Early intermediates in the photocycle of the Glu46Gln mutant of photoactive yellow protein: femtosecond spectroscopy

Transient absorption spectroscopy in the time range from -1 ps to 4 ns, and over the wavelength range from 420 to 550 nm, was applied to the Glu46Gln mutant of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. This has allowed us to elucidate the kinetic constants of excited state formation and decay and photochemical product formation, and the spectral characteristics of stimulated emission and the early photocycle intermediates. Both the quantum efficiency ( approximately 0.5) and the rate constants for excited state decay and the formation of the initial photochemical intermediate (I(0)) were found to be quite similar to those obtained for wild-type PYP. In contrast, the rate constants for the formation of the subsequent photocycle intermediates (I(0)(double dagger) and I(1)), as well as for I(2) and for ground state regeneration as determined in earlier studies, were found to be from 3- to 30-fold larger. The structural implications of these results are discussed.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A1AO ATP synthase of Methanosarcina mazei Gö1

The interaction of the nucleotide-binding subunit B with subunit F is essential in coupling of ion pumping and ATP synthesis in A₁A_O ATP synthases. Here we provide structural and thermodynamic insights on the nucleotide binding to the surface of subunits B and F of Methanosarcina mazei Gö1 A₁A_O ATP synthase, which initiated migration to its final binding pocket via two transitional intermediates on the surface of subunit B. NMR- and fluorescence spectroscopy as well as ITC data combined with molecular dynamics simulations of the nucleotide bound subunit B and nucleotide bound B-F complex in explicit solvent, suggests that subunit F is critical for the migration to and eventual occupancy of the final binding site by the nucleotide of subunit B. Rotation of the C-terminus and conformational changes in subunit B are initiated upon binding with subunit F causing a perturbation that leads to the migration of ATP from the transition site 1 through an intermediate transition site 2 to the final binding site 3. This mechanism is elucidated on the basis of change in binding affinity for the nucleotide at the specific sites on subunit B upon complexation with subunit F. The change in enthalpy is further explained based on the fluctuating local environment around the binding sites.     

Follicular and endocrinological turnover associated with GnRH induced follicular wave synchronization in Indian crossbred cows

The goal of this study was to record the hormonal and follicular turnover in Jersey crossbred cows when subjected for follicular wave synchronization using GnRH. Six healthy, non-lactating and regularly cycling Jersey crossbred cows (5-6 y) were used for the study. In the control group, the follicular wave pattern was ultrasonographically investigated in 18 cycles (3 cycles/cow). In the treatment group, GnRH analogue (buserelin acetate 10 μg im) was administered on Day 6 of the cycle and follicular wave pattern was studied in 12 cycles (2 cycles/animal). Follicular population was categorized based on their diameter Class I, ≤5 mm; Class II, >5-<9 mm; Class III, ≥9 mm) and the number of follicles in each category was determined on Day 6, Day 8 and Day 10. Plasma FSH and progesterone concentrations were estimated in both control and treatment groups. Out of 18 estrous cycles studied, 14 cycles (77.8%), three cycles (16.7%) and one cycle (5.6%) exhibited three-, two- and four-follicular waves per cycle, respectively. It was evident that the DF of Wave I established its dominance and was in the growing phase by Day 6 of the estrous cycle in all the normally cycling crossbred cows. The DF ovulated in all the animals (100%) in the mean interval of 27.7 ± 0.2 h after GnRH administration. A synchronized homogenous group of follicles emerged two days after GnRH injection (Day of 8.0 ± 0.0) in all the animals (100%). The combination of LH surge induced ovulation of DF (abrupt termination of Wave I) and FSH surge stimulated homogenous recruitment of Class I follicles, led to a synchronized emergence of follicular wave. All the GnRH treated cows had three follicular waves because of early emergence and short period of dominance of Wave II DF.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

A couple stress model of blood flow in the microcirculation

A simple mathematical model depicting blood flow in the capillary is developed with an emphasis on the permeability property of the blood vessel based on Starling's hypothesis. In this study the effect of inertia has been neglected in comparison with the viscosity on the basis of the smallness of the Reynolds number of the flow in the capillary. The capillary blood vessel is approximated by a circular cylindrical tube with a permeable wall. The blood is represented by a couple stress fluid. With such an ideal model the velocity and pressure fields are determined. It is shown that an increase in the couple stress parameter increases the resistance to the flow and thereby decreases the volume rate flow. A comparison of the results with those of the Newtonian case has also been made.     

Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Abeta(1-40) peptide in solid-supported lipid bilayers

We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid beta peptide(1-40) in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of approximately 2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mM zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.     

Plasmon-waveguide resonance studies of ligand binding to the human beta 2-adrenergic receptor

Plasmon-waveguide resonance (PWR) spectroscopy is an optical technique that can be used to probe the molecular interactions occurring within anisotropic proteolipid membranes in real time without requiring molecular labeling. This method directly monitors mass density, conformation, and molecular orientation changes occurring in such systems and allows determination of protein-ligand binding constants and binding kinetics. In the present study, PWR has been used to monitor the incorporation of the human beta(2)-adrenergic receptor into a solid-supported egg phosphatidylcholine lipid bilayer and to follow the binding of full agonists (isoproterenol, epinephrine), a partial agonist (dobutamine), an antagonist (alprenolol), and an inverse agonist (ICI-118,551) to the receptor. The combination of differences in binding kinetics and the PWR spectral changes point to the occurrence of multiple conformations that are characteristic of the type of ligand, reflecting differences in the receptor structural states produced by the binding process. These results provide new evidence for the conformational heterogeneity of the liganded states formed by the beta(2)-adrenergic receptor.