Onset Of Lysis Research Articles

Trends in the pathophysiology of Charcot neuroarthropathy

The pathophysiology of Charcot neuroarthropathy (CN) includes a number of gray areas, particularly regarding the onset of inflammation which induces the disruption of the bone remodeling factor responsible for the onset of bone lysis. This clinical insight highlights a potential link between this inflammation and the rapid correction of chronic hyperglycemia (Dardari et al., 2022), which is known to be responsible for a particular type of neuropathy known as treatment-induced neuropathy of diabetes (TIND). Our description makes an additional contribution to shed light on the mysterious physiopathology of CN.

The specifics of clotting and endogenic fibrinolysis in acute coronary syndrome patients

Aim. To assess the processes of clot formation in relation to endogenic fibrinolysis in patients with various types of acute coronary syndrome (ACS).Material and methods. To the study, 89 persons (40 males) included: 59 with ACS, 30 — control group. Among the ACS patients, 28 were ST-elevation (STEMI), 31 with non-ST-elevation ACS (NSTEACS). All participants were assessed for clotting formation parameters and thrombus lysis with rotational elastometry on ROTEM equipment (Germany) and “Hemacore” Thrombodynamics (Russia), before coronary arteriography.Results. Clot density and its size in 10 minutes were significantly higher in ACS comparing to control patients (2867 units vs 25084 units, p<0,001; 41,8 mm vs 36,4 mm, p<0,05, respectively). The time from onset of thrombus lysis was lower in ACS (79,4 min vs 69,3 min, p<0,05). The percent of lysis by the end of process registration was lower in ACS (20,1% vs 23,1%, p<0,05). Amplitude of clot formation in 20 minutes from the onset of clotting, as the maximum density of clot were significantly higher in NSTEACS comparing to STEMI (56,2 mm vs 49,7 mm, p=0,018; 59 mm vs 53,2 mm, p=0,011; respectively). Thrombus lysis was slower in NSTEACS patients comparing to STEMI (62,26 min vs 45,5 min, p=0,02).Conclusion. With the measurement in vitro, the process of clotting and thrombus lysis is more active in ACS patients comparing to controls, that point on a baseline activated hemostasis. Within the ACS group, higher the existing clot in vessel lumen, more active the shift of the balance to anticoagulation and fibrinolysis.

Sensing bacterial vibrations and early response to antibiotics with phase noise of a resonant crystal

The speed of conventional antimicrobial susceptibility testing (AST) is intrinsically limited by observation of cell colony growth, which can extend over days and allow bacterial infections to advance before effective antibiotics are identified. This report presents an approach for rapidly sensing mechanical fluctuations of bacteria and the effects of antibiotics on these fluctuations. Bacteria are adhered to a quartz crystal resonator in an electronic bridge that is driven by a high-stability frequency source. Mechanical fluctuations of cells introduce time-dependent perturbations to the crystal boundary conditions and associated resonant frequency, which translate into phase noise measured at the output of the bridge. In experiments on nonmotile E. coli exposed to polymyxin B, cell-generated frequency noise dropped close to zero with the first spectra acquired 7 minutes after introduction of the antibiotic. In experiments on the same bacterial strain exposed to ampicillin, frequency noise began decreasing within 15 minutes of antibiotic introduction and proceeded to drop more rapidly with the onset of antibiotic-induced lysis. In conjunction with cell imaging and post-experiment counting of colony-forming units, these results provide evidence that cell death can be sensed through measurements of cell-generated frequency noise, potentially providing a basis for rapid AST.

The Viral Gene ORF79 Encodes a Repressor Regulating Induction of the Lytic Life Cycle in the Haloalkaliphilic Virus ϕCh1.

In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea By random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus ϕCh1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing ϕCh1 as a provirus. This approach yielded mutant virus particles carrying a disrupted version of ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii, resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In the absence of gp79, onset of lysis and expression of viral proteins occurred prematurely compared to their timing in the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of ϕCh1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadiiIMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetics approach to identify the function of a yet undescribed gene of ϕCh1. We provide evidence that gp79, a currently unknown protein of ϕCh1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.

