrabdominal sepsis and allogeneic blood transfusion have been associated with a depression of the immune response in patients undergoing surgery. Some authors have considered that an already immunocompromised host is probably primed for a potential detrimental effect of allogeneic blood. The aim of this paper is to ascertain the effects of allogeneic blood transfusion on the lymphocyte subsets and cytokines in septic rats. MATERIALS AND METHODS: Thirty rats were allotted into three groups: Sham-CLP, anesthesia and laparotomy; CLP, cecal ligation and puncture; CLP+BT, CLP and allogeneic blood transfusion. Preoperatively and on the 1st, 3rd, and 7th postoperative days, the cell percentages of lymphocyte subpopulations, the IL-2 receptor expression, and the IL-1, IL-2, TNF-alpha and IFN-gamma were measured in blood by flow cytometry and ELISA: RESULTS: CLP+BT rats showed on Day 3 a decrease of the CD4+%, an increase of the IL-2R expression directly correlated to the increase of the CD8+% phenotype, a steady increase of IL-1 levels, a decrease of the TNF-alpha levels on the 1st and 3rd days, and a decrease of the IL-2 and IFN-gamma on Day 1. CONCLUSIONS: An accumulative effect of the immunodepression induced by sepsis was observed when allogeneic blood transfusion is added. Blood transfusion + sepsis induces an extensive impairment on cellular immune response and an initial cytokine downregulation, except for IL-1.
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By measuring steady-state rates of dinucleotide synthesis on double-stranded (d.s.) and partially single-stranded (p.s.s.) promoters, and topological unwinding due to open complex formation on plasmids, we have obtained evidence that open complex formation in bacteriophage T7 RNA polymerase:promoter binary complexes is thermodynamically disfavored and that the rate of collapse of the open complex is competitive with the rate of transcription initiation. It is suggested that open complex instability is a kinetic mechanism that allows T7 RNA polymerase (RNAP) to achieve promoter specificity while still allowing for efficient promoter release. Open complex instability could also provide a mechanism for modulating the KM for the initiating NTPs so as to allow different promoters to respond differently to physiological changes in NTP concentration.
The catalytic specificity of T7 RNA polymerase (RNAP) for ribonucleoside triphosphates vs deoxynucleoside triphosphates {(kcat/Km)rNTP/(kcat/Km)dNTP} during transcript elongation is approximately 80. Mutation of tyrosine 639 to phenylalanine reduces specificity by a factor of approximately 20 and largely eliminates the Km difference between rNTPs and dNTPs. The remaining specificity factor of approximately 4 is kcat-mediated and is nearly eliminated if Mn2+ is substituted for Mg2+ in the reaction. Mn2+ substitution does not significantly affect the Km difference between rNTPs and dNTPs. Mn2+ substitution also enhances the activity of poorly active mutant enzymes carrying nonconservative substitutions in the active site, and its effects are generally consistent with the Mn2+-catalyzed reaction being less restrictive in its requirements for alignment of the reactive groups. In addition to discrimination occurring at the level of nucleoside monophosphate (NMP) incorporation, it is also found that transcripts containing deoxynucleoside monophosphates (dNMPs) are more poorly extended than transcripts of canonical structure, though a severe barrier to transcript extension is seen only when the 3' region of the transcript is heavily substituted with dNMPs. The barrier to extension of transcripts heavily substituted with dNMPs is reduced for sequences known to be amenable to forming A-like helices and is larger for sequences that resist transformation from B-form DNA.DNA structures. The barrier to extension of dNMP-substituted transcripts is also reduced by solution conditions known to destabilize B-form DNA and to stabilize A-form structures. These observations imply a requirement for a non-B-form, possibly A-like, conformation in the transcript.template hybrid that is disrupted when the transcript is of predominantly deoxyribose structure.
The mechanism by which T7 RNA polymerase (RNAP) discriminates between rNTP and dNTP substrates has been characterized. During transcript elongation T7 RNAP uses rNTPs 70-80-fold more efficiently than dNTPs. Discrimination of the hydrogen-bonding character of the ribose 2'-substituent contributes a largely Km-mediated factor of approximately 20 to this preference for rNTPs. Discrimination of 2'-substituent H-bonding character appears to be made through a hydrogen bond to the hydroxyl group of tyrosine 639. This hydrogen bond makes little net contribution to either rNTP ground or transition state binding energy apparently because it is balanced by the energy of desolvation of the tyrosine hydroxyl. This mechanism may reflect a strategy to facilitate translocation by minimizing contributions from polymerase-NMP moiety interactions to NTP binding energy so as to minimize the affinity of the NTP binding site for the 3'-NMP of the product nucleic acid.
In vitro, after binding to the promoter to form a catalytically active complex, RNA polymerases abortively cycle over the first transcribed nucleotides (initial transcribed sequence or ITS) before leaving the promoter. With the bacteriophage T7 enzyme, the extent of abortive transcription varies with the nature of the ITS and with the elongation speed of the polymerase. Here, we compare in vitro and in vivo the yield of long transcripts from T7 promoters, with two different ITSs, the T7 gene10 and the lactose operon ITSs, and two different T7 RNA polymerases, the wild-type and a 2.7-fold slower mutant (G645A). The use of non-cognate ITS and/or slow polymerase decreases the yield of long transcripts in vitro and in vivo in a parallel fashion, with low polymerase speed and non-cognate ITS acting synergistically. In vitro, this decrease is mirrored by an increase in the average number of abortive cycles the enzyme undergoes before leaving the promoter; specifically, with the G645A mutant, transcript release is favored at any ITS position, whereas with the lac ITS it is particularly frequent at positions five and six following the incorporation of uridine residues. Hence, the more abortive cycles per long transcript synthesis in vitro, the lower the yield of long transcripts in vitro or in vivo. We conclude that the duration of abortive cycling can limit long transcript synthesis in vivo, as in vitro. Under conditions where cycling is minimal (wild-type polymerase, gene10 ITS), T7 promoter drives the synthesis of three long transcripts per second at 37 degrees C in vivo, a figure higher than for any Escherichia coli promoter.