Improving mass spectrometric sequencing of arginine-containing peptides by derivatization with acetylacetone

Modification of arginine residues in bradykinin, [1-5]-bradykinin, splenopentin and two synthetic pentapeptides with acetylacetone (pentane-2,4-dione) significantly increases the relative abundance of sequence-specific fragment ions produced by matrix-assisted laser desorption/ionization (MALDI). The fragmentation efficiency as measured by post-source decay in a reflectron time-of-flight mass spectrometer increases by a factor of 2-3.5. Peptide bonds adjacent to modified residues are more susceptible to cleavage than in the non-derivatized peptide ions. The increased lability of these bonds gives rise to more complete sequence information. In addition, the relative abundances of sequence-specific fragment ions are enhanced. This strategy makes it possible to obtain valuable structural information from arginine-containing peptides that otherwise do not fragment well.     

Progression of interproximal caries in the primary dentition

In the patients with invasion to the aortic window, we performed operation via median sternotomy combined with anteroaxillar thoracotomy. In such patients with T4 invasion, conventional pneumonectomy could not be performed because of the extensive invasion near the main pulmonary artery trunk. In these patients in this study, complete resection of the involved pulmonary artery could be performed using a vascular clamp without CP bypass. Operative technique was as follows: first, the pericardium was opened and taping of the aorta was applied. When the uninvolved part of the intrapericardial pulmonary artery was long enough to cut, we could use a stapling device, but the stapling device could not be used in many cases because the length of the uninvolved segment was too short to cut the left pulmonary artery. In order to carry out complete resection, it was necessary to clamp the central part of the main pulmonary artery diagonally from the left lower side to the right upper side. The pulmonary arterial stump was closed with continuous 4-0 monofilament mattress and over and over suture. We recommend an aggressive surgical approach for the tumor with invasion to the aortic window, because the prognosis is dismal in nonresected locally advanced lung cancer.     

Antimicrobial resistance and molecular subtyping of Campylobacter jejuni and Campylobacter coli from retail meats

Campylobacter isolates (n = 297; 202 C. jejuni and 95 C. coli isolates) recovered from 2,513 retail meat samples (chicken breasts, ground turkey, ground beef, and pork chops) were examined for antimicrobial susceptibility. The isolates were further analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE) using SmaI and KpnI restriction enzymes, and a subset of isolates (n = 174) were subtyped by multilocus sequence typing (MLST). The resistance most frequently observed was that to doxycycline (27.6%), followed by ciprofloxacin (13.8%) and erythromycin (6.4%). All isolates were susceptible to gentamicin and meropenem. C. coli showed higher resistance to doxycycline than did C. jejuni (42.1 versus 20.8%) and lower resistance to ciprofloxacin than did C. jejuni (10.5 versus 15.3%). Erythromycin resistance was only observed in C. coli. PFGE using SmaI plus KpnI digestion generated 168 clusters from 297 isolates: 115 from C. jejuni and 53 from C. coli. MLST revealed 44 sequence types (STs) under 10 clonal complexes from 120 C. jejuni and 27 STs under two clonal complexes from 54 C. coli. There was a positive association between PFGE and STs; however, PFGE showed greater discriminatory power than MLST. Subtyping data did not correlate with antimicrobial resistance phenotypes.     

Vacuum Technology Considerations For Mass Metrology

Vacuum weighing of mass artifacts eliminates the necessity of air buoyancy correction and its contribution to the measurement uncertainty. Vacuum weighing is also an important process in the experiments currently underway for the redefinition of the SI mass unit, the kilogram. Creating the optimum vacuum environment for mass metrology requires careful design and selection of construction materials, plumbing components, pumping, and pressure gauging technologies. We review the vacuum technology¹ required for mass metrology and suggest procedures and hardware for successful and reproducible operation.     

Proteolysis of native proteins. Trapping of a reaction intermediate

When limited proteolysis of the mouse major urinary proteins by trypsin was stopped by rapid denaturation of the proteinase, a covalent adduct of the two proteins was observed. The formation of this complex required active trypsin, was favored at low pH, and could be reversed by the addition of covalent or non-covalent trypsin inhibitors. Electrospray mass spectrometry of the complex demonstrated that it was an acyl-enzyme complex, formed after an unusual exopeptidase attack on the C-terminal-Arg-Glu-OH sequence by trypsin. The complex could sequester over 50% of the trypsin in a digestion mixture, and as anticipated, the protein was an effective trypsin inhibitor.     

Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo

Complexes composed of peptide ligand for the serpin enzyme complex receptor covalently coupled to poly-L-lysine condensed by charge interaction with plasmid DNA direct gene transfer into receptor bearing cells. We compared intensity and duration of reporter gene expression in vitro and in vivo from serpin-enzyme receptor-directed gene transfer complexes prepared with poly-L-lysine of different chain lengths. When substituted with linker and ligand to comparable extents, DNA complexes containing short chain poly-L-lysine were larger and gave higher peak expression but significantly shorter duration of expression than those containing long chain poly-L-lysine. Both peak expression and duration of expression exceeded that observed with Lipofectin. Neither naked DNA nor DNA complexed with unsubstituted polylysine was effective in gene transfer. For in vivo experiments, complexes containing optimal ligand and degree of substitution (based on in vitro data, peptide C105Y, 11 ligands/plasmid DNA molecule) were prepared with either short chain or long chain polylysine and a beta-galactosidase expression plasmid. Following injection into the tail veins of mice, longer chain complexes gave significantly higher expression of reporter gene in lung and spleen that lasted for a significantly longer period of time than the shorter chain complexes. The short chain poly-L-lysine-DNA complexes were larger in diameter, as assessed by electron microscopy or atomic force microscopy, and gave less protection against DNase digestion in vitro than longer chain complexes. Thus, for gene transfer complexes directed at the serpin enzyme complex receptor, longer chain poly-L-lysine gave a much longer duration of expression both in vitro and in vivo. We speculate that this may be due to protection against degradation afforded the plasmid DNA by the tighter compaction produced by long chain poly-L-lysine.     

Polymeric Multilayers that Contain Silver Nanoparticles can be Stamped onto Biological Tissues to Provide Antibacterial Activity

The design of polyelectrolyte multilayers (PEMs) that can be prefabricated on an elastomeric stamp and mechanically transferred onto biomedically‐relevant soft materials, including medical‐grade silicone elastomers (E’～450–1500 kPa; E’‐elastic modulus) and the dermis of cadaver skin (E’～200–600 kPa), is reported. Whereas initial attempts to stamp PEMs formed from poly(allylamine hydrochloride) and poly(acrylic acid) resulted in minimal transfer onto soft materials, we report that integration of micrometer‐sized beads into the PEMs (thicknesses of 6–160 nm) led to their quantitative transfer within 30 seconds of contact at a pressure of ～196 kPa. To demonstrate the utility of this approach, PEMs were impregnated with a range of loadings of silver‐nanoparticles and stamped onto the dermis of human cadaver skin (a wound‐simulant) that was subsequently incubated with bacterial cultures. Skin dermis stamped with PEMs that released 0.25 ± 0.01 μg cm^?2 of silver ions caused a 6 log₁₀ reduction in colony forming units of Staphylococcus epidermidis and Pseudomonas aeruginosa within 12 h. Significantly, this level of silver release is below that which is cytotoxic to NIH 3T3 mouse fibroblast cells. Overall, this study describes a general and facile approach for the functionalization of biomaterial surfaces without subjecting them to potentially deleterious processing conditions.