The Effector Domain Region of the Vibrio vulnificus MARTX Toxin Confers Biphasic Epithelial Barrier Disruption and Is Essential for Systemic Spread from the Intestine.

Vibrio vulnificus causes highly lethal bacterial infections in which the Multifunctional Autoprocessing Repeats-in-Toxins (MARTX) toxin product of the rtxA1 gene is a key virulence factor. MARTX toxins are secreted proteins up to 5208 amino acids in size. Conserved MARTX N- and C-terminal repeat regions work in concert to form pores in eukaryotic cell membranes, through which the toxin’s central region of modular effector domains is translocated. Upon inositol hexakisphosphate-induced activation of the of the MARTX cysteine protease domain (CPD) in the eukaryotic cytosol, effector domains are released from the holotoxin by autoproteolytic activity. We previously reported that the native MARTX toxin effector domain repertoire is dispensable for epithelial cellular necrosis in vitro, but essential for cell rounding and apoptosis prior to necrotic cell death. Here we use an intragastric mouse model to demonstrate that the effector domain region is required for bacterial virulence during intragastric infection. The MARTX effector domain region is essential for bacterial dissemination from the intestine, but dissemination occurs in the absence of overt intestinal tissue pathology. We employ an in vitro model of V. vulnificus interaction with polarized colonic epithelial cells to show that the MARTX effector domain region induces rapid intestinal barrier dysfunction and increased paracellular permeability prior to onset of cell lysis. Together, these results negate the inherent assumption that observations of necrosis in vitro directly predict bacterial virulence, and indicate a paradigm shift in our conceptual understanding of MARTX toxin function during intestinal infection. Results implicate the MARTX effector domain region in mediating early bacterial dissemination from the intestine to distal organs–a key step in V. vulnificus foodborne pathogenesis–even before onset of overt intestinal pathology.

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss.

Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016

The Association of Sacral Table Angle Measurements With Spondylolytic and Spondylolisthetic Defects at the Lumbosacral Articulation: A Radiographic Analysis

Study DesignRetrospective radiographic study of 6 patients with L5 spondylolysis observed prospectively before the onset of lysis through adulthood. A radiographic analysis of 50 pediatric control subjects was compared with the study group. ObjectiveTo determine whether sacral table angle (STA) measurements bear etiologic association with the development of spondylolysis and/or subsequent spondylolisthesis. Summary of Background DataAlthough radiographic parameters in association with spondylolysis and isthmic spondylolisthesis have been studied, no parameter has been shown to definitively have a role in development of this disease process. The STA is a recently described radiographic parameter useful in measuring anatomic changes across the lumbosacral articulation. This measurement's role as a predictor of pars lysis and subsequent slippage remains unknown. MethodsThe researchers examined the longitudinal plain radiographs of 6 patients observed from childhood, before the development of spondylolysis, through adulthood. Measurements of STA and percent slippage were performed. Fifty pediatric control subjects' radiographs were also examined with STA measurements. Statistical analysis was conducted on results. ResultsMean STA of the study group before the development of spondylolysis was 95° ± 5.5°. Mean STA from the control group was 97.5° ± 4.3°. No statistical difference was found between groups (p > .05). No index patient had an abnormal STA before spondylolysis (less than 89°, defined as being outside 2 standard deviations from the control mean). Four of 6 index patients with spondylolysis developed spondylolisthesis. A negative correlation (r = .54) was seen for STA as a function of increasing percent slip when assessed longitudinally. ConclusionsAbnormal STA measurement was not seen before the development of spondylolysis in this study population. Decreasing STAs were seen secondarily in patients with L5 spondylolisthetic progression. This finding points to anatomic change and secondary remodeling of the upper sacrum as a result of slippage.

