The structural basis of receptor-binding by Escherichia coli associated with diarrhea and septicemia

GafD in Escherichia coli G (F17) fimbriae is associated with diarrheal disease, and the structure of the ligand-binding domain, GafD1-178, has been determined at 1.7A resolution in the presence of the receptor sugar N-acetyl-D-glucosamine. The overall fold is a beta-barrel jelly-roll fold. The ligand-binding site was identified and localized to the side of the molecule. Receptor binding is mediated by side-chain as well main-chain interactions. Ala43-Asn44, Ser116-Thr117 form the sugar acetamide specificity pocket, while Asp88 confers tight binding and Trp109 appears to position the ligand. There is a disulfide bond that rigidifies the acetamide specificity pocket. The three fimbrial lectins, GafD, FimH and PapG share similar beta-barrel folds but display different ligand-binding regions and disulfide-bond patterns. We suggest an evolutionary path for the evolution of the very diverse fimbrial lectins from a common ancestral fold.     

Activation of TGF-beta by Leishmania chagasi: importance for parasite survival in macrophages

TGF-beta is a potent regulatory cytokine that suppresses expression of inducible NO synthase and IFN-gamma, and suppresses Th1 and Th2 cell development. We examined whether functionally active TGF-beta is present in the local environment surrounding the invading protozoan Leishmania chagasi. Our prior data showed that TGF-beta levels are significantly increased in L. chagasi-infected mice. In the current study, we found TGF-beta was also abundant in bone marrows of humans with acute visceral leishmaniasis but not in those of uninfected controls. Furthermore, L. chagasi infection caused an increase in biologically active TGF-beta in human macrophage cultures without changing the total TGF-beta. Therefore, we investigated the means through which leishmania could augment activated but not total TGF-beta. Incubation of latent TGF-beta with Leishmania sp. promastigotes caused active TGF-beta to be released from the latent complex. In contrast, the nonpathogenic protozoan Crithidia fasciculata could not activate TGF-beta. TGF-beta activation by leishmania was prevented by inhibitors of cysteine proteases and by the specific cathepsin B inhibitor CA074. Physiologic concentrations of TGF-beta inhibited killing of intracellular L. chagasi in macrophages, although the phagocytosis-induced respiratory burst remained intact. In contrast, supraphysiologic concentrations of TGF-beta had no effect on parasite survival. We hypothesize that the combined effect of abundant TGF-beta stores at extracellular sites during infection, and the ability of the parasite to activate TGF-beta in its local environment, leads to high levels of active TGF-beta in the vicinity of the infected macrophage. Locally activated TGF-beta could, in turn, enhance parasite survival through its effects on innate and adaptive immune responses.     

Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite

AMP-activated kinase (AMPK) is a fuel-sensing enzyme present in most mammalian tissue. In response to a decrease in the energy state of a cell AMPK is phosphorylated and activated by still poorly characterized upstream events. Exposure of bovine aortic endothelial cells (BAEC) to chemically synthesized ONOO- acutely and significantly increased phosphorylation of c-Src, PDK1, AMPK, and its downstream target, acetyl-CoA carboxylase (ACC), without affecting cellular AMP. This novel pathway for AMPK activation was confirmed by the use of pharmacological inhibitors and dominant-negative mutants. Exposure of BAEC to hypoxia-reoxygenation (H/R) caused a biphasic increase in AMPK and ACC phosphorylation, which was prevented by adenoviral overexpression of superoxide dismutase (SOD) or inhibition of nitric-oxide synthase (NOS) implicating a role of ONOO- formed during H/R. Furthermore, dominant-negative mutants of c-Src or kinase-defective PDK1 also blocked H/R-induced AMPK activation indicating that, as with addition of exogenous ONOO-, both c-Src and PI 3-kinase are upstream of AMPK. Moreover, H/R, like ONOO-, significantly increased co-immunoprecipitation of AMPK with c-Src, suggesting that ONOO- favors physical association of AMPK with upstream kinases. Taken together, our results indicate a novel pathway by which H/R via ONOO- activates AMPK in a c-Src-mediated, PI 3-kinase-dependent manner, and suggest that ONOO--induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC.     

Another fossil agaric from Dominican amber

We report the discovery of a fossil agaricoid homobasidiomycete from Dominican amber (ca 15-20 Ma). Aureofungus yaniguaensis appears to be a member of the euagarics clade, but its precise taxonomic placement is obscure. This is the fourth known fossil agaric and the third from Dominican amber.     

