ESCRT-0 assembles as a heterotetrameric complex on membranes and binds multiple ubiquitinylated cargoes simultaneously

The ESCRT machinery consists of multiple protein complexes that collectively participate in the biogenesis of multivesicular endosomes (MVEs). The ESCRT-0 complex is composed of two subunits, Hrs and STAM, both of which can engage ubiquitinylated substrates destined for lysosomal degradation. Here, we conduct a comprehensive analysis of ESCRT-0:ubiquitin interactions using isothermal titration calorimetry and define the affinity of each ubiquitin-binding domain (UBD) within the intact ESCRT-0 complex. Our data demonstrate that ubiquitin binding is non-cooperative between the ESCRT-0 UBDs. Additionally, our findings show that the affinity of the Hrs double ubiquitin interacting motif (DUIM) for ubiquitin is more than 2-fold greater than that of UBDs found in STAM, suggesting that Hrs functions as the major ubiquitin-binding protein in ESCRT-0. In vivo, Hrs and STAM localize to endosomal membranes. To study recombinant ESCRT-0 assembly on lipid bilayers, we used atomic force microscopy. Our data show that ESCRT-0 forms mostly heterodimers and heterotetramers of Hrs and STAM when analyzed in the presence of membranes. Consistent with these findings, hydrodynamic analysis of endogenous ESCRT-0 indicates that it exists largely as a heterotetrameric complex of its two subunits. Based on these data, we present a revised model for ESCRT-0 function in cargo recruitment and concentration at the endosome.     

Specific Initiation Site for Simian Virus 40 Deoxyribonucleic Acid Replication

Replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) molecules have been isolated under conditions in which the newly synthesized DNA is uniformly labeled with (3)H-thymidine. These newly synthesized strands are released from the replicative intermediate molecules by alkaline treatment, and it has been possible to isolate single-stranded SV40 DNA which varies in size from 157,000 daltons (from molecules that are 10% replicated) to 1,360,000 daltons (85% replicated). The rates of duplex formation of newly synthesized DNA have been used to relate their genetic complexity to the extent of DNA replication. As DNA replication proceeds, the time required to effect 50% renaturation of the newly synthesized DNA increases at a proportional rate. The data establish that DNA replication is not initiated at random, but rather that there is a single specific initiation site for DNA replication.     

Implication of C-Terminal Deletion on the Structure and Stability of Bovine β-casein

Bovine β-casein (β-CN) with its C-terminal truncated by chymosin digestion, β-CN-(f1-192), was examined and characterized using circular dichroism (CD) under various temperature conditions. CONTIN/LL analysis of the CD data revealed significant secondary structure disruption in β-CN-(f1-192) relative to its parent protein,β-CN, in the temperature range (5° to 70°C) studied. Near-UV CD spectra indicated significant temperature dependent structural changes. Analytical ultracentrifugation results showed significant reduction but not complete abolishment of self-association in β-CN-(f1-192) compared to whole β-casein at 2°–37°C. Furthermore, binding experiments with the common hydrophobic probe – 8-anilino-1- naphthalene sulfonic acid (ANS) illustrated that β-CN-(f1-192) is nearly incapable of binding to ANS relative to whole β-CN, suggesting a nearly complete open overall tertiary structure brought about by the C-terminal truncation. It has been demonstrated clearly that the tail peptide β-CN-(f193-209) is important in maintaining the hydrophobic core of β-CN but the residual association observed argues for a minor role for other sites as well.     

Presumptive lymph node organizers are differentially represented in developing mesenteric and peripheral nodes

During murine embryogenesis, the formation of Peyer's patches (PPs) is initiated by CD45(+)CD4(+)CD3(-) lymphoid tissue inducers that trigger adhesion molecule expression and specific chemokine production from an organizing stromal cell population through ligation of the lymphotoxin-beta receptor. However, the steps involved in the development of lymph nodes (LNs) are less clear than those of PPs, and the characteristics of the organizing cells within the LN anlagen have yet to be documented. In this study, we show for the first time that the early anlage is bordered by an endothelial layer that retains a mixed lymphatic and blood vascular phenotype up to embryonic day 16.5. This in turn encompasses CD45(+)CD4(+)CD3(-) cells interspersed with ICAM-1/VCAM-1/mucosal addressin cell adhesion molecule-1, lymphotoxin-beta receptor-positive, chemokine-producing cells analogous to the organizing population previously observed in PPs. Moreover, these LN organizers also express the TNF family member, TRANCE. Lastly, we show that the ICAM-1/VCAM-1/mucosal addressin cell adhesion molecule-1 cells present in peripheral and mesenteric LN form two discrete populations expressing either intermediate or high levels of these adhesion molecules but that the former population is specifically reduced in PLN. These findings provide a possible explanation for the well-known differences in developmental requirements for nodes at peripheral or mesenteric locations.     