Facilitation of urokinase‐mediated fibrinolysis by MST‐188 (833.7)

Introduction MST‐188 (Mast Therapeutics, San Diego, CA.) is a highly purified form of the , triblock copolymer poloxamer 188. It exhibits potent rheologic properties that result in improved blood flow especially under low shear conditions. Initial observations suggested that endogenous fibrinolysis is facilitated by this agent. In this study we investigate the effect of MST‐188 on urokinase induced fibrinolysis.Materials and Methods A dynamic fibrinokinetic assay was used to assess both the assembly and dissolution of fibrin clots. MST‐188 (0 ‐ 10 mg/ml) was added to citrated human plasma with and without urokinase at a concentrations between 0 ‐ 2,500 U/ml (N=10‐15 donors). Clotting was initiated with the sequential addition of and calcium chloride (0.25 uM) and then thrombin (0.5u/ml). Absorbance was monitored to detect the assembly and dissolution of fibrin.Results Control studies of MST‐188 alone had a dose dependent effect on fibrin assembly as reflected by both the rate of change and peak optical density. Urokinase alone showed no effect on clot formation but a dose dependent increase in clot lysis. Supplementation of MST‐188 to urokinase facilitated its lytic profile in a concentration dependent manner accelerating the time to onset of lysis and reducing the overall AUC for clot dissolution.Discussion These results demonstrate that MST‐ 188 increases urokinase mediated fibrinolysis. The increase in the susceptibility of the formed clot to lysis in the presence of MST‐188 may be due to structural modification of the clot. MST‐188 may bind to fibrin strands, thereby modifying their accessibility to the lytic action of generated plasmin.

Visualization of pinholin lesions in vivo

Lambdoid phage 21 uses a pinholin-signal anchor release endolysin strategy to effect temporally regulated host lysis. In this strategy, the pinholin S(21)68 accumulates harmlessly in the bilayer until suddenly triggering to form lethal membrane lesions, consisting of S(21)68 heptamers with central pores <2 nm in diameter. The membrane depolarization caused by these pores activates the muralytic endolysin, R(21), leading immediately to peptidoglycan degradation. The lethal S(21)68 complexes have been designated as pinholes to distinguish from the micrometer-scale holes formed by canonical holins. Here, we used GFP fusions of WT and mutant forms of S(21)68 to show that the holin accumulates uniformly throughout the membrane until the time of triggering, when it suddenly redistributes into numerous small foci (rafts). Raft formation correlates with the depletion of the proton motive force, which is indicated by the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol. By contrast, GFP fusions of either antiholin variant irsS(21)68, which only forms inactive dimers, or nonlethal mutant S(21)68(S44C), which is blocked at an activated dimer stage of the pinhole formation pathway, were both blocked in a state of uniform distribution. In addition, fluorescence recovery after photobleaching revealed that, although the antiholin irsS(21)68-GFP fusion was highly mobile in the membrane (even when the proton motive force was depleted), more than one-half of the S(21)68-GFP molecules were immobile, and the rest were in mobile states with a much lower diffusion rate than the rate of irsS(21)68-GFP. These results suggest a model in which, after transiting into an oligomeric state, S(21)68 migrates into rafts with heterogeneous sizes, within which the final pinholes form.

Hyperfibrinolysis in out of hospital cardiac arrest is associated with markers of hypoperfusion

Aim of the studyThis study investigated the incidence of hyperfibrinolysis upon emergency department (ED) admission in patients with out of hospital cardiac arrest (OHCA), and the association of the degree of hyperfibrinolysis with markers of hypoperfusion. MethodsFrom 30 OHCA patients, cardiopulmonary resuscitation (CPR) time, pH, base excess (BE), and serum lactate were measured upon ED admission. A 20% decrease of rotational thromboelastometry maximum clot firmness (MCF) was defined as hyperfibrinolysis. Lysis parameters included maximum lysis (ML), lysis onset time (LOT) and lysis index at 30 and 45min (LI30/LI45). The study was approved by the Human Subjects Committee. ResultsHyperfibrinolysis was present in 53% of patients. Patients with hyperfibrinolysis had longer median CPR times (36 (15–55) vs. 10 (7–18)min; P=0.001), a prolonged activated partial thromboplastin time (54±16 vs. 38±10s; P=0.006) and elevated D-dimers (6.1±2.1 vs. 2.3±2.0μg/ml; P=0.02) when compared to patients without hyperfibrinolysis. Hypoperfusion markers, including pH (6.96±0.11 vs. 7.17±0.15; P<0.001), base excess (−20.01±3.53 vs. −11.91±6.44; P<0.001) and lactate (13.1±3.7 vs. 8.0±3.7mmol/l) were more disturbed in patients with hyperfibrinolysis than in non-hyperfibrinolytic subjects, respectively. The LOT showed a good association with CPR time (r=−0.76; P=0.003) and lactate (r=−0.68; P=0.01), and was longer in survivors (3222±34s) than in non-survivors (1356±833; P=0.044). ConclusionA substantial part of OHCA patients develop hyperfibrinolysis in association with markers for hypoperfusion. Our data further suggest that the time to the onset of clot lysis may be an important marker for the severity of hyperfibrinolysis and patient outcome.

