Molecular modeling of the domain structure of C9 of human complement by neutron and X-ray solution scattering.

C9 is the most abundant component of the membrane attack complex of the complement system of immune defense. This is a typical mosaic protein with thrombospondin (TSR) and low density lipoprotein receptor (LDLr) domains at its N-terminus and an epidermal growth factor-like (EGF) domain at its C-terminus. Between these lies a perforin-like sequence. In order to define the arrangement in solution of these four moieties in C9, high-flux neutron and synchrotron X-ray solution scattering studies were carried out. The neutron radius of gyration RG at infinite contrast is 3.33 nm, and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtained by synchrotron X-ray scattering after allowance for radiation effects. Stuhrmann analyses showed that the neutron radial inhomogeneity of scattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5) from the RXS data. These values are typical for soluble glycoproteins and show no evidence for the existence of any large hydrophobic surface patches on free C9 that might form contacts with lipids. Indirect transformation of the neutron and X-ray scattering curves into real space showed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and this suggests that the lengths of 7-8 nm deduced from previous electron microscopy studies in vacuo are underestimated. Molecular modeling of the C9 scattering curves utilized small spheres in the Debye equation, in which the analyses were constrained by the known volumes of the four moieties of C9 and the known sizes of the TSR and EGF-like domains. The most likely models for C9 suggest that these four regions of C9 are arranged in a V-shaped structure, with an angle of 10 degrees between the two arms, each of length 11.1 nm. This structure has a more hydrophobic character between the two arms. The scattering model is fully consistent with hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamic models could be developed for C6, C7, C8, and C9 of complement. Such a compact structure is atypical of other multidomain complement proteins so far studied by solution scattering and is fully compatible with mechanisms in which C9 is postulated, on activation, to undergo a drastic unfolding of its domain structure and to expose a more hydrophobic surface which can be embedded into lipid bilayers.     

Crystal structure at 1.5-A resolution of d(CGCICICG), an octanucleotide containing inosine, and its comparison with d(CGCG) and d(CGCGCG) structures.

The octadeoxyribonucleotide d(CGCICICG) has been crystallized in space group P(6)5(22) with unit cell dimensions of a = b = 31.0 A and c = 43.7 A, and X-ray diffraction data have been collected to 1.5-A resolution. Precession photographs and the self-Patterson function indicate that 12 base pairs of Z-conformation DNA stack along the c-axis, and the double helices pack in a hexagonal array similar to that seen in other crystals of Z-DNA. The structure has been solved by both Patterson deconvolution and molecular replacement methods and refined in space group P(6)5 to an R factor of 0.225 using 2503 unique reflections greater than 3.0 sigma (F). Comparison of the molecules within the hexagonal lattice with highly refined crystal structures of other Z-DNA reveals only minor conformational differences, most notably in the pucker of the deoxyribose of the purine residues. The DNA has multiple occupancy of C:I and C:G base pairs, and C:I base pairs adopt a conformation similar to that of C:G base pairs.     

The flavivirus envelope protein E: isolation of a soluble form from tick-borne encephalitis virus and its crystallization.

By the use of limited trypsin digestion of purified virions, we generated a membrane anchor-free and crystallizable form of the tick-borne encephalitis virus envelope glycoprotein E. It retained its reactivity with a panel of monoclonal antibodies, and only subtle structural differences from the native protein E were recognized. Treatment with the bifunctional cross-linker dimethylsuberimidate resulted in the formation of a dimer. Crystallization experiments yielded hexagonal rod-shaped crystals suitable for X-ray diffraction analysis.     

Iron (III) can be transferred between ferritin molecules.

The iron-storage molecule ferritin can sequester up to 4500 Fe atoms as the mineral ferrihydrite. The iron-core is gradually built up when FeII is added to apoferritin and allowed to oxidize. Here we present evidence, from M?ssbauer spectroscopic measurements, for the surprising result that iron atoms that are not incorporated into mature ferrihydrite particles, can be transferred between molecules. Experiments were done with both horse spleen ferritin and recombinant human ferritin. M?ssbauer spectroscopy responds only to 57Fe and not to 56Fe and can distinguish chemically different species of iron. In our experiments a small number of 57FeII atoms were added to two equivalent apoferritin solutions and allowed to oxidize (1-5 min or 6 h). Either ferritin containing a small iron-core composed of 56Fe, or an equal volume of NaCl solution, was added and the mixture frozen in liquid nitrogen to stop the reaction at a chosen time. Spectra of the ferritin solution to which only NaCl was added showed a mixture of species including 57FeIII in solitary and dinuclear sites. In the samples to which 150 56FeIII-ferritin had been added the spectra showed that all, or almost all, of the 57FeIII was in large clusters. In these solutions 57FeIII initially present as intermediate species must have migrated to molecules containing large clusters. Such migration must now be taken into account in any model of ferritin iron-core formation.     

