Mutant Study of Sinorhizobium meliloti Proline Utilization A (PutA)

The purpose of this project is to purify and characterize the reaction kinetics of mutant versions the enzyme Proline Utilization A (PutA) in Sinorhizobium meliloti. The enzyme catalyzes the first step in proline metabolism. It has two active sites. The first is proline dehydrogenase (PRODH) which converts proline to pyrroline-5-carboxylate (P5C). The second is P5C dehydrogenase (P5CDH) which converts P5C to glutamate. Although many bacterial organisms have PutA, there are still significant interspecies variations, resulting in an entire family of PutA enzymes. The main difference is the length of the amino acid sequence. This affects the protein’s structure or its shape, and the protein’s kinetics or how it behaves in reactions. In order to have a complete understanding of proline metabolism, all the variations of PutA must be characterized both structurally and kinetically. The version of PutA found in S. meliloti (SmPutA) has been categorized structurally but not kinetically. This project aims to fill this gap in our knowledge of proline metabolism and PutA from S. meliloti

Slowly cycling Rho kinase-dependent actomyosin cross-bridge slippage explains intrinsic high compliance of detrusor smooth muscle

Biological soft tissues are viscoelastic because they display timeindependent pseudoelasticity and time-dependent viscosity. However, there is evidence that the bladder may also display plasticity, defined as an increase in strain that is unrecoverable unless work is done by the muscle. In the present study, an electronic lever was used to induce controlled changes in stress and strain to determine whether rabbit detrusor smooth muscle (rDSM) is best described as viscoelastic or viscoelastic plastic. Using sequential ramp loading and unloading cycles, stress-strain and stiffness-stress analyses revealed that rDSM displayed reversible viscoelasticity, and that the viscous component was responsible for establishing a high stiffness at low stresses that increased only modestly with increasing stress compared with the large increase produced when the viscosity was absent and only pseudoelasticity governed tissue behavior. The study also revealed that rDSM underwent softening correlating with plastic deformation and creep that was reversed slowly when tissues were incubated in a Ca2+ -containing solution. Together, the data support a model of DSM as a viscoelastic-plastic material, with the plasticity resulting from motor protein activation. This model explains the mechanism of intrinsic bladder compliance as slipping cross bridges, predicts that wall tension is dependent not only on vesicle pressure and radius but also on actomyosin cross-bridge activity, and identifies a novel molecular target for compliance regulation, both physiologically and therapeutically

Low prevalence of fibrosis in thalassemia major assessed by late gadolinium enhancement cardiovascular magnetic resonance

<p>Abstract</p> <p>Background</p> <p>Heart failure remains a major cause of mortality in thalassaemia major. The possible role of cardiac fibrosis in thalassemia major in the genesis of heart failure is not clear. It is also unclear whether cardiac fibrosis might arise as a result of heart failure.</p> <p>Methods</p> <p>We studied 45 patients with thalassaemia major who had a wide range of current cardiac iron loading and included patients with prior and current heart failure. Myocardial iron was measured using T2* cardiovascular magnetic resonance (CMR), and following this, late gadolinium enhancement (LGE) was used to determine the presence of macroscopic myocardial fibrosis.</p> <p>Results</p> <p>The median myocardial T2* in all patients was 22.6 ms (range 5.3-58.8 ms). Fibrosis was detected in only one patient, whose myocardial T2* was 20.1 ms and left ventricular ejection fraction 57%. No fibrosis was identified in 5 patients with a history of heart failure with full recovery, in 3 patients with current left ventricular dysfunction undergoing treatment, or in 18 patients with myocardial iron loading with cardiacT2* < 20 ms at the time of scan.</p> <p>Conclusion</p> <p>This study shows that macroscopic myocardial fibrosis is uncommon in thalassemia major across a broad spectrum of myocardial iron loading. Importantly, there was no macroscopic fibrosis in patients with current or prior heart failure, or in patients with myocardial iron loading without heart failure. Therefore if myocardial fibrosis indeed contributes to myocardial dysfunction in thalassemia, our data combined with the knowledge that the myocardial dysfunction of iron overload can be reversed, indicates that any such fibrosis would need to be both microscopic and reversible.</p