LytM-Domain Factors Are Required for Daughter Cell Separation and Rapid Ampicillin-Induced Lysis inEscherichia coli

Bacterial cytokinesis is coupled to the localized synthesis of new peptidoglycan (PG) at the division site. This newly generated septal PG is initially shared by the daughter cells. In Escherichia coli and other gram-negative bacteria, it is split shortly after it is made to promote daughter cell separation and allow outer membrane constriction to closely follow that of the inner membrane. We have discovered that the LytM (lysostaphin)-domain containing factors of E. coli (EnvC, NlpD, YgeR, and YebA) are absolutely required for septal PG splitting and daughter cell separation. Mutants lacking all LytM factors form long cell chains with septa containing a layer of unsplit PG. Consistent with these factors playing a direct role in septal PG splitting, both EnvC-mCherry and NlpD-mCherry fusions were found to be specifically recruited to the division site. We also uncovered a role for the LytM-domain factors in the process of beta-lactam-induced cell lysis. Compared to wild-type cells, mutants lacking LytM-domain factors were delayed in the onset of cell lysis after treatment with ampicillin. Moreover, rather than lysing from midcell lesions like wild-type cells, LytM(-) cells appeared to lyse through a gradual loss of cell shape and integrity. Overall, the phenotypes of mutants lacking LytM-domain factors bear a striking resemblance to those of mutants defective for the N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC. E. coli thus appears to rely on two distinct sets of putative PG hydrolases to promote proper cell division.

Corn trypsin inhibitor decreases tissue-type plasminogen activator-mediated fibrinolysis of human plasma

Corn trypsin inhibitor (CTI) has been considered the molecule of choice to inhibit activated factor XII (FXIIa) during the conduct of experimentation focusing on tissue factor-initiated coagulation. However, CTI-mediated attenuation of fibrinolysis following celite activation of coagulation was observed in pilot studies with the clot lifespan model. The goal of the present study was thus to characterize the mechanism(s) responsible for CTI-mediated hypofibrinolysis. Normal plasma was exposed to 0 or 49.6 microg/ml CTI, with coagulation initiated with celite or tissue factor. Fibrinolysis was initiated with tissue-type plasminogen activator (tPA). Additional experiments utilized plasminogen activator inhibitor 1 deficient or alpha2-antiplasmin-deficient plasma. Coagulation/fibrinolysis kinetics were monitored with the thrombelastography-based clot lifespan model. In addition to delaying the initiation of coagulation, CTI prolonged clot growth time, delayed the onset of lysis, and prolonged clot lysis time in normal plasma after celite activation. Conversely, CTI increased the speed of clot growth, clot strength, and prolonged clot lysis time after tissue factor activation. Experiments with plasma deficient in antifibrinolytic proteins supported a primary inhibition of tPA by CTI. In addition to anti-FXIIa effects following celite activation, CTI likely exerts an anti-tPA effect, which contributed to hypofibrinolysis in this model.

