Chicken joint amyloid protein is of the AA-type. I. Characterization of the amyloid protein

Amyloid fibrils were extracted from deposits in joint tissue of heavy breed layers with spontaneous amyloid arthropathy and characterized as being of the AA-type. Amino acid sequencing revealed a pattern quite similar to duck AA. Acute phase sera of chicken experimentally injected with Enterococcus faecalis showed a SAA-protein like band cross reacting with anti-chicken AA in immunoblot.     

An Integrated Force Probe and Quartz Crystal Microbalance for High-Speed Microtribology

We have developed a technique for measuring frictional forces and contact areas, over a wide range of applied loads, at microscopic contacts reaching high sliding speeds near 1 m/s. Our approach is based on integrating two stand-alone methods: nanoindentation and quartz crystal microbalance (QCM). Energy dissipation and lateral contact stiffness are monitored by a transverse shear quartz resonator, while a spherical indenter probe is loaded onto its surface. Variations in these two quantities as functions of shear amplitude, with the normal load held fixed, reveal a transition from partial to full slip at a critical amplitude. Average values of both the threshold force for full slip and the kinetic friction during sliding are determined from these trends, and the contact area is inferred from the lateral stiffness at low shear amplitudes. Measurements are performed at loads ranging from 5 µN to 8 mN using an electrostatically actuated indenter probe. For the materials chosen in this study, we find that the full slip threshold force is about a factor of two larger than kinetic friction. The forces increase sublinearly with load in close correspondence with the contact area, and the shear strengths are found to be relatively insensitive to pressure. The threshold shear amplitude scales in proportion to the contact radius. These results demonstrate that the probe–QCM technique is a versatile and full-featured platform for microtribology in the speed range relevant to practical applications.     

From mechanism to mouse: a tale of two bioorthogonal reactions

Bioorthogonal reactions are chemical reactions that neither interact with nor interfere with a biological system. The participating functional groups must be inert to biological moieties, must selectively reactive with each other under biocompatible conditions, and, for in vivo applications, must be nontoxic to cells and organisms. Additionally, it is helpful if one reactive group is small and therefore minimally perturbing of a biomolecule into which it has been introduced either chemically or biosynthetically. Examples from the past decade suggest that a promising strategy for bioorthogonal reaction development begins with an analysis of functional group and reactivity space outside those defined by Nature. Issues such as stability of reactants and products (particularly in water), kinetics, and unwanted side reactivity with biofunctionalities must be addressed, ideally guided by detailed mechanistic studies. Finally, the reaction must be tested in a variety of environments, escalating from aqueous media to biomolecule solutions to cultured cells and, for the most optimized transformations, to live organisms. Work in our laboratory led to the development of two bioorthogonal transformations that exploit the azide as a small, abiotic, and bioinert reaction partner: the Staudinger ligation and strain-promoted azide-alkyne cycloaddition. The Staudinger ligation is based on the classic Staudinger reduction of azides with triarylphosphines first reported in 1919. In the ligation reaction, the intermediate aza-ylide undergoes intramolecular reaction with an ester, forming an amide bond faster than aza-ylide hydrolysis would otherwise occur in water. The Staudinger ligation is highly selective and reliably forms its product in environs as demanding as live mice. However, the Staudinger ligation has some liabilities, such as the propensity of phosphine reagents to undergo air oxidation and the relatively slow kinetics of the reaction. The Staudinger ligation takes advantage of the electrophilicity of the azide; however, the azide can also participate in cycloaddition reactions. In 1961, Wittig and Krebs noted that the strained, cyclic alkyne cyclooctyne reacts violently when combined neat with phenyl azide, forming a triazole product by 1,3-dipolar cycloaddition. This observation stood in stark contrast to the slow kinetics associated with 1,3-dipolar cycloaddition of azides with unstrained, linear alkynes, the conventional Huisgen process. Notably, the reaction of azides with terminal alkynes can be accelerated dramatically by copper catalysis (this highly popular Cu-catalyzed azide-alkyne cycloaddition (CuAAC) is a quintessential "click" reaction). However, the copper catalysts are too cytotoxic for long-term exposure with live cells or organisms. Thus, for applications of bioorthogonal chemistry in living systems, we built upon Wittig and Krebs' observation with the design of cyclooctyne reagents that react rapidly and selectively with biomolecule-associated azides. This strain-promoted azide-alkyne cycloaddition is often referred to as "Cu-free click chemistry". Mechanistic and theoretical studies inspired the design of a series of cyclooctyne compounds bearing fluorine substituents, fused rings, and judiciously situated heteroatoms, with the goals of optimizing azide cycloaddition kinetics, stability, solubility, and pharmacokinetic properties. Cyclooctyne reagents have now been used for labeling azide-modified biomolecules on cultured cells and in live Caenorhabditis elegans, zebrafish, and mice. As this special issue testifies, the field of bioorthogonal chemistry is firmly established as a challenging frontier of reaction methodology and an important new instrument for biological discovery. The above reactions, as well as several newcomers with bioorthogonal attributes, have enabled the high-precision chemical modification of biomolecules in vitro, as well as real-time visualization of molecules and processes in cells and live organisms. The consequence is an impressive body of new knowledge and technology, amassed using a relatively small bioorthogonal reaction compendium. Expansion of this toolkit, an effort that is already well underway, is an important objective for chemists and biologists alike.     