Mechanism of inhibition of skeletal muscle actomyosin by N-benzyl-p-toluenesulfonamide

N-Benzyl-p-toluenesulfonamide (BTS) is a small organic molecule that specifically inhibits the contraction of fast skeletal muscle fibers. To determine the mechanism of inhibition by BTS, we performed a kinetic analysis of its effects on the elementary steps of the actomyosin subfragment-1 ATPase cycle. BTS decreases the steady-state acto-S1 ATPase rate approximately 10-fold and increases the actin concentration for half-maximal activation. BTS primarily affects three of the elementary steps of the reaction pathway. It decreases the rate of P(i) release >20-fold in the absence of actin and >100-fold in the presence of actin. It decreases the rate of S1.ADP dissociation from 3.9 to 0.8 s(-)(1) while decreasing the S1.ADP dissociation constant from 2.3 to 0.8 microM. BTS weakens the apparent affinity of S1.ADP for actin, increasing the K(d) from 7.0 to 29.5 microM. ATP binding to S1, hydrolysis, and the affinity of nucleotide-free S1 for actin are unaffected by BTS. Kinetic modeling indicates that the binding of BTS to myosin depends on actin association/dissociation and on nucleotide state. Our results suggest that the reduction of the acto-S1 ATPase rate is due to the inhibition of P(i) release, and the suppression of tension is due to inhibition of P(i) release in conjunction with the decreased apparent affinity of S1.ADP.P(i) and S1.ADP for actin.     

Structure and function of a vimentin-associated matrix adhesion in endothelial cells

The alpha4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the alpha4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by alphav and beta3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the alpha4 laminin subunit G domain in an alphavbeta3-integrin-dependent manner. The alphavbeta3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the alphavbeta3 integrin-positive focal contacts. We have investigated the function of alpha4-laminin and alphavbeta3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.     

Expression and glycosylation studies of human FGF receptor 4

Fibroblast growth factor receptor subtype 4 (FGFR4) has been shown to have special activation properties and just one splicing form, unlike the other FGFRs. FGFR4 overexpression is correlated with breast cancer and therefore FGFR4 is a target for drug design. Our aim is to overexpress high amounts of homogeneous FGFR4 extracellular domain (FGFR4(ed)) for structural studies. We show that baculovirus-insect cell-expressed FGFR4(ed) is glycosylated on three (N88, N234, and N266) of the six possible N-glycosylation sites but is not O-glycosylated. The deglycosylated triple mutant was expressed and had binding properties similar to those of glycosylated FGFR4(ed), but was still heterogeneous. Large amounts of FGFR4(ed) have been produced into inclusion bodies in Escherichia coli and refolded at least partly correctly but the refolded E. coli-produced FGFR4(ed) still aggregates.     

Thermal and Bioenergetics of Elasmobranchs: Bridging the Gap

Physiological telemetry is a powerful tool in studying the thermal biology and energetics of elasmobranchs in the laboratory and field. Controlled laboratory studies have increased our understanding of the physiology and behavior of many elasmobranchs, but have focused primarily on small, slow moving species. Extrapolating results from these laboratory studies to free-swimming animals in the field or to other unstudied species may be problematic, due to laboratory constraints or species specific differences. Some elasmobranchs are too large or logistically difficult to maintain in captivity, making them extremely difficult to study in the laboratory, and thus can only be studied in the field. Physiological telemetry offers a bridge between the laboratory and the field providing an opportunity to elucidate similarities and differences. Previous studies have coupled a variety of sensors with ultrasonic transmitters to relay information on epaxial muscle and stomach temperatures of free-swimming lamnid sharks. Even though these studies indicate lamnids exhibit elevated body temperatures, the degree to which these sharks may control body temperature is still not fully understood. Telemetry of heart rate, swimming speed, muscle contraction rate, and tail beat frequency has been used to estimate energy consumption of free-swimming elasmobranchs with varying success. Based on recent advances in technology, several hypotheses regarding thermoregulation, cardiac output, and obligate ram ventilation are discussed. Although many telemetry studies have been restricted by logistical difficulties in conducting long-term tracks, recent developments such as acoustic modems, underwater listening stations and satellite telemetry may significantly increase the amount and types of physiological data that can be collected. These improvements in technology and captive animal husbandry techniques will help to bridge the gap between the laboratory and the field.     

Perineal muscles and motoneurons are sexually monomorphic in the naked mole-rat (Heterocephalus glaber)

Naked mole-rats are eusocial mammals that live in colonies with a single breeding female and one to three breeding males. All other members of the colony, known as subordinates, are nonreproductive and exhibit few sex differences in behavior or genital anatomy. This raises questions about the degree of sexual differentiation in subordinate naked mole-rats. The striated perineal muscles associated with the phallus [the bulbocavernosus (BC), ischiocavernosus (IC), and levator ani (LA) muscles], and their innervating motoneurons, are sexually dimorphic in all rodents examined to date. We therefore asked whether perineal muscles and motoneurons were also sexually dimorphic in subordinate naked mole-rats. Muscles similar to the LA and IC of other rodents were found in naked mole-rats of both sexes. No clear BC muscle was identified, although a large striated muscle associated with the urethra in male and female naked mole-rats may be homologous to the BC of other rodents. There were no sex differences in the volumes of the LA, IC, or the urethral muscles. Motoneurons innervating the perineal muscles were identified by retrograde labeling with cholera-toxin-conjugated horseradish peroxidase. All perineal motoneurons were found in a single cluster in the ventrolateral lateral horn, in a position similar to that of Onuf's nucleus of carnivores and primates. There was no sex difference in the size or number of motoneurons in Onuf's nucleus of naked mole-rats. Thus, unlike findings in any other mammal, neither the perineal muscles nor the perineal motoneurons appear to be sexually differentiated in subordinate naked mole-rats.