Bacteriophage-Modified Microarrays for the Direct Impedimetric Detection of Bacteria

A novel method is presented for the specific and direct detection of bacteria using bacteriophages as recognition receptors immobilized covalently onto functionalized screen-printed carbon electrode (SPE) microarrays. The SPE networks were functionalized through electrochemical oxidation in acidic media of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) by applying a potential of +2.2 V to the working electrode. Immobilization of T4 bacteriophage onto the SPEs was achieved via EDC by formation of amide bonds between the protein coating of the phage and the electrochemically generated carboxylic groups at the carbon surface. The surface functionalization with EDC, and the binding of phages, was verified by time-of-flight secondary ion mass spectrometry. The immobilized T4 phages were then used to specifically detect E. coli bacteria. The presence of surface-bound bacteria was verified by scanning electron and fluorescence microscopies. Impedance measurements (Nyquist plots) show shifts of the order of 10(4) Omega due to the binding of E. coli bacteria to the T4 phages. No significant change in impedance was observed for control experiments using immobilized T4 phage in the presence of Salmonella. Impedance variations as a function of incubation time show a maximum shift after 20 min, indicating onset of lysis, as also confirmed by fluorescence microscopy. Concentration-response curves yield a detection limit of 10(4) cfu/mL for 50-microL samples.

Solubilization of human erythrocyte membranes by ASB detergents

Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 microM and ASB-16 = 10 microM) was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (Re sat) and total lysis (Re sol) were calculated, allowing the determination of the membrane binding constants (Kb). ASB-14 presented lower membrane affinity (Kb = 7050 M(-1)) than ASB-16 (Kb = 15610 M(-1)). The amount of proteins and cholesterol solubilized by both ASB detergents was higher while Re sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively) were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

BackgroundSignal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression) or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus.ResultsHere we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20) was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis.ConclusionThe SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.

ε-Aminocaproic acid inhibition of fibrinolysis in vitro: should the ‘therapeutic’ concentration be reconsidered?

The therapeutic concentration of epsilon-aminocaproic acid (EACA) has been 130 microg/ml or greater for nearly 50 years. We tested the effects on clot growth/disintegration of EACA with a plasmatic model of hyperfibrinolysis in vitro. Human plasma was exposed to 1000 U/ml tissue-type plasminogen activator containing 0, 13, 65 or 130 microg/ml EACA, with clot growth/disintegration kinetics quantified via thrombelastography. Data were analyzed with one-way analysis of variance or Kruskal-Wallis analysis of variance as appropriate. Exposure of plasma to 1000 U/ml tissue-type plasminogen activator resulted in a brief-lived clot, lasting 2 min. EACA at all concentrations tested significantly increased the rate of clot growth compared with samples with 0 microg/ml EACA. Clot strength was significantly increased by EACA in a concentration-dependent fashion. Similarly, EACA significantly prolonged the time of onset of clot lysis and decreased the rate of lysis. Samples with 130 microg/ml EACA had no sign of lysis present for 30 min. Subtherapeutic to therapeutic concentrations of EACA significantly attenuated or abolished fibrinolysis in the presence of a concentration of tissue-type plasminogen activator more than 2000-fold that encountered systemically during cardiopulmonary bypass. Further clinical investigation is warranted to determine whether smaller concentrations of EACA could provide a reduction in bleeding with a concomitant decrease in thrombotic complications.

Bortezomib-Induced Tumor Lysis Syndrome in Multiple Myeloma

Tumor lysis syndrome (TLS) is exceedingly rare in multiple myeloma because of the relatively slow proliferation and response of the malignant cells. Bortezomib is a novel agent that inhibits proteasome and has shown activity against multiple myeloma. We report 8 episodes of TLS seen in 7 patients with bortezomib therapy, with or without dexamethasone, among 496 patients treated on 3 phase II multicenter studies. Biochemical abnormalities resolved with supportive therapy in 6 patients (including hemodialysis in 2) but proved fatal in 1. Clinicians should be alert for TLS in patients with myeloma with significant disease burden treated with bortezomib because of the potential for rapid onset of cell lysis with this agent.