First Evidence for Substrate Channeling between Proline Catabolic Enzymes \u3ci\u3eA VALIDATION OF DOMAIN FUSION ANALYSIS FOR PREDICTING PROTEIN-PROTEIN INTERACTIONS\u3c/i\u3e

Background: PRODH and P5CDH from Thermus thermophilus are monofunctional enzymes in proline catabolism. Results: Steady-state kinetics and intermediate trapping data show the PRODH and P5CDH reactions are coupled by a channeling step. Conclusion: Substrate channeling in monofunctional enzymes is achieved via weak interactions. Significance: Evidence for substrate channeling between monofunctional proline catabolic enzymes is shown and confirms the Rosetta Stone hypothesis

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenaseand ∆\u3csup\u3e1\u3c/sup\u3e-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline UtilizationA(PutA)

Background: PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism. Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step. Conclusion: Substrate channeling in PutA exhibits hysteretic behavior. Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation

Neutron Vibrational Spectroscopy and First-Principles Calculations of the Ternary Hydrides Li\u3csub\u3e4\u3c/sub\u3eSi\u3csub\u3e2\u3c/sub\u3eH(D) and Li\u3csub\u3e4\u3c/sub\u3eGe\u3csub\u3e2\u3c/sub\u3eH(D): Electronic Structure and Lattice Dynamics

Using combined neutron spectroscopy and first-principles calculations, we investigated the electronic structure and vibrational dynamics of the recently discovered class of ternary hydrides Li4Tt2H (Tt=Si and Ge). In these compounds, all hydrogen atoms are located in a single type of Li6-defined octahedral site. The Tt atoms form long-range Tt-Tt chains sandwiched between each Li6-octahedra layer. The Li-H interactions are strongly ionic, with bond lengths comparable to those in LiH. Our density functional theory calculations indicate that Li atoms transfer their electrons to both H and Tt atoms. Tt atoms within the Tt-Tt chain are bonded covalently. The electronic density of states reveals that both hydrides exhibit metallic behavior. The observed vibrational spectra of these hydrides are in good overall agreement with the calculated phonon modes. There is evidence of dispersion induced splitting in the optical phonon peaks that can be ascribed to the coupling of H vibrations within the Li6-octahedra layers

Small-angle X-ray Scattering Studies of the Oligomeric State and Quaternary Structure of the Trifunctional Proline Utilization A (PutA) Flavoprotein from \u3ci\u3eEscherichia coli\u3c/i\u3e

Background: Trifunctional proline utilization A (PutA) proteins are multifunctional flavoproteins that catalyze two reactions and repress transcription of the put regulon. Results: PutA from Escherichia coli is a V-shaped dimer, with the DNA-binding domain mediating dimerization. Conclusion: Oligomeric state and quaternary structures are not conserved by PutAs. Significance: The first three-dimensional structural information for any trifunctional PutA is reported

Small-angle X-ray Scattering Studies of the Oligomeric State and Quaternary Structure of the Trifunctional Proline Utilization A (PutA) Flavoprotein from \u3ci\u3eEscherichia coli\u3c/i\u3e

Background: Trifunctional proline utilization A (PutA) proteins are multifunctional flavoproteins that catalyze two reactions and repress transcription of the put regulon. Results: PutA from Escherichia coli is a V-shaped dimer, with the DNA-binding domain mediating dimerization. Conclusion: Oligomeric state and quaternary structures are not conserved by PutAs. Significance: The first three-dimensional structural information for any trifunctional PutA is reported

Structure and Interstitial Deuterium site of ß-phase ZrNi Deuteride

ß-ZrNiD1-x (for x≈0.1, near the ß-γ phase boundary) was found to possess a triclinic P1(overline) structure as determined by high-resolution neutron power diffraction. This is very different from the widely accepted orthohombic and distorted orthorhombic Cmcm structures previously proposed. In contrast to the single type of D site associated with these latter structures, the true ß-ZrNiD1-x structure contains two crystallographically distinct interstitial D sites: Zr4Ni2 octahedral sites and Zr4 tetrahedral sites, alternately ordered along the a direction. From first-principles calculations, the total energy of the P1(overline) structure was found to be ≈0.24 eV per unit cell lower than Cmcm-symmetry ZrNiD and could be rationalized in terms of different D local-bonding configurations and metal-deuterium interactions. Resultant phonon calculations based on this structure were also consistnet with the measured neutron vibrational spectrum