Respiratory behavior in the pond snail Lymnaea stagnalis

Summary This study describes the neural basis of respiratory behavior in a pulmonate mollusc, Lymnaea stagnalis. We describe and identify muscles of the respiratory orifice (pneumostome) and mantle cavity as well as relevant motor neurons innervating these muscles. All of these identified motor neurons are active during spontaneously occurring respiratory behavior and a sporadically occurring synaptic input, termed Input 3, controls the activities of these motor neurons. This spontaneous input can also be recorded from isolated brain preparations, suggesting that the respiratory motor program is generated centrally. However, evidence is also presented that in semi-intact preparations the role of peripheral feedback is important for the initiation and termination of respiratory behavior in Lymnaea.     

Antimicrobial effect of fermented Ghanaian maize dough

Unhygienic conditions of a typical rural community in a developing country were simulated in the laboratory by inoculating fermented maize dough porridge with Shigella flexneri and enterotoxigenic Escherichia coli (ETEC). The antimicrobial effects of the different processes involved in the preparation of fermented maize dough porridge were assessed. The soaking process reduced the pH but no antimicrobial effect against shigella and ETEC was noted. Unfermented maize dough did not inhibit any of the test strains. When the fermentation process had become established, half of the strains tested were inhibited by the fermented maize dough when examined 8 h after inoculation. Cooking the fermented maize dough into porridge reduced the antimicrobial effect but there was still significant inhibition of pathogens. This suggests that the antimicrobial effect of fermented maize dough is not due to pH per se. Fermentation of maize dough appears to be a useful strategy for reducing contamination of weaning foods by Sh. flexneri and ETEC. The possible nature of the antimicrobial agent(s) produced during the fermentation of maize dough is discussed.     

Second serogroup of Legionella quinlivanii isolated from two unrelated sources in the United Kingdom.

A series of strains, presumptively identified as legionellas on the basis of their nutritional requirements and biochemical reactivity, were isolated from two unrelated environmental sources in the UK. Representatives of each of these series had a restriction endonuclease digest pattern indistinguishable from that of the Legionella quinlivanii type strain (1442-AUS-E) and the identity of these strains was confirmed by DNA homology studies. Serological examination of the two strains showed that they were distinct from the type strain 1442-AUS-E but indistinguishable from each other. A second serogroup, L. quinlivanii serogroup 2 (type strain LC870; NCTC 12434), is proposed to accommodate these strains.     

Mucosubstance histochemistry of pleomorphic adenoma of parotid and submandibular salivary glands of man: light and electron microscopy

Summary Lumina and adluminal cells in human salivary gland pleomorphic adenomas were found to contain neutral, carboxylated, and occasionally sulphated glycoproteins. A variable component of luminal contents and secretory granules did not appear to contain glycoprotein and possibly consisted of protein. Glycosaminoglycans, which appeared to be hyaluronic acid and chondroitin sulphate, were demonstrated rarely in lumina, often between epithelial cells, and forming the matrix of myxoid tissue and, together with collagen, chondroid tissue. No differences were seen between tumours from parotid glands and those from submandibular glands. Glycoproteins demonstrated in the epithelium are similar to those of intercalary ducts of parotid and submandibular glands, and may represent a primitive form of salivary secretion. Glycosaminoglycans secreted intercellularly by epithelial cells cause their increasing separation to form myxoid or chondroid tissue. This stromalization extends to lumina to produce a loss of epithelium. Pleomorphic adenoma appears to be a manifest example of variable derepression of the genotype.     

B lymphocyte activation is accompanied by phosphorylation of a 72-kDa protein-tyrosine kinase

The cross-linking of membrane IgM on the surface of splenic B lymphocytes or WEHI 231 cells leads to the rapid phosphorylation on tyrosine of a 72-kDa protein as detected in Western blotting experiments using anti-phosphotyrosine antibodies. The 72-kDa phosphoprotein detected in this manner comigrates, in both one- and two-dimensional polyacrylamide gel electrophoresis systems, with PTK72, a 72-kDa protein-tyrosine kinase characterized previously in this laboratory (Zioncheck, T. F., Harrison, M. L., Isaacson, C. C., and Geahlen, R. L. (1988) J. Biol. Chem. 263, 19195-19202). Anti-phosphotyrosine antibodies and anti-PTK72 antibodies immunoprecipitate the same protein-tyrosine kinase from extracts of anti-IgM-activated cells as determined by immune complex kinase assays and one-dimensional phosphopeptide mapping. These results indicate that the tyrosine phosphorylation of a 72-kDa protein-tyrosine kinase is an early event in the activation of B lymphocytes via the antigen receptor.