Response of cytochrome a,a3 to carbon monoxide in canine hearts with prior infarcts

The effects of moderate levels of carbon monoxide (CO) on the oxidation-reduction state of cytochrome a,a3 (cyt a,a3) were examined in the hearts of twelve dogs with a prior myocardial infarction. Exposure to ten minutes CO produced a carboxyhemoglobin (CO-Hb) level of 9.4%, a level experienced by heavy smokers. Accompanying the exposure to CO, cyt a,a3 became more reduced; 17.4% +/- 4.7%. Exposure to CO was accompanied by an increase of 33% +/- 4% in the rate of cyt a,a3 reduction following occlusion of the left circumflex coronary artery and a decrease of 24% +/- 8% in the rate of cyt a,a3 oxidation with release. There was also a decrease in the magnitude of cyt a,a3 reduction from 86% +/- 9% to 70% +/- 11%. These results indicate that moderate levels of CO trap cyt a,a3 in the reduced state which impairs the ability of the heart to recover from transient ischemic episodes.     

Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes

Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.     

The Function of Vacuolar ATPase (V-ATPase) a Subunit Isoforms in Invasiveness of MCF10a and MCF10CA1a Human Breast Cancer Cells

The vacuolar H⁺ ATPases (V-ATPases) are ATP-driven proton pumps that transport protons across both intracellular and plasma membranes. Previous studies have implicated V-ATPases in the invasiveness of various cancer cell lines. In this study, we evaluated the role of V-ATPases in the invasiveness of two closely matched human breast cancer lines. MCF10a cells are a non-invasive, immortalized breast epithelial cell line, and MCF10CA1a cells are a highly invasive, H-Ras-transformed derivative of MCF10a cells selected for their metastatic potential. Using an in vitro Matrigel assay, MCF10CA1a cells showed a much higher invasion than the parental MCF10a cells. Moreover, this increased invasion was completely sensitive to the specific V-ATPase inhibitor concanamycin. MCF10CA1a cells expressed much higher levels of both a1 and a3 subunit isoforms relative to the parental line. Isoforms of subunit a are responsible for subcellular localization of V-ATPases, with a3 and a4 targeting V-ATPases to the plasma membrane of specialized cells. Knockdown of either a3 alone or a3 and a4 together using isoform-specific siRNAs inhibited invasion by MCF10CA1a cells. Importantly, overexpression of a3 but not the other a subunit isoforms greatly increased the invasiveness of the parental MCF10a cells. Similarly, overexpression of a3 significantly increased expression of V-ATPases at the plasma membrane. These studies suggest that breast tumor cells employ particular a subunit isoforms to target V-ATPases to the plasma membrane, where they function in tumor cell invasion.     

Preferential expression of anti-azobenzenearsonate antibodies of heterozygous a1a3 rabbits in the a1 allotype.

The preferential expression of anti-As antibodies in the allotype a1 of heterozygous a1a3 rabbits immunized with As-TMA-BSA has been investigated by means of quantitative methods. The average content of the anti-As antibodies in a1 and a3 allotypes was 84 and 11%, respectively; the analogous values for anti-TMA antibodies were 41 and 56%, and for anti-BSA antibodies they were 54 and 41%. The molar anti-As/anti-TMA ratios in the heterozygous a1a1 rabbits sensitized with As-TMA-BSA. The very low yields of anti-As-antibodies of allotype a3 cannot be caused by a lack of genes for the production of anti-As antibodies of allotype a3 because a3a3 homozygotes produce considerable amounts of anti-As antibody of allotpye a3. Competition between lymphoid cells having anti-As receptors of different allotype and different affinity for the antigenic p-azobenzenearsonate determinant is discussed as a possible cause for the preferential expression in the a1 allotype.     

