Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography

Weak protein interactions are often characterized in terms of the osmotic second virial coefficient (B(22)), which has been shown to correlate with protein phase behavior, such as crystallization. Traditional methods for measuring B(22), such as static light scattering, are too expensive in terms of both time and protein to allow extensive exploration of the effects of solution conditions on B(22). In this work we have measured protein interactions using self-interaction chromatography, in which protein is immobilized on chromatographic particles and the retention of the same protein is measured in isocratic elution. The relative retention of the protein reflects the average protein interactions, which we have related to the second virial coefficient via statistical mechanics. We obtain quantitative agreement between virial coefficients measured by self-interaction chromatography and traditional characterization methods for both lysozyme and chymotrypsinogen over a wide range of pH and ionic strengths, yet self-interaction chromatography requires at least an order of magnitude less time and protein than other methods. The method thus holds significant promise for the characterization of protein interactions requiring only commonly available laboratory equipment, little specialized expertise, and relatively small investments of both time and protein.     

Correlation of diafiltration sieving behavior of lysozyme-BSA mixtures with osmotic second virial cross-coefficients

The role of protein-protein interactions in membrane separations of protein mixtures remains incompletely understood, largely due to the difficulty of characterizing protein self- and, especially, cross-association. Recently, a novel technique, cross-interaction chromatography, has been developed to measure weak protein cross-association in terms of the osmotic second virial cross-coefficient. In this work the relationship between protein cross-association and the sieving behavior of lysozyme in the presence of BSA has been investigated. Sieving coefficients were measured using a stirred diafiltration cell over a range of pH and ionic strength, and a striking correlation between the lysozyme sieving and second virial cross-coefficients for BSA/lysozyme mixtures has been found: when the protein cross-interactions are most attractive (negative second virial cross-coefficient), the lysozyme sieving coefficients are lowest, and vice versa. The correlation between the sieving and second virial cross-coefficients may be due to the physically similar environments in the chromatography and filtration experiments since one protein is passed through a concentrated region of the second protein either immobilized on the column or accumulated at the membrane surface, and the migration rate of the mobile protein in both cases is influenced by protein cross-association. This study represents the first time that molecular interactions in binary mixtures have been related directly to filtration behavior, and may provide a useful approach to optimize the separation of other binary protein mixtures.     

Opioid receptor-coupled second messenger systems

Although pharmacological data provide strong evidence for different types of opioid receptors (e.g., mu, delta, and kappa), they share many common properties in their ability to couple to second messenger systems. All opioid receptor types are coupled to G-proteins, since agonist binding is diminished by guanine nucleotides and agonist-stimulated GTPase activity has been identified in several preparations. Moreover, all three types inhibit adenylyl cyclase. This second messenger system has been identified for opioid receptors in both isolated brain membranes and in transformed cell culture. Studies with chronic treatment with opioid agonists suggest that the coupling of receptors with G-proteins and second messenger effectors may play important roles in development of opioid tolerance.     

Effects of second order photobleaching on recovered diffusion parameters from fluorescence photobleaching recovery

In the original theoretical development of fluorescence photobleaching recovery with circular or Gaussian laser intensity profiles (Axelrod et al., 1976, Biophys. J.) the bleaching process is assumed to obey first order kinetics in the fluorescent probe. While this is reasonable in most cases where oxygen participates in the photolysis reaction, some processes may obey second order kinetics in the fluorophore concentration due to dimerization. Accordingly, we present here an analysis of the fluorescence recovery when the photobleaching process is taken to be second order in the probe. Analytical solutions for small bleaching levels indicate that the fluorescence recovery curve is very similar to that measured following a bleaching process first order in the probe. Numerical solutions for moderate bleaching levels show that the recovery is qualitatively similar, but quantitatively different. Because the shape of the recovery curve provides no evidence as to the order of photobleaching, we recommend continued use of the previous theoretical analysis. However, it must be borne in mind that the diffusion coefficient is increasingly underestimated as the extent of photobleaching is increased. The true diffusion coefficient is obtained in the limit of small levels of photobleaching. Estimates of the fractional recovery are not affected by this approach.     

Combined second and third toe transfer

Historically, restoration of hand function following multiple digital amputation has been unsatisfactory. The evolution of digital reconstruction with toe transfer has enabled surgeons to reestablish prehension in these severely injured hands. A 4-year experience with 26 consecutive combined second and third toe transfers to replace missing adjacent fingers was reviewed in order to delineate the indications and technical considerations and to emphasize prevention of donor-site complications. Combined second and third toe transfer is reserved for adjacent finger amputations proximal to the digital web space with remaining fingers no longer than the small finger. Radial amputations are replaced with contralateral combined toe units, while ipsilateral toes are more ideal for ulnar amputations. Limited dorsal and plantar skin flaps extending only to the midpoint of the first and third digital web spaces allow for direct donor-site closure and uncomplicated healing. Maintenance of the plantar metatarsal arch by avoiding metatarsal shaft osteotomies or bone grafting-shortened metatarsals eliminates potential gait disturbances. When properly applied in selected patients, this single-stage microsurgical procedure can restore prehensile function, improve the appearance of the hand with multiple digital amputations, and preserve near-normal donor-foot function.     