Enabling Technologies in Carbohydrate Chemistry: Automated Glycan Assembly,Flow Chemistry and Data Science

The synthesis of defined oligosaccharides is a complex task. Several enabling technologies have been introduced in the last two decades to facilitate synthetic access to these valuable biomolecules. In this concept, we describe the technological solutions that have advanced glycochemistry using automated glycan assembly, flow chemistry and data science as examples. We highlight how the synergies between these different technologies can further advance the field, with progress toward the realization of a self-driving lab for glycan synthesis.     

Fragments of the constant region of immunoglobulin light chains are constituents of AL-amyloid proteins

Immunoglobulin light chains are the precursor proteins of AL-amyloidosis. In the fibril formation process properties of the variable part of the immunoglobulin light chains are believed to be of major importance. In this work it is shown that fragments of the constant part of the immunoglobulin light chain are a constituent of the AL-amyloid proteins of kappa type. A specific antiserum has identified these fragments in gel filtration fractions where the absorbance approached the base line after the main retarded peak. The fragments are small and have been overlooked previously in the purification process. The significance of the constant part in AL-proteins is unclear, but adds new aspects to the discussion of pre- or post-fibrillogenic cleavage of the immunoglobulin light chains.     

NMR Investigation of the Copper(II)-Ciprofloxacin System

A proton NMR study was performed on the copper(ll)-ciprofloxacin system. The proton relaxation times (T(1)) were determined from the titration data in acidic and basic media. In acidic medium the H5 signal is dramatically affected and it is assumed that copper is bonded to the quinolone through carbonyl and one of the carboxyl oxygens. Such bonding is in agreement with the X-ray literature data for the complex [Cu(cf)(1)]Cl(2).6H(2)O isolated from the slightly acidic solution. There are additional significant changes in (1)T(1) of H3' and H5' atoms which suggest that the terminal nitrogen atom of the piperazine ring system-N4' also interacts with copper in the basic conditions. Thus it is plausible that more than one species are present in the solution at high pH values.     

Resolution of a phase ambiguity in a calibration procedure forpolarimetric radar systems

A cross-pol/co-pol phase ambiguity in a previously published calibration procedure for polarimetric radar systems is discussed. The original procedure is modified to resolve the ambiguity while still retaining insensitivity to calibration target orientation. The modified form is then generalized and applied to an ultrawideband radar system for which the ambiguity in the original procedure is particularly evident     

Purification and characterization of plantaricin A, a Lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides

A Lactobacillus plantarum bacteriocin, plantaricin A, has been purified to homogeneity by ammonium sulphate precipitation, binding to cation exchanger and Octyl-Sepharose, and reverse-phase chromatography. The bacteriocin activity was associated with two peptides, termed alpha and beta, which were separated upon reverse-phase chromatography. Bacteriocin activity required the complementary action of both the alpha and beta peptides. From the N-terminal end, 21 and 22 amino acid residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acid residues, suggesting that either the C terminus had been reached or that modifications in the next amino acid residue blocked the sequencing reaction. Judging from their amino acid sequence, alpha and beta may be encoded by the same gene, since alpha appeared to be a truncated form of beta. Alanine, the first amino acid residue at the N-terminal end of beta was not present at this position in alpha. Otherwise the sequences of alpha and beta appeared to be identical. The calculated molecular masses of the sequenced part of alpha and beta were 2426 and 2497 Da, respectively. The molecular masses of alpha and beta as determined by mass spectroscopy were 2687 +/- 30 and 2758 +/- 30 Da, respectively, indicating that (i) the only difference between alpha and beta was the presence of the N-terminal alanine residue in beta, and that (ii) in addition to the sequenced residues, two to three unidentified amino acid residues are present at the C-terminal ends of the alpha and beta peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extended analysis of AL-amyloid protein from abdominal wall subcutaneous fat biopsy: kappa IV immunoglobulin light chain

OBJECTIVE: HIV-associated nephropathy is an important cause of morbidity that is characterized clinically by uremia and proteinuria and histologically by focal segmental glomerulosclerosis. In the largest series yet analyzed to our knowledge, we describe new sonographic findings and record the prevalence of other findings. We review the sonographic findings in a large group of HIV-infected patients. MATERIALS AND METHODS: Seventy-six consecutive HIV-infected patients underwent renal sonography. Abnormalities seen on sonography were recorded. RESULTS: Of 152 kidneys imaged, sonography showed that 30 kidneys (20%) were enlarged. Abnormal echogenicity was present in 136 kidneys (89%). Eighty-one kidneys (53%) were globular; 58 (38%) had decreased corticomedullary definition; 74 (49%) had decreased renal sinus fat; and 66 (43%) had heterogeneous parenchyma, some with echogenic striations. CONCLUSION: Our data reveal several sonographic abnormalities that have not previously been described: decreased corticomedullary definition, decreased renal sinus fat, parenchymal heterogeneity, and globular renal configuration. These new findings were found mainly in patients with advanced HIV infection.