Fast ignition upon the implosion of a thin shell onto a precompressed deuterium-tritium ball

Fast ignition of a precompressed inertial confinement fusion (ICF) target by a hydrodynamic material flux is investigated. A model system of hydrodynamic objects consisting of a central deuterium-tritium (DT) ball and a concentric two-layer shell separated by a vacuum gap is analyzed. The outer layer of the shell is an ablator, while the inner layer consists of DT ice. The igniting hydrodynamic flux forms as a result of laser-driven acceleration and compression of the shell toward the system center. A series of one-dimensional numerical simulations of the shell implosion, the collision of the shell with the DT ball, and the generation and propagation of thermonuclear burn waves in both parts of the system are performed. Analytic models are developed that describe the implosion of a thin shell onto a central homogeneous ball of arbitrary radius and density and the initiation and propagation of a thermonuclear burn wave induced by such an implosion. Application of the solution of a model problem to analyzing the implosion of a segment of a spherical shell in a conical channel indicates the possibility of fast ignition of a spherical ICF target from a conical target driven by a laser pulse with an energy of 500?C700 kJ.     

Characterization of the basis of lipoprotein [a] lysine-binding heterogeneity

Although elevated plasma concentrations of lipoprotein [a] (Lp[a]) are considered to be a risk factor for atherosclerosis, the mechanisms by which Lp[a] mediates its pathogenic effects have not been conclusively determined. The apolipoprotein [a] (apo[a]) component of Lp[a] confers unique structural properties to this lipoprotein, including the ability to bind to lysine residues in biological substrates. It has been shown, however, that only a fraction of plasma Lp[a] (Lp[a]-Lys(+)) binds to lysine-Sepharose in vitro. The nature of the non-lysine-binding Lp[a] fraction in plasma (Lp[a]-Lys(-)) is currently unknown. In the present study, the Lp[a]-Lys(+) fraction was determined in the plasma of six unrelated individuals; the Lp[a]-Lys(+) fraction in these plasma samples ranged from approximately 37 to approximately 48%. Interestingly, purification of the Lp[a] by density gradient ultracentrifugation followed by gel filtration and ion-exchange chromatography resulted in progressive increases in the Lp[a]-Lys(+) fraction. Addition of either purified low density lipoprotein (LDL) or fibronectin to the purified Lp[a] at a 1:1 molar ratio reduced the Lp[a]-Lys(+) fraction (maximal decrease of 34 and 20%, respectively) whereas addition of both fibronectin and LDL to the purified Lp[a] resulted in a further decrease (45% maximally) in this fraction. Similar results were obtained by using a recombinant expression system for apo[a]: addition of a 4-fold molar excess of either LDL or fibronectin to conditioned medium containing metabolically labeled recombinant apo[a] reduced the Lys(+) fraction by 49 and 23%, respectively.Taken together, our data suggest that the lysine-binding heterogeneity of plasma Lp[a] is not primarily an intrinsic property of the lipoprotein, but rather results in large part from its ability to noncovalently associate with abundant plasma components such as LDL and fibronectin. These interactions appear to mask the lysine-binding site in apo[a] kringle IV type 10, which mediates the interaction of Lp[a] with lysine-Sepharose. The contribution of these interactions to the function of Lp[a] in vivo remains to be investigated.     

6-Aminohexanoic acid as a chemical chaperone for apolipoprotein(a)

Apolipoprotein (a) (apo(a)) is a component of the atherogenic lipoprotein, Lp(a). The efficiency with which apo(a) escapes the endoplasmic reticulum (ER) and is secreted by the liver is a major determinant of plasma Lp(a) levels. Apo(a) contains a series of domains homologous to plasminogen kringle (K) 4, each of which possesses a potential lysine-binding site. By using primary mouse hepatocytes expressing a 17K4 human apo(a) protein, we found that high concentrations (25-200 mM) of the lysine analog, 6-aminohexanoic acid (6AHA), increased apo(a) secretion 8-14-fold. This was accompanied by a decrease in apo(a) presecretory degradation. 6AHA inhibited accumulation of apo(a) in the ER induced by the proteasome inhibitor, lactacystin. Thus, 6AHA appeared to inhibit degradation by increasing apo(a) export from the ER. Significantly, 6AHA overcame the block in apo(a) secretion induced by the ER glucosidase inhibitor, castanospermine. 6AHA may therefore circumvent the requirement for calnexin and calreticulin interaction in apo(a) secretion. Sucrose gradients and a gel-based folding assay were unable to detect any influence of 6AHA on apo(a) folding. However, non-covalent or small, disulfide-dependent changes in apo(a) conformation would not be detected in these assays. Proline also increased the efficiency of apo(a) secretion. We propose that 6AHA and proline can act as chemical chaperones for apo(a).     