Potent second generation vinyl sulfonamide inhibitors of the trypanosomal cysteine protease cruzain

A new family of potent N-alkoxyvinylsulfonamide inhibitors of cruzain have been developed. Inhibitor 13 has a second order inactivation rate constant of 6,480,000s(-1)M(-1) versus cruzain, and is also highly effective against Trypanosoma cruzi trypomastigotes in a tissue culture assay.     

Hand dominance and bilateral asymmetry in the structure of the second metacarpal

Bilateral asymmetry in the structure of the second metacarpal was examined in relation to functional hand dominance in a large, clinically nonselected, healthy population sample from the Baltimore Longitudinal Study of Aging. Bilateral bone measurements were made from anteroposterior hand radiographs of a total of 992 individuals, 609 males and 383 females, with an age range of 19–94 years. Hand dominance was determined on the basis of personal impression. Total width and medullary width at the midshaft of the second metacarpal were measured to 0.05 mm using a Helios caliper. These two measurements were used to derive cortical thickness, cortical bone area, periosteal (total) area, medullary area, percent cortical area, and the second moment of area in the mediolateral plane. In both right and left-handed individuals, statistically significant side differences were found in the calculated bone areas and the second moment of area, with the dominant hand being larger. Cortical thickness did not show significant side-related differences for either handedness. These results show that functional handedness leads to periosteal and endosteal expansion of the second metacarpal cortex on the dominant side, increasing bone strength without increasing cortical thickness. This is the first time this pattern of asymmetry has been reported in left-handers as well as right-handers. Our results argue for the primacy of environmental (mechanical) effects in determining bilateral asymmetry of limb bone structural properties. © 1994 Wiley-Liss, Inc.     

Rethinking transnationalism through the second generation

The concept of transnationalism was developed to describe the ties maintained by migrants with their homelands and, until recently, little attention was given to the question of whether these ties would be continued by the migrants’ children, the ‘second generation’. This article discusses the transnational practices of second‐generation Tongans in Australia, using the research findings to reconsider some of the existing assumptions about what constitutes transnationalism. To fully understand second‐generation transnationalism, three forms of transnationalism are described: intradiasporic, indirect and forced transnationalism. Taking these forms into account reveals a more complex picture of the transnational ties maintained by migrants and their children than has been drawn in previous accounts.     

Intracellular stainings of the large ocellar second order neurons in the cockroach

Summary The large ocellar second order neurons (L-neurons) in the cockroach,Periplaneta americana have been studied physiologically by intracellular recordings and morphologically by intracellular and whole nerve cobalt stainings. All the recorded L-neurons showed similar light responses, i.e., light on-hyperpolarization and a small number of off-spikes. All the stained L-neurons had an ocellar arborization covering the whole region of the ocellar neuropile and an central arborization in the region posterior to the protocerebral bridge.     

A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.     

A second extensin-like hydroxyproline-rich glycoprotein from carrot cell walls

The insoluble extensin matrix of dicot cell walls has been studied most fruitfully by examining the salt-extractable precursors to this matrix. Multiple extensin-like hydroxyproline-rich glycoproteins (HRGPs) have been isolated, or their existence inferred, from tomato, potato, bean, soybean, melon, carrot, and other plants. We and others previously have studied a carrot extensin which we call extensin-1. Here we report on the properties of extensin-2, a second salt-extractable hydroxyproline-rich glycoprotein from carrot. Like extensin-1, extensin-2 contains large amounts of hydroxyproline, serine, histidine, and lysine. In contrast, its tyrosine content is only about one-third that of extensin-1. Arabinose and galactose are the most abundant neutral sugars in both proteins, and nearly identical buoyant densities in CsCl suggest a similar proportion of carbohydrate in each. The size of extensin-2 is about half the size of extensin-1 based on: (a) the measured lengths of shadowed molecules (about 40 versus 84 nanometers); (b) the migration of extensin-2 in acid-urea gels relative to monomers, dimers, and trimers of extensin-1; and (c) the Stokes' radii of these molecules as determined by gel filtration chromatography. Electron microscopy of shadowed extensin-2 molecules indicates that they contain kinks, which may indicate the presence of intramolecular isodityrosine cross-links, but intermolecular cross-links, either with other extensin-2 molecules or extensin-1 molecules, are observed rarely if ever.     

