Mutagenesis of the crystal contact of acidic fibroblast growth factor

Several mutations at Glu81 located on the crystal contact of human acidic fibroblast growth factor were studied in an effort to improve crystal growth. Mutation to Ser and Thr resulted in crystallization of a rather bulky form of the wild type, whereas mutation to Val prohibited crystallization. These results suggest that crystal growth may be controlled by designing a new interface by protein engineering

Very low-density lipoprotein-apoprotein CI is increased in diabetic nephropathy: Comparison with apoprotein CIII

Very low-density lipoprotein-apoprotein CI is increased in diabetic nephropathy: Comparison with apoprotein CIII.BackgroundRecent studies have suggested that apoprotein (apo) CI in very low-density lipoprotein (VLDL) plays an important role in causing hypertriglyceridemia independent of apo CIII, which is associated with coronary heart disease (CHD). Because the incidence of CHD is increased in diabetic patients and is even higher when diabetic nephropathy is developed, we measured apo CI levels in VLDL from type 2 diabetic patients, with various degree of nephropathy, and compared the results with those for healthy controls or nondiabetic patients with chronic renal failure (CRF).MethodsThis study enrolled healthy control subjects, type 2 diabetic patients with normoalbuminuria, microalbuminuria, overt proteinuria, and CRF on hemodialysis and nondiabetic hemodialyis patients. VLDL (density <1.006) was separated by ultracentrifugation. Then the apo CI, CIII, and B concentrations in VLDL were measured by enzyme-linked immunosorbent assay (ELISA).ResultsThe apo CI, CIII, and B concentrations in VLDL were respectively 3-, 2-, and 2-fold higher, respectively, in diabetic patients with overt proteinuria than in controls. Hemodialysis patients with diabetic nephropathy had levels of apo CI, CIII, and B in VLDL that were 2.6-, 2.7- and 2-fold higher, respectively, than those in controls. Nondiabetic hemodialysis patients also had a 2.7-fold higher level of VLDL apo CIII, whereas VLDL apo CI and VLDL apo B were not significantly increased. VLDL apo CI was significantly correlated with VLDL apo B independently of VLDL apo CIII level.ConclusionAn increase of VLDL apo CIII is a prominent feature of dyslipidemia in CRF patients, regardless of whether they are diabetic or nondiabetic, whereas an increase of VLDL apo CI is more specific to diabetic nephropathy and is closely associated with an increase of VLDL particle numbers, a new risk factor for CHD

Long-term effectiveness of right septal pacing vs. right apical pacing in patients with atrioventricular block

AbstractBackgroundLong-term right ventricular apical (RVA) pacing increases the risk of heart failure (HF) by inducing ventricular dyssynchronization. Although recent studies suggest that right ventricular septal (RVS) pacing results in improved short-term outcomes, its long-term effectiveness remains unclear.Methods and resultsThis study investigated 149 consecutive patients who underwent implantation of a dual chamber pacemaker for atrioventricular block with either RVS-pacing between July 2007 and June 2010 or RVA-pacing between January 2003 and June 2007. The endpoint was defined as death and hospitalization due to heart failure (HF). The rates of mortality and hospitalization due to HF were significantly lower in the RVS-pacing group than that in the RVA-pacing group (event free RVS: 1 year, 98% and 2 years, 98%; RVA: 1 year, 85% and 2 years, 81%; p<0.05). None of the patients died from HF in the RVS-pacing group, while 4 patients died from HF in the RVA-pacing group within 2 years after pacemaker implantation. The paced QRS interval was significantly shorter with RVS pacing than with RVA pacing at different times after pacemaker implantation (RVS: immediately 157.8±24.0ms, after 3 months 157.3±17.5ms, after 6 months 153.6±21.7ms, after 12 months 153.6±19.4ms, after 24 months 149.3±24.0ms vs. RVA: immediately 168.3±23.7ms, after 3 months 168.7±26.0ms, after 6 months 168.0±22.8ms, after 12 months 171.2±22.3ms, after 24 months 176.1±25.5ms; p<0.05).ConclusionsRVS pacing is feasible and safe with more favorable clinical benefits than RVA pacing

Developmental genetic bases behind the independent origin of the tympanic membrane in mammals and diapsids

