Direct amidation of poly(gamma-glutamic acid) with benzylamine in dimethyl sulfoxide

Partially benzylamidated, amphipathic poly(gamma-glutamic acid) (BzPGA) was synthesized from poly(gamma-glutamic acid) (PGA) and benzylamine by direct amidation in dimethyl sulfoxide (DMSO). Benzylamine and PGA were heated in DMSO for 1 to 26 h at temperatures between 110 and 130 degrees C, producing derivatives of various degrees of benzylamidation as a function of the reaction time and temperature. Neither any carboxyl-activating agent nor catalyst is needed for the reaction to proceed. After purification by dialysis, the product was identified by 1H and 13C 1D and 2D NMR in DMSO-d(6). BzPGA prepared by the new direct amidation method was identical to that obtained with a conventional carbodiimide-mediated reaction in water. The one-pot amidation procedure described in the present article can probably be applied to the synthesis of amides from other amines and carboxylic acids.     

Sequential unfolding of the hemolysin two‐partner secretion domain from Proteus mirabilis

Protein secretion is a major contributor to Gram‐negative bacterial virulence. Type Vb or two‐partner secretion (TPS) pathways utilize a membrane bound β‐barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C‐proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N‐proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site‐specific mutants of HpmA265. By correlating the effect of individual site‐specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C‐terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C‐terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N‐proximal polar core subdomain.     

Tracing the genetic origin of Europe's first farmers reveals insights into their social organization

Farming was established in Central Europe by the Linearbandkeramik culture (LBK), a well-investigated archaeological horizon, which emerged in the Carpathian Basin, in today''s Hungary. However, the genetic background of the LBK genesis is yet unclear. Here we present 9 Y chromosomal and 84 mitochondrial DNA profiles from Mesolithic, Neolithic Starčevo and LBK sites (seventh/sixth millennia BC) from the Carpathian Basin and southeastern Europe. We detect genetic continuity of both maternal and paternal elements during the initial spread of agriculture, and confirm the substantial genetic impact of early southeastern European and Carpathian Basin farming cultures on Central European populations of the sixth–fourth millennia BC. Comprehensive Y chromosomal and mitochondrial DNA population genetic analyses demonstrate a clear affinity of the early farmers to the modern Near East and Caucasus, tracing the expansion from that region through southeastern Europe and the Carpathian Basin into Central Europe. However, our results also reveal contrasting patterns for male and female genetic diversity in the European Neolithic, suggesting a system of patrilineal descent and patrilocal residential rules among the early farmers.     

Behavior of the ATP grasp domain of biotin carboxylase monomers and dimers studied using molecular dynamics simulations

The enzyme biotin carboxylase (BC) uses adenosine triphosphate (ATP) to carboxylate biotin and is involved in fatty acid synthesis. Structural evidence suggests that the B domain of BC undergoes a large hinge motion of ～45° when binding and releasing substrates. Escherichia coli BC can function as a natural homodimer and as a mutant monomer. Using molecular dynamics simulations, we evaluate the free energy profile along a closure angle of the B domain of E. coli BC for three cases: a monomer without bound Mg(2)ATP, a monomer with bound Mg(2)ATP, and a homodimer with bound Mg(2)ATP in one subunit. The simulation results show that a closed state is the most probable for the monomer with or without bound Mg(2)ATP. For the dimer with Mg(2)ATP in one of its subunits, communication between the two subunits was observed. Specifically, in the dimer, the opening of the subunit without Mg(2)ATP caused the other subunit to open, and hysteresis was observed upon reclosing it. The most stable state of the dimer is one in which the B domain of both subunits is closed; however, the open state for the B domain without Mg(2)ATP is only approximately 2k(B)T higher in free energy than the closed state. A simple diffusion model indicates that the mean times for opening and closing of the B domain in the monomer with and without Mg(2)ATP are much smaller than the overall reaction time, which is on the order of seconds.     

Mechanism of regulation of autoimmunity by iNKT cells

iNKT cells, CD1d dependent natural killer T cells are a unique population of T cells. The capacity of iNKT cells to produce regulatory cytokines first provided an indication of their regulatory potential. Later on, in experimental models as well as in patients afflicted with an auto-immune disease, such as Type 1 diabetes mellitus, multiple sclerosis, and systemic lupus erythematosus along with others, a deficit in iNKT cell number was observed, suggesting the role these cells may possibly have in the prevention of auto-immune diseases. More importantly, experimental strategies which focused on increasing the volume or stimulation of iNKT cells in laboratory animals, demonstrated an improved level of protection against the development of auto-immune diseases. This article reviews the mechanism of protection against autoimmunity by iNKT cells, discusses the obstacles against and indications for the potential use of iNKT cell manipulation in the treatment of human auto-immune diseases.     

Spatial scaling of arbuscular mycorrhizal fungal diversity is affected by farming practice

Evidence suggests that microbial communities show patterns of spatial scaling which can be driven by geographical distance and environmental heterogeneity. Here we demonstrate that human management can have a major impact on microbial distribution patterns at both the local and landscape scale. Mycorrhizal fungi are vital components of terrestrial ecosystems, forming a mutualistic symbiosis with plant roots which has a major impact on above ground ecology and productivity. We used contrasting agricultural systems to investigate the spatial scaling of the most widespread mycorrhizal fungus group, the arbuscular mycorrhizal fungi (AMF). Using multiple sampling sites with a maximum separation of 250 km we describe for the first time the roles which land management, environmental heterogeneity and geographical distance play in determining spatial patterns of microbial distribution. Analysis of AMF taxa–area relationships at each sampling site revealed that AMF diversity and spatial turnover was greater under organic relative to conventional farm management. At the regional scale (250 km) distance–decay analyses showed that there was significant change in AMF community composition with distance, and that this was greater under organic relative to conventional management. Environmental heterogeneity was found to be the major factor determining turnover of AMF taxa at the landscape scale. Overall we demonstrate that human management can play a key role in determining the turnover of microbial communities at both the local and regional scales.     

Stromal down-regulation of macrophage CD4/CCR5 expression and NF-κB activation mediates HIV-1 non-permissiveness in intestinal macrophages

Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.     

Grp78, Grp94, and Grp170 interact with alpha1-antitrypsin mutants that are retained in the endoplasmic reticulum

In alpha1-antitrypsin (alpha1-AT) deficiency, a mutant form of alpha1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant alpha1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The alpha1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which alpha1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant alpha1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of approximately 110-130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic alpha1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the alpha1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that approximately 85% of alpha1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and approximately 15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of alpha1-AT Z as well as on its residual secretion.