Tick histamine-binding proteins: lipocalins with a second binding cavity

Tick histamine-binding proteins (HBPs) are lipocalins with two binding pockets. One of these binds histamine with a high affinity and is found at the position expected from other lipocalins, adjacent to the omega-loop at the open-end of the beta-barrel. A second binding cavity, which is a low-affinity site for histamine in one of the HBPs, is located at the end of the barrel that is closed off in other lipocalins. In order to create the second site, the 'closed-end' region has undergone a major reconstruction. Typical lipocalin characteristics, such as the 3(10) helix and a structural cluster of highly conserved residues, have been lost, while an alpha-helix now shields the cavity from the exterior. The prominence of acidic residues in the binding pockets is another distinctive characteristic of HBPs. Whereas most lipocalins have highly hydrophobic binding cavities designed to bind lipophilic compounds, HBPs have evolved to trap cationic, hydrophilic molecules.     

Bimetallic Cobalt‐Based Phosphide Zeolitic Imidazolate Framework: CoPx Phase‐Dependent Electrical Conductivity and Hydrogen Atom Adsorption Energy for Efficient Overall Water Splitting

Cobalt‐based bimetallic phosphide encapsulated in carbonized zeolitic imadazolate frameworks has been successfully synthesized and showed excellent activities toward both hydrogen evolution reaction (HER) and oxygen evolution reaction (OER). Density functional theory calculation and electrochemical measurements reveal that the electrical conductivity and electrochemical activity are closely associated with the Co₂P/CoP mixed phase behaviors upon Cu metal doping. This relationship is found to be the decisive factor for enhanced electrocatalytic performance. Moreover, the precise control of Cu content in Co‐host lattice effectively alters the Gibbs free energy for H* adsorption, which is favorable for facilitating reaction kinetics. Impressively, an optimized performance has been achieved with mild Cu doping in Cu_0.3Co_2.7P/nitrogen‐doped carbon (NC) which exhibits an ultralow overpotential of 0.19 V at 10 mA cm^–2 and satisfying stability for OER. Cu_0.3Co_2.7P/NC also shows excellent HER activity, affording a current density of 10 mA cm^–2 at a low overpotential of 0.22 V. In addition, a homemade electrolyzer with Cu_0.3Co_2.7P/NC paired electrodes shows 60% larger current density than Pt/RuO₂ couple at 1.74 V, along with negligible catalytic deactivation after 50 h operation. The manipulation of electronic structure by controlled incorporation of second metal sheds light on understanding and synthesizing bimetallic transition metal phosphides for electrolysis‐based energy conversion.     

The second human calcitonin/CGRP gene is located on chromosome 11

Summary A second human calcitonin/calcitonin gene related peptide (hCT/CGRP) gene has been identified. This second hCT/CGRP gene has been shown to contain sequences highly homologous to exons 3, 5 (CGRP-encoding), and 6 of the first hCT/CGRP gene, but sequences closely related to exon 4 (CT-encoding) could not be demonstrated. Southern blot hybridization analysis of DNA from human-rodent somatic cell hybrids showed that the second hCT/CGRP gene is located in the q12-pter region of chromosome 11. The first hCT/CGRP gene has previously been assigned to the p13–p15 region of chromosome 11.     

Direct measurement of protein osmotic second virial cross coefficients by cross-interaction chromatography

The importance of weak protein interactions, such as protein self-association, is widely recognized in a variety of biological and technological processes. Although protein self-association has been studied extensively, much less attention has been devoted to weak protein cross-association, mainly due to the difficulties in measuring weak interactions between different proteins in solution. Here a framework is presented for quantifying the osmotic second virial cross coefficient directly using a modified form of self-interaction chromatography called cross-interaction chromatography. A theoretical relationship is developed between the virial cross coefficient and the chromatographic retention using statistical mechanics. Measurements of bovine serum albumin (BSA)/lysozyme cross-association using cross-interaction chromatography agree well with the few osmometry measurements available in the literature. Lysozyme/alpha-chymotrypsinogen interactions were also measured over a wide range of solution conditions, and some counterintuitive trends were observed that may provide new insight into the molecular origins of weak protein interactions. The virial cross coefficients presented in this work may also provide insight into separation processes that are influenced by protein cross-interactions, such as crystallization, precipitation, and ultrafiltration.     