International audienceThe amniote middle ear is a classical example of the evolutionary novelty. Although paleontological evidence supports the view that mammals and diapsids (modern reptiles and birds) independently acquired the middle ear after divergence from their common ancestor, the developmental bases of these transformations remain unknown. Here we show that lower-to-upper jaw transformation induced by inactivation of the Endothelin1-Dlx5/6 cascade involving Goosecoid results in loss of the tympanic membrane in mouse, but causes duplication of the tympanic membrane in chicken. Detailed anatomical analysis indicates that the relative positions of the primary jaw joint and first pharyngeal pouch led to the coupling of tympanic membrane formation with the lower jaw in mammals, but with the upper jaw in diapsids. We propose that differences in connection and release by various pharyngeal skeletal elements resulted in structural diversity, leading to the acquisition of the tympanic membrane in two distinct manners during amniote evolution

Recurrence of Atrial Fibrillation within Three Months after Pulmonary Vein Isolation in Patients with Paroxysmal Atrial Fibrillation : Analysis Using an External Loop Recorder with Auto-trigger Function

Pulmonary vein isolation (PVI) via catheter ablation has been shown to be a highly effective option for patients with symptomatic paroxysmal atrial brillation (AF). The recurrence of AF within 3 months after PVI is not considered a failure of the ablation procedure because early recurrence of AF is not always associated with late recurrence. We examined the usefulness of an external loop recorder with auto-trigger function (ELR-AUTO) to detect AF following PVI to characterize early recurrence and determine the implication of AF within 3 months after PVI. The study included 53 consecutive patients with symptomatic paroxysmal AF (age, 61.6 ± 12.6 years ; 77％ male) who underwent PVI, and were fitted with an ELR-AUTO for 7 ± 2 days within 3 months after PVI. Of the 33 patients(62.2％) who did not have AF within the 3-month period, only 1 patient had AF recurrence at 12 months. Seven of 20 patients (35％) who experienced AF within 3 months had symptomatic AF recurrence at 12 months. The sensitivity, specificity, positive predictive value, and negative predictive value of early AF recurrence for late recurrence was 87.5％, 71.1％, 35.0％, and 96.9％, respectively. Thus, AF recurrence detected by ELR-AUTO within 3 months after PVI can predict late AF recurrence. Freedom from AF in the firrst 3 months following ablation significantly predicts long-term freedom from AF. An ELR-AUTO is useful for detecting symptomatic and asymptomatic AF

Highly Efficient Ultracentrifugation-free Chromatographic Purification of Recombinant AAV Serotype 9

Recombinant adeno-associated virus serotype 9 (rAAV9) can specifically transduce muscle and neuronal tissues; thus, rAAV9 can potentially be used in gene therapy. However, rAAV9 is the most challenging rAAV serotype to purify. Traditionally, rAAV9 has been purified by ultracentrifugation, which is not scalable. We recently described a chromatographic purification protocol for rAAV1; this protocol can achieve scalable purifications. In this study, we attempted to optimize this protocol for purifying rAAV9 preparations, and we developed a novel, effective method for high-yield purification of rAAV9 using quaternary ammonium anion exchangers and size-exclusion chromatography. The final purified rAAV9 contained mainly three capsid proteins, as observed by SDS-PAGE. Furthermore, negative-stain electron microscopy demonstrated that 96.1% ± 1.1% of rAAV9 particles carried the viral genome containing the EGFP transgene, indicating that impurities and empty capsids can be eliminated with our purification protocol. The final rAAV9 titer obtained by our protocol totaled 2.5 ± 0.4 × 1015 viral genomes produced from ∼3.2 × 109 HEK293EB cells. We confirmed that our protocol can also be applied to purify other varied AAV genome constructs. Our protocol can scale up production of pure rAAV9, in compliance with current good manufacturing practice, for clinical applications in human gene therapy

Efficient synthesis of 6-O-palmitoyl-1,2-O-isopropylidene-α-D-glucofuranose in an organic solvent system by lipase-catalyzed esterification.

In order to synthesize a sugar ester at high concentration, 1, 2-O-isopropylidene-α-D-glucofuranose (IpGlc), which is one of the sugar acetals and is more hydrophobic than unmodified glucose, was esterified with palmitic acid at 40°C using immobilized lipase from Candida antarctica in some organic solvents or their mixtures. Acetone + t-butyl alcohol (3:1 v/v) improved both the initial reaction rate and yield after 80 h: the product reached its maximum value (240 mmol/kg solvent; ca. 110 g/kg solvent) when 400 mmol IpGlc/kg solvent and 1, 200 mmol palmitic acid/kg solvent were used in this solvent mixture