Attraction of the amphipod <Emphasis Type="Italic">Gammarus pulex</Emphasis> to water-borne cues of food

We examined the ability of the amphipod Gammarus pulexto detect chemical cues released from potential food sources. Therefore, response of G. pulex to chemical cues from food was tested in paired-choice laboratory experiments. Comparisons were made between artificial and natural leaves, with and without the importance of aufwuchs, and with different components of the aufwuchs community. Our study demonstrated that G. pulex actively chose its food and that G. pulex is most strongly attracted to the aufwuchs on discs rather than to the leaf itself. Fungi and bacteria are more important in the food selection process than algae probably because fungal and bacterial cues are more specific cues for decaying leaves than algal cues, since algae also grow on mineral substrates and then do not contribute to leaf decomposition.     

Non-specific effects of 4-chloro-m-cresol may cause calcium flux and respiratory burst in human neutrophils

We examined the effects of 4-chloro-m-cresol (4-CmC, a potent and specific activator of ryanodine receptors) on Ca(2+)-release/influx and respiratory burst in freshly isolated human PMN as well as HL60 cells. 4-CmC induces Ca(2+) store-depletion in a dose-dependent manner at concentrations between 400muM and 3mM, however no dose-dependent effect on Ca(2+)-influx was found. 4-CmC depleted Ca(2+) stores that were shared with the GPC agonists such as fMLP and PAF, and therefore 4-CmC presumably depletes Ca(2+) from ER. Since the authentic ligand for RyR is cyclic ADP-ribose (cADPR), we assessed the functional relevance of RyR in PMN by studying the presence and function of membrane-bound ADP-ribosyl cyclase (CD38) in PMN. First, expression of CD38 was confirmed by RT-PCR using cDNA from HL60 cells. Second, PMN from trauma patients showed significantly enhanced CD38 expression than those from healthy volunteers. In addition, although no chemotaxis effect was detected by 4-CmC, it stimulated respiratory burst in PMN in a dose-dependent manner. Our findings suggest that RyRs exist in human PMN and that RyR pathway may play an active role in inflammatory PMN calcium signaling. 8-Br-cADPR and cyclic 3-deaza-ADP did not have inhibitory effects either on 4-CmC-induced Ca(2+) store-depletion or on respiratory burst, on the other hand, PLC inhibitor, U73122, completely attenuated both 4-CmC-induced Ca(2+) store-depletion and respiratory burst. Although it has been used as a specific activator of RyR, 4-CmC has non-specific effects which cause Ca(2+) store-depletion and respiratory burst at least in human PMN.     

Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin

Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation. In many multidomain proteins these conformational changes involve reorientation of domains within the protein. Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent. The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond (15)N-H(N), (15)N-(13)C', (13)C(alpha)-(13)C', two-bond (13)C'-H(N), and three-bond (13)C(alpha)-H(N) dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings. Consistent structures have been derived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/-1) degrees domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5 degrees uncertainty in orientation of internuclear vectors within domains). In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein.     

A DNA polymorphism influencing α(1,2)fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18 adhesion

The alpha(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions. In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine. FUT1 polymorphisms influence adhesion of F18 fimbriated Escherichia coli (ECF18) to intestinal mucosa, and FUT2 is associated with expression of erythrocyte antigen 0. The FUT1 polymorphisms result in amino acid substitutions at positions 103 (Ala-->Thr) and 286 (Arg-->Glu). Tightly controlled expression of the FUT2 gene results in either an abundance or an absence of mRNA in small intestinal mucosa. ECF18-resistant animals were shown to be homozygous for threonine at amino acid 103 of the FUT1 enzyme. Susceptibility to ECF18 adhesion appeared to be solely dependent on the activity of FUT1 in intestinal epithelia. In intestinal mucosae of ECF18-resistant pigs which expressed FUT1 but not FUT2 RNA, the levels of alpha(1,2)fucosyltransferase activity were significantly lower (28- to 45-fold, P<0.001) than in susceptible pigs. Moreover, lysates of CHO cells transfected with FUT1 constructs encoding threonine at amino acid position 103 also showed significantly reduced enzyme activity compared with constructs encoding alanine at this position. Our genetic and enzymatic studies support the hypothesis that the FUT1 enzyme, and particularly the amino acid at position 103, is likely important in the synthesis of a structure that enables adhesion of ECF18 bacteria to small intestinal mucosa.