Phase-Transfer Identification of Core-Shell Structures in Bimetallic Nanoparticles

On the basis of a combination of previously published experimental procedures, ultraviolet–visible spectroscopy, transmission electron microscopy, and energy-dispersive X-ray measurements, a systematic investigation was carried out on the phase-transfer characteristics of different bimetallic nanoparticles (Ag–Au, Ag–Pt, Ag–Ru, Au–Pt, Au–Ru, and Pt–Ru) formed by the seed-mediated growth reactions. The different phase-transfer characteristics of the monometallic nanoparticles of Au, Ag, Pt, and Ru were used to form the basis of differentiation between various possible structures existing in the bimetallic systems (core-shell particles or a physical mixture of nanoparticles). The experimental results indicate clearly the formation of core-shell nanoparticles of Ag–Au, Ag–Pt, Ru–Ag, Pt–Au, Au–Ru, and Pt–Ru when the nanoparticles of the first metal were used as the seeds in the seed-mediated growth reactions. However, when the order of the synthesis was reversed using the nanoparticles of the second metal as the seeds, only a physical mixture of the two metal nanoparticles was obtained instead.Parts of the data on Au–Ru and Ag–Pt systems have been published in Analytica Chimica Acta (2005, 537, 279–284) and Journal of Physical Chemistry B (2005, 109, 5468–5472), respectively.     

Luteinizing hormone secretion as a response to a second naltrexone administration

Previous studies with naltrexone (Nalt), a "long-lasting" opioid antagonist, demonstrated a rapid increase in luteinizing hormone (LH) secretion which gradually declined, reaching baseline values after 1 hr. A second Nalt challenge, 120 min later, caused only a blunted response. This poor reaction has been shown in this study not to be due to lack of pituitary responsiveness, because LH-releasing hormone treatment revealed a normal response. A time-response study was carried out in order to establish the refractory period length, by administering a second Nalt injection at 0 hr (immediately after the first injection) and at 2, 4, 8, 16, and 24 hr after the first bolus. Partial responsiveness could be achieved 2 and 4 hr after the first challenge. However, only after 8 hr was a full response recorded. The diurnal changes in serum LH (nadir at 18.00 hr) did not affect the response to Nalt challenge. It is suggested that in the presence of a Nalt blockade, nonopioid systems are able to "normalize" LH blood levels. However, when Nalt blood levels have fallen sufficiently to allow the endogenous opioid system to take primary control again, then a second Nalt injection will provoke a renewed response.     

The novel application of Benford's second order analysis for monitoring radiation output in interventional radiology

Benford's law is an empirical observation which predicts the expected frequency of digits in naturally occurring datasets spanning multiple orders of magnitude, with the law having been most successfully applied as an audit tool in accountancy. This study investigated the sensitivity of the technique in identifying system output changes using simulated changes in interventional radiology Dose-Area-Product (DAP) data, with any deviations from Benford's distribution identified using z-statistics.The radiation output for interventional radiology X-ray equipment is monitored annually during quality control testing; however, for a considerable portion of the year an increased output of the system, potentially caused by engineering adjustments or spontaneous system faults may go unnoticed, leading to a potential increase in the radiation dose to patients. In normal operation recorded examination radiation outputs vary over multiple orders of magnitude rendering the application of normal statistics ineffective for detecting systematic changes in the output. In this work, the annual DAP datasets complied with Benford's first order law for first, second and combinations of the first and second digits. Further, a continuous ‘rolling’ second order technique was devised for trending simulated changes over shorter timescales. This distribution analysis, the first employment of the method for radiation output trending, detected significant changes simulated on the original data, proving the technique useful in this case. The potential is demonstrated for implementation of this novel analysis for monitoring and identifying change in suitable datasets for the purpose of system process control.     

Reversible inhibition of the second step of splicing suggests a possible role of zinc in the second step of splicing

A multicomponent complex of proteins and RNA is assembled on the newly synthesized pre-mRNA to form the spliceosome. This complex catalyzes a two-step transesterification reaction required to remove the introns and ligate the exons. To date, only six proteins have been found necessary for the second step of splicing in yeast, and their human homologs have been identified. We demonstrate that the addition of the selective chelator of zinc, 1,10-phenanthroline, to an in vitro mRNA splicing reaction causes a dose-dependent inhibition of the second step of splicing. This inhibition is accompanied by the accumulation of spliceosomes paused before completion of step two of the splicing reaction. The inhibition effect on the second step is due neither to snRNA degradation nor to direct binding to the mRNA, and is reversible by dialysis or add-back of zinc, but not of other divalent metals, at the beginning of the reaction. These findings suggest that the activity of a putative zinc-dependent metalloprotein(s) involved in the second step of splicing is affected. This study outlines a new method for specific reversible inhibition of the second step of splicing using external reagents, and suggests a possible role of divalent cations in the second step of mRNA splicing, most likely zinc.