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Notes: CHEKANOV, V., et al.: Transplantation of Autologous Endothelial Cells Induces Angiogenesis. This study examined the feasibility and efficacy of autologous endothelial cell (EC) transplantation using a fibrin matrix in the ischemic myocardium of sheep. Four weeks after placing an ameroid constrictor in the circumflex artery of 12 adult sheep, four animals (EC group) were subjected to EC transplantation. In four others (saline [SAL] group) saline with added inactivated cells was injected and four animals served as controls. Eight weeks after treatment the animals were sacrificed to assess histology and ultrastructure. Eight weeks after injection, ventricular function was markedly improved in the EC transplant group, but had deteriorated in the SAL and control groups. Myocardial blood flow was also increased in the EC group. Histology and electron microscopy revealed extensive neovascularization after EC transplantation and improved myocardial appearance. Heterotopic transplantation of EC within a fibrin matrix enhances neovascularization, increases myocardial blood flow, and improves left ventricular function. (PACE 2003; 26[Pt. II]:496–499)

Notes: Background: Patient and procedural characteristics associated with major adverse cardiac events following balloon angioplasty have been identified. Factors predictive of angiographic restenosis following coronary stent implantation have been reported, although patient variables associated with adverse clinical outcome are not well defined. Hypothesis and Methods: To identify predictors of adverse clinical outcome following NIR stent implantation, clinical and angiographic characteristics of patients enrolled in the FINESS Trial were subjected to stepwise logistic regression analysis. From December 1995 through March 1996, NIR stent implantation was attempted in 255 patients (341 lesions) enrolled prospectively in a multicenter registry with broad entry criteria. Results: On stepwise logistic regression analysis, the presence of multivessel disease, diabetes, and the total length of the stented segment were predictive of major adverse cardiac events during 6-month follow-up. For every 1 mm increase in stent length, the risk for the combined end point of death or myocardial infarction increased by 3%. Lesion length was not predictive of clinical events on multivariate analysis. Conclusions: Our data raise the possibility that an attempt to use shorter stents to cover significant stenoses, but not adjacent areas of visible narrowing, may improve outcome.

Notes: Coronary flow reserve (CFR) and fractional flow reserve (FFRmyo) are two guidewire-based methods currently used to assess the functional severity of coronary artery lesions. Acquiring both measurements simultaneously may provide complementary information, but would require the passage of two different guidewires and complex instrumentation for their calculation. This study assessed the procedural safety and performance of a novel personal computer-based algorithm, the SmartFlow Intravascular Processor (SFIP), which utilizes a single conventional pressure wire for obtaining simultaneous CFR and FFRmyo measurements for the assessment of coronary artery lesion severity. In 20 consecutive patients with 21 lesions, pressure-derived CFR, FFRmyo and SFIP-FFRmyo measurements were obtained during adenosine-induced hyperemia. Intravascular ultrasound and quantitative coronary angiography lesion analysis was done off-line at the Washington Core Laboratory. Mean FFRmyo was 0.83 ± 0.11, SFIP-FFRmyo was 0.86 ± 0.06, and CFR was 1.74 ± 0.46. Pairwise correlation analysis showed excellent correlation between the FFRmyo and the FFRmyo-SFIP (r2= 0.83, P 〈 0.0001) and a fair degree of relationship between the CFR and minimal lumen diameter (r2= 0.43, P = 0.07). We could not find a correlation between CFR and FFRmyo (r2= 0.22, P = 0.37), between CFR and FFR-SFIP (r2= 0.05, P = 0.86), or between the hemodynamic measurements and any of the IVUS-derived measurements. In conclusion, the SFIP is u novel algorithm for obtaining simultaneous CFR and FFRmyo that may provide valuable information for the assessment of lesion severity and clinical decision making.