Increased virulence and competitive advantage of a/alpha over a/a or alpha/alpha offspring conserves the mating system of Candida albicans

The majority of Candida albicans strains in nature are a/alpha and must undergo homozygosis to a/a or alpha/alpha to mate. Here we have used a mouse model for systemic infection to test the hypothesis that a/alpha strains predominate in nature because they have a competitive advantage over a/a and alpha/alpha offspring in colonizing hosts. Single-strain injection experiments revealed that a/alpha strains were far more virulent than either their a/a or alpha/alpha offspring. When equal numbers of parent a/alpha and offspring a/a or alpha/alpha cells were co-injected, a/alpha always exhibited a competitive advantage at the time of extreme host morbidity or death. When equal numbers of an engineered a/a/alpha2 strain and its isogenic a/a parent strain were co-injected, the a/a/alpha2 strain exhibited a competitive advantage at the time of host morbidity or death, suggesting that the genotype of the mating-type (MTL) locus, not associated genes on chromosome 5, provides a competitive advantage. We therefore propose that heterozygosity at the MTL locus not only represses white-opaque switching and genes involved in the mating process, but also affects virulence, providing a competitive advantage to the a/alpha genotype that conserves the mating system of C. albicans in nature.     

Cell Proliferation and Oxygen Diffusion in a Vascularising Scaffold

The supply of oxygen to proliferating cells within a scaffold is a key factor for the successful building of new tissue in soft tissue engineering applications. A recent in vivo model, where an arteriovenous loop is placed in a scaffold, allows a vascularising network to form within a scaffold, establishing an oxygen source within, rather than external, to the scaffold. A one-dimensional model of oxygen concentration, cell proliferation and cell migration inside such a vascularising scaffold is developed and investigated. In addition, a vascularisation model is presented, which supports a vascularisation front which moves at a constant speed. The effects of vascular growth, homogenous and heterogenous seeding, diffusion of cells and critical hypoxic oxygen concentration are considered. For homogenous seeding, a relationship between the speed of the vascular front and a parameter defining the rate of oxygen diffusion relative to the rate of oxygen consumption determines whether a hypoxic region exists at some time. In particular, an estimate of the length of time that a fixed point in the scaffold will remain under hypoxic conditions is determined. For heterogenous seeding, a Fisher-like travelling wave of cells is established behind the vascular front. These findings provide a fundamental understanding of the important interplay between the parameters and allows for a theoretical assessment of a seeding strategy in a vascularising scaffold.     

淀粉样蛋白Aβ的插膜作用可以抑制其形成纤维

作为老年性痴呆(AD)患者脑中淀粉样斑块的核心蛋白,β-淀粉样蛋白(Aβ)是从淀粉样前体蛋白(APP)水解而来。该蛋白是多种长度多肽的混合物,其中Aβ40和Aβ42是主要组分。分别研究了膜中胆固醇含量及溶液pH对Aβ40和Aβ42形成纤维的影响。电镜观察发现,含有胆固醇的脂质体几乎可以完全抑制Aβ40的纤维形成,而低pH只能部分地抑制Aβ42的纤维形成。单层膜的实验证明这两种因素都有利于Aβ40和Aβ42的插膜。构象研究表明插膜会诱导Aβ40和Aβ42的二级结构发生不同的变化。结果说明,Aβ40和Aβ42的插膜作用能够在一定程度上抑制蛋白形成纤维,但两者具有不同的抑制机制。     

Morphology and development of the reproductive shoots of Lilaea scilloides (Poir.) Hauman (Alismatidae)

Shoots of adult plants of Lilaea scilloides have a sympodial form. Each unit of the sympodium bears a single sheathing prophyll (which is the only kind of foliage leaf produced in the adult) and terminates in an inflorescence. The prophyll subtends the next unit of the sympodium. A further accessory bud can form in association with each unit. This bud repeats the pattern of the main sympodium, giving the plant a tufted habit. Five different kinds of flower can be identified in the inflorescence: a unisexual male flower with a single perianth member and adnate stamen; a bisexual flower, with a single perianth member and adnate stamen, and a single carpel with an anatropous bitegmic ovule; a unisexual female flower with a single perianth member and carpel; a unisexual female flower comprising only a single carpel; and a female flower comprising only a single carpel with a very long filamentous style. The first four kinds occur in the upper part of the inflorescence which is normally elevated on a scape, while the last kind is restricted to the base of the inflorescence. In the position of the basal flowers several variations have been observed in cultivated material. These include branching associated with the basal flowers, which results in the development of additional basal flowers or inflorescences, and even total replacement of a basal flower by an inflorescence or a branching structure bearing flowers. A review of past literature includes a clarification of some persistent errors which have confused the taxonomic position of the plant and the morphological interpretation of the reproductive appendages.     

