Diagnostic Laparoscopy for Small Intestinal Intussusception in a Horse

Laparoscopy is a low-invasive diagnostic and surgical technique for examining and performing surgical procedures in the equine peritoneal cavity. This article is a case study of a horse with weakly expressed, irregular symptoms of colic occurring over a period of four weeks. Diagnostic laparoscopy was performed, and liver and spleen tissue samples were collected for a histopathological analysis. An endoscopic examination of the abdominal cavity ruled out small intestinal intussusception, and a histopathological analysis supported the identification of the causes of colic

Structure and multiple conformations of the Kunitz-type domain from human type VI collagen alpha3(VI) chain in solution.

BACKGROUND: The Kunitz-type inhibitor motif is found at the C terminus of the human collagen alpha3(VI) chain. This 76-residue module (domain C5) was prepared in recombinant form and showed high stability against proteases; however, it lacked any inhibitory activity against trypsin, thrombin, kallikrein and several other proteases. We have undertaken the determination of the three-dimensional (3D) structure of domain C5 in solution, by nuclear magnetic resonance (NMR), in order to establish the structural basis for the properties of this protein. RESULTS: The 7 N-terminal and 12 C-terminal residues of domain C5 are disordered in the solution structure. The 55-residue core, which shows high homology to bovine pancreatic trypsin inhibitor, retains the characteristic fold of all members of the Kunitz-type inhibitor family. 24 residues of this main structural body show more than one resonance, symptomatic of multiple conformations slowly exchanging on the NMR time scale. In addition, significant proton chemical exchange line broadening is observed for residues in the vicinity of the disulfide bridge between residues 20 and 44: this indicates interconversion, on the micro- to millisecond time scale, between multiple conformations. CONCLUSION: The NMR study demonstrates that domain C5 is a highly dynamic molecule at temperatures studied (between 10 and 30 degrees C). Indeed, some 44% of the main body structure of C5 showed multiple conformations. The existence of multiple conformations was not necessarily expected in view of the conformational constraints imposed by the 3D structure of proteins as rigid as C5; it should therefore be considered in the interpretation of its structural and dynamical properties. The accessibility of the inhibitory binding loop (Gly18 [P4] to Leu25 [P4']) should be relatively unaffected by this conformational exchange and thus would not explain the unusual specificity of C5. Most serine proteinase inhibitors that, like C5, have an arginine at the P1 position inhibit trypsin; the lack of trypsin inhibition of C5 must therefore arise from the amino-acid side-chain composition of the adjoining positions in the binding loop

CS23D: a web server for rapid protein structure generation using NMR chemical shifts and sequence data

CS23D (chemical shift to 3D structure) is a web server for rapidly generating accurate 3D protein structures using only assigned nuclear magnetic resonance (NMR) chemical shifts and sequence data as input. Unlike conventional NMR methods, CS23D requires no NOE and/or J-coupling data to perform its calculations. CS23D accepts chemical shift files in either SHIFTY or BMRB formats, and produces a set of PDB coordinates for the protein in about 10–15 min. CS23D uses a pipeline of several preexisting programs or servers to calculate the actual protein structure. Depending on the sequence similarity (or lack thereof) CS23D uses either (i) maximal subfragment assembly (a form of homology modeling), (ii) chemical shift threading or (iii) shift-aided de novo structure prediction (via Rosetta) followed by chemical shift refinement to generate and/or refine protein coordinates. Tests conducted on more than 100 proteins from the BioMagResBank indicate that CS23D converges (i.e. finds a solution) for >95% of protein queries. These chemical shift generated structures were found to be within 0.2–2.8 Å RMSD of the NMR structure generated using conventional NOE-base NMR methods or conventional X-ray methods. The performance of CS23D is dependent on the completeness of the chemical shift assignments and the similarity of the query protein to known 3D folds. CS23D is accessible at http://www.cs23d.ca

Design, Synthesis, and Biological Evaluation of 2-Hydroxy-4-phenylthiophene-3-carbonitrile as PD-L1 Antagonist and Its Comparison to Available Small Molecular PD-L1 Inhibitors

In search of a potent small molecular PD-L1 inhibitor, we designed and synthesized a compound based on a 2-hydroxy-4-phenylthiophene-3-carbonitrile moiety. Ligand's performance was tested in vitro and compared side-by-side with a known PD-L1 antagonist with a proven bioactivity BMS1166. Subsequently, we modified both compounds to allow 18F labeling that could be used for PET imaging. Radiolabeling, which is used in drug development and diagnosis, was applied to investigate the properties of those ligands and test them against tissue sections with diverse expression levels of PD-L1. We confirmed biological activity toward hPD-L1 for this inhibitor, comparable with BMS1166, while holding enhanced pharmacological properties. </p

Eur. J. Biochem.

The alpha-phycoerythrocyanin subunits of the different phycoerythrocyanin complexes of the phycobilisomes from the cyanobacterium Mastigocladus laminosus perform a remarkable photochemistry. Similar to phytochromes - the photoreceptors of higher plants - the spectral properties of the molecule reversibly change according to the irradiation wavelength. To enable extensive analyses, the protein has been produced at high yield by improving purification protocols. As a result, several comparative studies on the Z - and E -configurations of the intact alpha-subunit, and also on photoactive peptides originating from nonspecific degradations of the chromoprotein, were possible. The analyses comprise absorbance, fluorescence and CD spectroscopy, crystallization, preliminary X-ray measurements, mass spectrometry, N-terminal amino acid sequencing and 1D NMR spectroscopy. Intact alpha- phycoerythrocyanin aggregates significantly, due to hydrophobic interactions between the two N-terminal helices. Removal of these helices reduces the aggregation but also destabilizes the protein fold. The complete subunit could be crystallized in its E -configuration, but the X-ray measurement conditions must be improved. Nevertheless, NMR spectroscopy on a soluble photoactive peptide presents the first insight into the complex chromophore protein interactions that are dependent on the light induced state. The chromophore environment in the Z - configuration is rigid whereas other regions of the protein are more flexible. In contrast, the E -configuration has a mobile chromophore, especially the pyrrole ring D, while other regions of the protein rigidified compared to the Z -configuration

J. Mol. Biol.

P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D H-1-N-15 HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30% near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(- 1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83% native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions. (C) 2002 Academic Press