Expression of HGF/SF,HGFI/MSP,and c-met suggests new functions during early chick development

We report the cloning of full-length cDNAs for a plasminogen-related growth factor, hepatocyte growth factor/scatter factor (HGF/SF), its tyrosine kinase receptor, c-met, and a close member of the same family, hepatocyte growth factor-like/macrophage stimulating protein (HGFI/MSP), from the chick. We have used these cDNAs to provide the first report of the expression of this family of growth factors and the c-met receptor at early stages of vertebrate development. RNAase protection and wholemount in situ hyb ridization were used on chick embryos between formation of the primitive streak and early organogenesis. We find patterns of expression for HGF/SF and its receptor c-met consistent with their known roles in ep ithelial-mesenchymal transformation and angiogenesis. In addition, these genes and HGFI/MSP are expressed in discrete locations within developing somites, suggesting a role in paraxial mesodermal development. Very strong and early expression of HGF/SF in the elevating limb buds suggests its involvement in limb outgrowth. HGFI/MSP is expressed in the notochord and then in the prospective floor plate region and could play a role in development of the neural tube. Interestingly, c-met is often more closely as sociated with HGFI/MSP than with its known ligand, HGF/SF, raising the possibility that c-met expression may be induced by HGFI/MSP. © 1995 Wiley-Liss, Inc.     

Addition of allochthonous fungi to a historically contaminated soil affects both remediation efficiency and bacterial diversity

Botryosphaeria rhodina DABAC P82 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow and to degrade aromatic hydrocarbons in an aged contaminated soil. To evaluate the impact of indigenous microflora on the overall process, incubations were performed on both fumigated and nonfumigated soils. Fungal colonization by B. rhodina was unexpectedly lower in the fumigated than in the nonfumigated soil while the growth of P. pulmonarius showed an opposite response. Degradation performances and detoxification by both fungi in the nonfumigated soil were markedly higher than those observed in the fumigated one. Heterotrophic bacterial counts in nonfumigated soil augmented with either B. rhodina or P. pulmonarius were significantly higher than those of the corresponding incubation control (6.7 ± 0.3 × 10⁸ and 8.35 ± 0.6 × 10⁸, respectively, vs 9.2 ± 0.3 × 10⁷). Bacterial communities of both incubation controls and fungal-augmented soil were compared by numerical analysis of denaturing gradient gel electrophoresis profiles of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA (rRNA) genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Besides increasing overall diversity, fungal augmentation led to considerable qualitative differences with respect to the pristine soil.     

Entwicklungsmechanische Studien

Schlußfolgerung Wir prüften die wichtigsten Möglichkeiten, mit deren Hilfe man künstlich auf die Eier einwirken kann, um die Richtung der Furchungsebenen abzuändern, und kamen zu dem Schlusse, daß die bisher ausgeführten Ergebnisse mit unseren theoretischen vollständig übereinstimmten. Wir können also behaupten, daß unsere Erklärung der Zellteilung dasExperimentum crucis siegreich bestand.Wir glauben, damit die Zellteilung wissenschaftlich und positiv erörtert zu haben, welche an sich eine sehr einfache Erscheinung ist und deren Ursachen nicht in innewohnenden mysteriösen Kräften zu suchen sind, sondern einfach in den mechanischen Bedingungen, welchen das Ei und die Blastomeren sukzessiv ausgesetzt sind. In der Veränderung dieser und in der Abschätzung ihrer wirklichen Werte liegt die Komplikation und die Schwierigkeit der Probleme, welche die Natur uns aufgibt, nicht aber im innersten Wesen der Zellteilung.Dem Forscher können sich noch verschiedene andere Probleme darbieten; wir hoffen aber, daß es in dieser Arbeit gelungen ist, die Grundlagen zu formulieren, welche ihre Lösung ohne Schwierigkeiten ermöglichen.Im VI. Teile werden wir die ontogenetische Entwicklung vom biologischen Standpunkte behandeln und hoffen beweisen zu können, daß zwar das Ei mechanischen Gesetzen folgt, die Entwicklungsmöglichkeiten aber letzten Endes davon unabhängig sind.Dies vollbringen wir, wie bisher, ohne spezielle Hypothesen oder Annahme von mehr oder weniger mysteriösen Kräften. Unser Zweck ist bloß eine wissenschaftliche und rationelle Erklärung der grundlegenden biologischen Erscheinungen zu finden, ohne solche Prinzipien zu Hilfe zu nehmen, welche bei den Manifestationen anderer Körper unbekannt sind.     

Oxidative stress in subjects affected by celiac disease

In order to study the role of oxidative stress in celiac disease, protein carbonyl groups, thiobarbituric acid-reactive substance and pentosidine were evaluated in the plasma of nine patients with asymptomatic celiac disease and in a control group (n = 25). Plasma alpha-tocopherol, retinol and lipids were determined in the same samples. The levels of markers of oxidative stress derived from both protein (carbonyl groups) and lipids (thiobarbituric acid-reactive substances) were significantly higher in celiac disease patients, whereas lipoproteins and alpha-tocopherol were significantly lower. These data indicate that in celiac disease, even when asymptomatic, a redox imbalance persists; this is probably caused by an absorption deficiency, even if slight. Dietary supplementation with antioxidant molecules may offer some benefit and deserves further investigation.     