Notes: Abstract  Background: Controversy exists whether transmyocardial laser revascularization (TMR) is associated with angiogenesis or neuromodulation and whether these are time-dependent phenomena. Accordingly, we performed a time-course analysis of the expression of angiogenic and neuronal factors following experimental percutaneous TMR. Methods and Results: Five weeks after placing ameroid constrictors on the circumflex coronary artery, 16 pigs underwent left ventricular mapping guided TMR using Ho:YAG laser (2 J × 1 pulse) at 30 sites directed at the ischemic zones and 11 animals were ischemic controls. Histology and immunostaining were obtained at 1 and 2 weeks (4 TMR and 3 controls at each time point) and at 4 weeks (8 TMR and 5 controls) for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), nerve growth factor (βNGF), substance P (SP), and monocyte chemoattractant protein-1 (MCP-1). Immunoreactivity was scored using a digital image analysis system. Factor VIII staining was used for blood vessel counting. Enhanced regional expression of VEGF, bFGF and MCP-1 in the TMR group was noted at 1 and 2 weeks with a threefold increase at 4 weeks following TMR compared to controls. βNGF expression in the TMR group was enhanced at 1 and 2 weeks with subsequent decline at 4 weeks to the controls level. SP expression was not significantly different between groups at all time points. There was a twofold increase in the number of blood vessels in the TMR group at 4 weeks, which was not apparent earlier. Conclusions: These immunohistological findings suggest that cytokines expression compatible with angiogenesis and neuromodulation occurs early after TMR. Up-regulation of angiogenic and inflammatory cytokines may be more sustained than neuromodulation.

Notes: Three experiments were conducted that were designed to evaluate our ability to predict essential amino acid (EAA) needs of hybrid striped bass using the quantified lysine requirement and whole-body amino acid concentrations. In the first experiment, six diets containing various amino acid profiles were fed to triplicate groups of fish initially weighing 7.7 g per fish. At the end of the 8-week experiment, no significant differences were detected in growth rates or feed efficiencies (FE) between fish fed a practical diet containing 510 g kg−1 herring fish meal (FM) and fish fed a purified diet containing the amino acid profile of herring fish meal (CAA-FM). Growth responses of fish fed purified diets containing 100 (HSB), 110 (HSB110), 120 (HSB120) or 140 g 100 g−1 (HSB140) of the amino acid profile of hybrid striped bass whole-bodies were significantly lower than those of fish fed diet FM. In the second experiment, triplicate groups of fish (5.6 g per fish) were fed diets containing various energy : protein (E : P) ratios (34.8, 41.2, 47.5 and 53.9 kJ g−1 protein) and one of two amino acid profiles (CAA-FM and HSB120) in a 4 × 2 factorial design. Carbohydrate concentration was varied to achieve the desired energy concentrations. At the end of the 8-week experiment, weight gain and FE were significantly higher in fish fed diets formulated to simulate the amino acid profile of herring fish meal (CAA-FM) compared with fish fed diets formulated to contain 120 g 100 g−1 of the amino acid profile of hybrid striped bass whole-bodies (HSB120). Weight gain, FE and survival data indicated the optimum dietary E : P was 41.2 kJ g−1 protein. Dietary treatments in the final experiment included three amino acid profiles and four levels of lipid in a 3 × 4 incomplete factorial design. Dietary amino acid treatments included the amino acid profile of herring fish meal (CAA-FM) or 120 g 100 g−1 of the predicted EAA requirement profile for hybrid striped bass (HSB120). The amino acid profile of the remaining dietary treatment (PRED+) was similar to that of the HSB120 treatment, but contained additional threonine, isoleucine and tryptophan. Diets CAA-FM and HSB120 contained either 90, 130, 170 or 210 g kg−1 lipid, whereas diet PRED+ contained 130 g kg−1 lipid. Dietary treatments were fed for 10 weeks to triplicate groups of fish initially weighing 81.0 g per fish. Weight gain and FE were not significantly affected by dietary amino acid profile. Feed efficiency was significantly reduced in fish fed diets containing 210 g kg−1 lipid compared with fish fed diets containing 90–170 g kg−1 lipid. Intraperitoneal fat (IPF) ratio and hepatosomatic index (HSI) values generally increased as dietary lipid concentrations increased. Total liver lipid concentrations were significantly reduced in fish fed diets containing 210 g kg−1 lipid compared with those of fish fed 90–130 g kg−1 lipid. Results of this study indicate an appropriate dietary amino acid profile can be predicted for hybrid striped bass using the quantified lysine requirement and whole-body amino acid concentrations. Further, the optimum E : P appears to be 40 kJ g−1 protein.

