Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

Stopped-flow kinetic data were obtained for the iron-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase) using methacrylonitrile as the substrate. Multiple turnover experiments suggest a three-step kinetic model that allows for the reversible binding of substrate, the presence of an intermediate, and the formation of product. Microscopic rate constants determined from these data are in good agreement with steady state data confirming that the stopped-flow method used was appropriate for the reaction. Single turnover stopped-flow experiments were used to identify catalytic intermediates. These data were globally fit confirming a three-step kinetic model. Independent absorption spectra acquired between 0.005 and 0.5 s of the reaction reveal a significant increase in absorbance at 375, 460, and 550 nm along with the hypsochromic shift of an Fe³⁺←S ligand-to-metal charge transfer band from 700 to 650 nm. The observed UV-visible absorption bands for the Fe³⁺-nitrile intermediate species are similar to low spin Fe³⁺-enzyme and model complexes bound by NO or N₃⁻. These data provide spectroscopic evidence for the direct coordination of the nitrile substrate to the nitrile hydratase active site low spin Fe³⁺ center.     

Internal forces and moments in the femur of the rat during gait

The rat is of increasing importance for experimental studies on fracture healing. The healing outcome of long bone fractures is strongly influenced by mechanical factors, such as the interfragmentary movement. This movement depends on the stability of the fracture fixation and the musculoskeletal loads. However, little is known about these loads in rats.The musculoskeletal loads during gait were estimated using an inverse-dynamic musculoskeletal model of the right hindlimb of the rat. This model was based on a micro-CT scan of the lower extremities and an anatomical study using 15 rat cadavers. Kinematics were reconstructed from X-ray movies, taken simultaneously from two perpendicular directions during a gait cycle. The ground reaction forces were taken from the literature. The muscle forces were calculated using an optimization procedure.The internal forces and moments varied over the gait cycle and along the femoral axis. The greatest internal force (up to 7 times bodyweight) acted in the longitudinal direction. The greatest internal moment (up to 13.8 bodyweight times millimeter) acted in the sagittal plane of the femur. The validity of the model was corroborated by comparing the estimated strains caused by the calculated loads on the surface of the femoral mid-shaft with those from the literature.Knowledge of the internal loads in the femur of the rat allows adjustment of the biomechanical properties of fixation devices in fracture healing studies to the desired interfragmentary movement.     

Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence

Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 μg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo.     

Inhibition of ATPase from chloroplasts by a hydroxamic acid from the gramineae

-DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), a hydroxamic acid from the Gramineae involved in the resistance of cereals to aphids, inhibits     

ent-labdane glycosides from Baccharis pingraea.

The aerial parts of Baccharis pingraea, collected in Chile, afforded, in addition to two diterpene glycosides isolated previously, 14 new ones and two of the corresponding desacyl aglycones. The structures were elucidated by high field 1H NMR spectroscopy. The differences between the collections from Argentina and Chile are discussed.     

Neuroprotective effects of genistein in VSC4.1 motoneurons exposed to activated microglial cytokines

Pro-inflammatory cytokines released from activated microglia may be responsible for neuronal damage and resulting motor deficits associated with CNS disorders such as spinal cord injury, Parkinson’s disease, and multiple sclerosis. Estrogen (17β-estradiol) is capable of ameliorating motoneuron death following spinal cord injury, but has a number of deleterious side effects. Genistein (GEN), an estrogen receptor beta agonist and potent antioxidant, may represent an alternative to estrogen in treating neurodegenerative disorders. However, little is known about the neuroprotective effects of GEN. We therefore tested whether GEN would prevent apoptosis in cultured motoneurons following exposure to pro-inflammatory cytokines released from IFN-γ activated microglia. Exposure of ventral spinal cord 4.1 motoneurons to microglial cytokine supernatant in vitro caused significant apoptosis and reduced mitochondrial membrane potential. An increase in reactive oxygen species, intracellular Ca²⁺, calpain, caspases, cytochrome c, and the bax:bcl-2 ratio were also noted. GEN treatment reversed apoptotic death and cellular changes following cytokine exposure and was associated with increased expression of estrogen receptor β suggesting that GEN may promote neuroprotection via receptor-mediated pathways. The addition of ICI 182, 780, an estrogen receptor antagonist following GEN treatment attenuated neuroprotection, suggesting that GEN may act mainly via estrogen receptor β to protect VSC4.1 motoneurons. We conclude that GEN protects cultured ventral spinal cord 4.1 cells from inflammatory insult and thus may represent a potential beneficial therapy in the treatment of neurodegenerative disorders.