Oxidation of apolipoprotein(a) inhibits kringle-associated lysine binding: the loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site.

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide-linked to apolipoprotein B-100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2'-azobis-(amidinopropane) HCl (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r-apo(a), to bind through their LBS domains. AAPH treatment caused a time-dependent decrease in the number of functional Lp(a) or r-apo(a) molecules capable of binding to fibrin or lysine-Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r-apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle-containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r-apo(a) was observed as assessed by native or SDS-PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl-containing compounds increase the LBS affinity of Lp(a) for fibrin.     

A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly

Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.     

Apolipoprotein(a): expression and characterization of a recombinant form of the protein in mammalian cells

We have stably expressed a recombinant form of apo(a) in a human embryonic kidney cell line. The engineered protein (predicted mass of 250 kDa) contains 17 copies of the apo(a) domain, which resembles kringle 4 of plasminogen, followed by the plasminogen-like kringle 5 and protease-like domain of apo(a). The recombinant protein [r-apo(a)] was isolated from cell culture media by immunoaffinity chromatography, and its physical properties were studied. As is the case for apo(a) isolated from plasma-derived Lp(a), r-apo(a) is highly glycosylated (23% by weight), containing both N- and O-linked glycans, which results in an observed molecular mass of 500 kDa by SDS-PAGE. The high sialic acid content was reflected in a pI of 4.3 for the r-apo(a). Two subpopulations of r-apo(a) secreted by the permanent cell line were identified with respect to lysine-Sepharose binding; the majority of the r-apo(a) bound specifically to this matrix and was eluted with epsilon-aminocaproic acid (epsilon-ACA). When the r-apo(a) plasmid was used to transfect a human hepatoma cell line, lipoprotein particles were secreted containing the disulfide-linked complex of apoB-100 and the r-apo(a). The density of these particles was shown to be heterogeneous, with the majority of the r-Lp(a) floating in the density range of plasma-derived Lp(a).     

Effects of Epidemic Diseases on the Distribution of Bonobos

This study examined how outbreaks and the occurrence of Anthrax, Ebola, Monkeypox and Trypanosomiasis may differentially affect the distribution of bonobos (Pan paniscus). Using a combination of mapping, Jaccard overlapping coefficients and binary regressions, the study determined how each disease correlated with the extent of occurrence of, and the areas occupied by, bonobos. Anthrax has only been reported to occur outside the range of bonobos and so was not considered further. Ebola, Monkeypox and Trypanosomiasis were each reported within the area of occupancy of bonobos. Their respective overlap coefficients were: J = 0.10; Q_{α = 0.05} = 2.00 (odds ratios = 0.0001, 95% CI = 0.0057; Z = −19.41, significant) for Ebola; J = 1.00; Q_{α = 0.05} = 24.0 (odds ratios = 1.504, 95% CI = 0.5066–2.6122) for Monkeypox; and, J = 0.33; Q_{α = 0.05} = 11.5 (Z = 1.14, significant) for Trypanosomiasis. There were significant relationships for the presence and absence of Monkeypox and Trypanosomiasis and the known extent of occurrence of bonobos, based on the equations y = 0.2368Ln(x)+0.8006 (R² = 0.9772) and y = −0.2942Ln(x)+0.7155 (R² = 0.698), respectively. The positive relationship suggested that bonobos tolerated the presence of Monkeypox. In contrast, the significant negative coefficient suggested that bonobos were absent in areas where Trypanosomiasis is endemic. Our results suggest that large rivers may have prevented Ebola from spreading into the range of bonobos. Meanwhile, Trypanosomiasis has been recorded among humans within the area of occurrence of bonobos, and appears the most important disease in shaping the area of occupancy of bonobos within their overall extent of occupancy.