Chitinolytic activity in Chromobacterium violaceum: substrate analysis and regulation by quorum sensing

Quorum sensing control mediated by N-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In Chromobacterium violaceum ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL N-hexanoyl-L-homoserine lactone (HHL). In this study we show that C. violaceum produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using p-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of N-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of N-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two N-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn5 mutant of C. violaceum (CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the C. violaceum wild-type strain or 10 microM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.     

Repair of left ventricular aneurysm: long-term results of linear repair versus endoaneurysmorrhaphy

BACKGROUND: Recently, endoaneurysomorrhaphy has been proposed as a more physiologic repair of postinfarction left ventricular aneurysm than is linear repair. There are only a few studies comparing the short-term and long-term results of the two techniques. METHODS: Clinical outcomes and echocardiographic measurements of left ventricular volume and sphericity in 27 patients who underwent endoaneurysmorrhaphy were compared with those in 20 patients who had linear repair. RESULTS: The two groups were matched with respect to age, gender, comorbid risk factors, functional class, urgency of the operation, and concomitant procedures. Preoperatively, left ventricular ejection fraction was lower in the endoaneurysmorrhaphy group (0.25 +/- 0.08 versus 0.30 +/- 0.09; p = 0.03). Follow-up was available in 44 patients (94%) and ranged from 2 to 86 months (mean, 41.0 +/- 26.5 months). Thirty-day operative mortality, perioperative complications, 5-year survival, and freedom from cardiac death were similar. Early postoperative percentage increase in left ventricular ejection fraction was greater after endoaneurysmorrhaphy (0.51 +/- 0.64 versus 0.18 +/- 0.48; p = 0.036). Long-term functional improvement was significantly better in the endoaneurysmorrhaphy group: At the time of last follow-up, 88% of patients were in New York Heart Association class I/II, compared with 53% after linear repair (p = 0.01). There were no measurable differences between the groups with respect to left ventricular ejection fraction (0.28 +/- 0.11 versus 0.27 +/- 0.11; p = 0.90), left ventricular volume (171.6 +/- 59.1 versus 169.9 +/- 54.4 mL; p = 0.94), and sphericity index (0.61 +/- 0.09 versus 0.61 +/- 0.12; p = 1.0). CONCLUSIONS: Despite having a similar effect on left ventricular geometry, endoaneurysmorrhaphy resulted in a greater increase in postoperative left ventricular ejection fraction and a substantially improved long-term clinical outcome.     

Monitoring of radio frequency tissue ablation in an interventional magnetic resonance environment. Preliminary ex vivo and in vivo results

RATIONALE AND OBJECTIVES: The authors evaluate the feasibility of monitoring radio frequency (RF) ablation in an interventional, open-configuration, 0.5-tesla magnetic resonance (MR) environment. METHODS: Ex vivo and in vivo RF coagulation necrosis were induced in porcine paraspinal muscle tissue using a 300 kHz monopolar RF generator applying 5 to 20 W over 3 to 9 minutes. Images were acquired simultaneous to RF application, after RF application, and in an intermittent mode (60 seconds of RF followed by 15 seconds of MR imaging). Temperature changes were monitored based on amplitude (ex vivo) and phase alterations (in vivo) of a T1-weighted graded refocused echo (GRE) sequence enabling an update every 2.5 seconds. A standardized color-coded subtraction technique enhanced signal changes. Additionally, T2- and T1-weighted spin echo (SE) images were acquired with and without intravenous contrast. Macroscopic coagulation size was compared with lesion size seen on MR images. RESULTS: Although lesion diameters were related directly to applied RF power, the application mode had no significant impact on coagulation size (P > 0.05). As could be expected, MR imaging during RF ablation resulted in major image distortion. Radio frequency effects were seen on images acquired in the continuous and intermittent modes. Coagulation size seen on GRE images correlated well with macroscopy both ex vivo (r = 0.89) and in vivo (r = 0.92). Poorer correlation was found with postinterventional SE sequences (r = 0.78-0.84). CONCLUSIONS: Magnetic resonance monitoring of RF effects is feasible both ex vivo as well as in vivo using temperature-sensitive sequences in an open-configuration MR environment.     

Pregnancy loss and autoantibodies against phospholipid-binding proteins

We investigated the utility of two approaches for exploiting pleiotropy to search for genes influencing related traits. To do this we first assessed the genetic correlations among a set of five closely related quantitative traits (Q1, Q2, Q3, Q4, Q5). We then used the genetic correlations among these five traits both to remove the common genetic effects of the four remaining traits, thereby identifying the unique genetic contribution to each trait, and to extract a synthetic phenotype which exploits the shared genetic information (pleiotropy) among these five traits. After obtaining these conditional traits, we then searched for evidence of quantitative trait loci (QTLs) (using variance component linkage) influencing the unique residual genetic component for each trait as well as those influencing the expression of the synthetic traits. From this work, we conclude that the removal of the common genetic effects of other traits in a group may be of greater utility when the majority of the pleiotropy initially detected between traits is attributable to the shared additive effects of polygenes, rather than to those of major loci. By contrast, decomposition of the genetic covariance matrix to its principal components is a greater utility when the majority of pleiotropy is attributable to major loci.     

Adenovirus-mediated gene transfer of inhibitors of apoptosis protein delays apoptosis in cerebellar granule neurons

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Clinical utility of human polymerized hemoglobin as a blood substitute after acute trauma and urgent surgery

We have previously documented the safety of 1 unit (50 gram) of human polymerized hemoglobin (Poly SFH-P) in healthy volunteers. This report describes the first patient trial to assess the therapeutic benefit of Poly SFH-P in acute blood loss. Thirty-nine patients received 1 (n = 14), 2 (n = 2), 3 (n = 15), or 6 (n = 8) units of Poly SFH-P instead of red cells as part of their blood replacement after trauma and urgent surgery. There were no safety issues related to the infusion of Poly SFH-P. The plasma hemoglobin concentration ([Hb]) after the infusion of 6 units (300 gram) of Poly SFH-P was 4.8 +/- 0.8 g/dL (mean +/- SD). Although the red cell [Hb] fell to 2.9 +/- 1.2 g/dL, the total [Hb] was maintained at 7.5 +/- 1.2 g/dL. Poly SFH-P maintained total [Hb], despite the marked fall in red cell [Hb] due to blood loss. The utilization of O2 (extraction ratio) was 27 +/- 16% from the red cells and 37 +/- 13% from the Poly SFH-P. Twenty-three patients (59%) avoided allogeneic transfusions during the first 24 hours after blood loss. Poly SFH-P effectively loads and unloads O2 and maintains total hemoglobin in lieu of red cells after acute blood loss, thereby reducing allogeneic transfusions. Poly SFH-P seems to be a clinically useful blood substitute.