Human cytomegalovirus regulates surface expression of the viral protein UL18 by means of two motifs present in the cytoplasmic tail

UL18 is a trans-membrane viral protein expressed on human cytomegalovirus (HCMV)-infected cells, and its surface expression determines the interaction of infected cells with lymphocytes expressing the CD85j (LIR-1/ILT2) receptor. We previously showed that the UL18-CD85j interaction elicits activation of T lymphocytes. However, in in vitro cell models UL18 displays mostly undetectable surface expression. Thus, we asked how surface expression of UL18 is regulated. Domain-swapping experiments and construction of specific mutants demonstrated that two motifs on its cytoplasmic tail, homologous to YXXPhi and KKXX consensus sequences, respectively, are responsible for impairing UL18 surface expression. However, the presence of the whole HCMV genome, granted by HCMV infection of human fibroblasts, restored surface expression of either UL18 or chimeric proteins carrying the UL18 cytoplasmic tail, starting from the third day after infection. It is of note that the two motifs responsible for cytoplasmic retention are identical in all 17 HCMV strains examined. We disclosed a control mechanism used by the HCMV to regulate the availability of UL18 on the infected-cell surface to allow interaction with its ligand on T and NK cells.     

Modelling of full-length human alpha4beta2 nicotinic receptor by fragmental approach and analysis of its binding modes

The objective of the study was to generate a full-length model for the heteropentameric structure of human α4β2 nicotinic receptor. The monomers structure was derived using a fragmental approach and the pentamer was assembled by protein-protein docking. The reliability of the model was assessed docking a representative set of known nicotinic ligands. Docking results unveiled that the ligand affinity depends on key interactions that the ligand’s charged moiety realizes with conserved apolar residues of α4 monomer, whereas the H-bond acceptor group interacts with a less conserved and more heterogeneous subpocket, involving polar residues of β2 subunit. The consistency of docking results and the agreement with the experimental data afford an encouraging validation for the proposed model and emphasize the soundness of such a fragmental approach to model any transmembrane protein.     

Engineering the NK1 fragment of hepatocyte growth factor/scatter factor as a MET receptor antagonist

The growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor MET, the tyrosine kinase encoded by the c-MET proto-oncogene, exert major roles in cancer invasion and metastasis and are key targets for therapy. NK1 is an alternative spliced variant of HGF/SF that consists of the N-terminal (N) and first kringle (K1) domains and has partial agonistic activity. NK1 crystallises as a head-to-tail dimer with an extensive inter-protomeric interface resulting from contacts between the two short interdomain linkers and reciprocal contacts between the N and K1 domains. Here we show that a subset of mutants at the NK1 dimer interface, such as the linker mutants Y124A or N127A or the kringle mutant V140A:I142A, bind the MET receptor with affinities comparable to wild-type NK1 but fail to assemble a dimeric, signalling competent NK1-MET complex. These NK1 variants have no detectable agonistic activity on, behave as bona fide receptor antagonists by blocking cell migration and DNA synthesis in target cells and have strong prospects as therapeutics for human cancer.     

Interactions of hepatocyte growth factor/scatter factor with various glycosaminoglycans reveal an important interplay between the presence of iduronate and sulfate density

Hepatocyte growth factor/scatter factor (HGF/SF) has a cofactor requirement for heparan sulfate (HS) and dermatan sulfate (DS) in the optimal activation of its signaling receptor MET. However, these two glycosaminoglycans (GAGs) have different sugar backbones and sulfation patterns, with only the presence of iduronate in common. The structural basis for GAG recognition and activation is thus very unclear. We have clarified this by testing a wide array of natural and modified GAGs for both protein binding and activation. Comparisons between Ascidia nigra (2,6-O-sulfated) and mammalian (mainly 4-O-sulfated) DS species, as well as between a panel of specifically desulfated heparins, revealed that no specific sulfate isomer, in either GAG, is vital for interaction and activity. Moreover, different GAGs of similar sulfate density had comparable properties, although affinity and potency notably increase with increasing sulfate density. The weaker interaction with CS-E, compared with DS, shows that GlcA-containing polymers can bind, if highly sulfated, but emphasizes the importance of the flexible IdoA ring. Our data indicate that the preferred binding sites in DS in vivo will be comprised of disulfated, IdoA(2S)-containing motifs. In HS, clustering of N-/2-O-/6-O-sulfation in S-domains will lead to strong reactivity, although binding can also be mediated by the transition zones where sulfates are mainly at the N- and 6-O- positions. GAG recognition of HGF/SF thus appears to be primarily driven by electrostatic interactions and exhibits an interesting interplay between requirements for iduronate and sulfate density that may reflect in part a preference for particular sugar chain conformations.