Notes: Calmodulin is a ubiquitous highly conserved calcium binding protein involved in cell signalling. Previous studies in our laboratory suggested a role for calmodulin in prepenetration morphogenesis in Colletotrichum trifolii, the causal agent of alfalfa anthracnose. In this report, we describe the cloning, sequencing and partial characterization of the calmodulin gene from C. trifolii. The gene is present as a single copy in the genome of C. trifolii and its predicted amino acid sequence shows considerable homology to other fungal calmodulins. The gene is most highly expressed during conidial germination and appressorial development. Using a Neurospora crassa inducible promoter driving the calmodulin gene in antisense orientation, transformants were obtained with constitutive levels of antisense calmodulin expression. Upon induction, transformants did not develop appressoria and were not pathogenic on alfalfa plants.

Notes: Ras is a small monomeric GTP binding protein that transduces signals for growth and differentiation of eukaryotic organisms. Previously, a unique ras gene, designated Ct-ras, was cloned from the alfalfa fungal phytopathogen, Colletotrichum trifolii. Expression of Ct-Ras in mouse fibroblast cells (NIH3T3) demonstrated that Ct-ras is functionally similar to the mammalian ras genes since activating mutations of Ct-ras caused oncogenic phenotypes in nu/nu mice, including tumors. In C. trifolii, activated ‘oncogenic’ Ras (Val2) induced abnormal hyphal proliferation, defects in polarized growth and significantly reduced differentiation such as conidiation and appressorium formation in a nutrient dependent manner. Gene disruption of ct-ras was lethal. To further evaluate the function of Ct-Ras in C. trifolii, three different approaches were used: overexpression of cytosolic Ras by CAAX box deletion; expression of dominant negative Ct-RasT22N; and antisense ct-ras expression. Results showed that suppression of Ct-Ras activity significantly decreases fungal germination frequencies and hyphal growth rates. Taken together, these data suggest involvement of Ct-Ras in regulation of fungal cell growth and differentiation.

Notes: The small G-protein superfamily is an evolutionarily conserved group of GTPases that regulate diverse signalling pathways including pathways for growth and development in eukaryotes. Previously, we showed that dominant active mutation in the unique Ras gene (DARas) of the fungal phytopathogen Colletotrichum trifolii displays a nutrient-dependent phenotype affecting polarity, growth and differentiation. Signalling via the MAP kinase pathway is significantly impaired in this mutant as well. Here we describe the cloning and functional characterization of Rac (Ct-Rac1), a member of the Rho family of G proteins. Ct-Rac1 expression is downregulated by DARas under limiting nutrition. Co-expression of DARas with dominant active Rac (DARac) stimulates MAPK activation and restores the wild-type phenotype. Inhibition of MAPK activation suppresses phenotypic restoration suggesting Rac-mediated MAPK activation is responsible for reversion to the wild-type phenotype. We also examined the role of reactive oxygen species (ROS) in these genetic backgrounds. The DARas mutant strain generates high levels of ROS as determined by DCFH-DA fluorescence. Co-expression with DNRac decreases ROS generation to wild-type levels and restores normal fungal growth and development. Pretreatment of DARas with antioxidants or a cytosolic phospholipase A2 inhibitor also restores the wild-type phenotype. These findings suggest that Ras-mediated ROS generation is dependent on a Rac–cPLA2-linked signalling pathway. Taken together, this study provides evidence that Rac functions to restore the hyphal morphology of DARas by regulating MAPK activation and intracellular ROS generation.