Contact Activation Prolongs Clot Lysis Time in Human Plasma: Role of Thrombin-activatable Fibrinolysis Inhibitor and Factor XIII

Contact activation system proteins (e.g., Factor XII, kallikrein) have been implicated as direct or indirect activators of plasminogen. However, contact activation and Factor XI have enhanced thrombin-activatable fibrinolysis inhibitor (TAFI) activation and decreased fibrinolysis, and Factor XIII (FXIII) also delays fibrinolysis via alpha(2)-anti-plasmin deposition on fibrin polymers. Thus, the goals of this study were to define how fibrinolysis is modulated in human plasma by contact or tissue factor (TF) activation, and what role TAFI and FXIII plays in this system. Normal, TAFI-deficient and TAFI-deficient/FXIII-supplemented plasma was exposed to tissue-type plasminogen activator and activated with either celite or TF. Clot growth/disintegration kinetics were documented with thrombelastography. Normal plasma activated with celite had significantly prolonged onset and duration of clot lysis compared with samples activated with TF. TAFI-deficient plasma activated with celite was noted to have a duration of clot lysis not different from samples activated with TF, but a significant difference in time to onset of lysis persisted. Celite activation of TAFI-deficient/FXIII-supplemented plasma showed significantly prolonged onset and duration of clot lysis compared with samples activated with TF. Primarily TAFI, and to a lesser extent FXIII, contributed to contact system protein-mediated attenuation of fibrinolysis. Clinical investigation of these phenomena is warranted in clinical settings involving contact activation (e.g., intra-aortic balloon pumps and ventricular assist devices) to determine whether these devices modulate fibrinolysis and perhaps contribute to thromboembolic morbidity.

P2 growth restriction on an rpoC mutant is suppressed by alleles of the Rz1 homolog lysC.

Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase beta' subunit and is nonpermissive for plating of bacteriophage P2. P2 gor mutants productively infect 397c and define a new gene, lysC, encoded by a reading frame that extensively overlaps the P2 lysis accessory gene, lysB. The unusual location of lysC with respect to lysB is reminiscent of the Rz/Rz1 lysis gene pair of phage lambda. Indeed, coexpression of lysB and lysC complemented the growth defect of lambda Rz/Rz1 null mutants, indicating that the LysB/C pair is similar to Rz/Rz1 in both gene arrangement and function. Cells carrying the rpoC397 mutation exhibited an early onset of P2-induced lysis, which was suppressed by the gor mutation in lysC. We propose that changes in host gene expression resulting from the rpoC397 mutation result in changes in the composition of the bacterial cell wall, making the cell more susceptible to P2-mediated lysis and preventing accumulation of progeny phage sufficient for plaque formation.

Functional regulation of the Listeria monocytogenes bacteriophage A118 holin by an intragenic inhibitor lacking the first transmembrane domain.

We have dissected the functional properties of the holin encoded by Listeria monocytogenes bacteriophage A118. Native hol118 was cloned into lambdaDeltaSthf, devoid of the S holin, and tested in an E. coli background. Surprisingly, it caused very late cell lysis, beginning at 80 min after induction. Immunological analyses demonstrated that Hol118 appears in the cytoplasmic membrane shortly after infection. The hol118 gene features a dual start motif similar to lambda S. Therefore, different N-terminally modified Hol118 variants were tested. However, in contrast to lambda S, inactivation of AUG-1 or AUG-2 showed no significant influence on lysis timing. In addition, Hol118-mediated lysis could not be triggered by energy poisons, indicating a functional regulation different from that of S. Toeprinting assays on hol118 mRNA revealed an unexpected translational start codon (AUG-3) at nucleotide position 40. We demonstrated by in vitro and in vivo approaches that the predicted Hol118(83) product is actually produced together with the full-length polypeptide. However, although the truncated holin lacking its first transmembrane domain appeared in the cytoplasmic membrane, it was shown to be functionally deficient and unable to support lambda R-mediated lysis. In contrast, specific mutations introduced to abolish translation initiation at AUG-3 drastically accelerated lysis, pointing to an inhibitor function of Hol118(83). This hypothesis was supported by the observation that hol118(83) inhibited holin function when expressed in trans. A deviation from the lambda S paradigm is proposed, which represents a new model of holin functional regulation: the intragenic, in frame translated Hol118(83) product, which is devoid of its first transmembrane domain, acts as a functional inhibitor and constitutes a key part of the lysis clock of A118. Presence of the dominant inhibitor function also explains the long latent period of A118, where the onset of lysis takes about 70 min, more than twice the time needed by lambda.