Demonstration of growth in porcine thyroid cell culture

Eagle's minimum essential medium supplemented with 20 per cent newborn calf serum (N.C.S.) allows porcine thyroid cell survival but not cell growth in vitro. In NCTC 109 medium supplemented with 20 per cent N.C.S. these cells actively grow and may be serially propagated. Cell population doubling time expressed as DNA doubling value is 3.5 days at 37 degrees C in 95 per cent air-5 per cent CO2. Thyrotropin does not affect porcine thyroid cell multiplication in vitro but stimulates the plating efficiency in primary cultures to about 130 per cent of controls. Cell selection was obtained by replacing media with Earle's balanced salt solution. This operation provoked death of nearly all cells by day 18 but subsequent addition of growth medium resulted in proliferation of epithelial cell clones. From generation 2 to generation 8, cells produce thyroglobulin but they do not actively trap iodide nor form follicles when thyrotropin is added to the media. Cell selection, demonstration of growth, as well as freeze-storage techniques described in this paper permit selection and storage of porcine thyroid cells and the potential constitution of cell collections.     

Neuron firing in driven nonlinear integrate-and-fire models

Statistical properties of neuron firing are studied in the framework of a nonlinear leaky integrate-and-fire model that is driven by a slow periodic subthreshold signal. The firing events are characterized by first passage time densities. The experimentally better accessible interspike interval density generally depends on the sojourn times in a refractory state of the neuron. This aspect is not part of the integrate-and-fire model and must be modelled additionally. For a large class of refractory dynamics, a general expression for the interspike interval density is given and further evaluated for the two cases with an instantaneous resetting (i.e. no refractory state) and a refractory state possessing a deterministic lifetime. First passage time densities and interspike interval densities following from the proposed theory compare favorably with precise numerical simulations.     

Antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, Halocynthia aurantium

Halocidin is an antimicrobial peptide isolated from the hemocytes of the tunicate. Among the several known synthetic halocidin analogues, di-K19Hc has been previously confirmed to have the most profound antibacterial activity against antibiotic-resistant bacteria. This peptide has been considered to be an effective candidate for the development of a new type of antibiotic. In this study, we have assessed the antifungal activity of di-K19Hc, against a panel of fungi including several strains of Aspergillus and Candida. As a result, we determined that the MICs of di-K19Hc against six Candida albicans and two Aspergillus species were below 4 and 16 microg/ml, respectively, thereby indicating that di-K19Hc may be appropriate for the treatment of several fungal diseases. We also conducted an investigation into di-K19Hc's mode of action against Candida albicans. Our colony count assay showed that di-K19Hc killed C. albicans within 30s. Di-K19Hc bound to the surface of C. albicans via a specific interaction with beta-1,3-glucan, which is one of fungal cell wall components. Di-K19Hc also induced the formation of ion channels within the membrane of C. albicans, and eventually observed cell death, which was confirmed via measurements of the K+ released from C. albicans cells which had been treated with di-K19Hc, as well as by monitoring of the uptake of propidium iodide into the C. albicans cells. This membrane-attacking quality of di-K19Hc was also visualized via confocal laser and scanning electron microscopy.     

B型肉毒毒素重链C端基因的表达与保护作用的初步研究

肉毒毒素是肉毒梭状杆菌产生的外毒素,有7种血清型(A～G),是目前已知的毒性最强的细菌蛋白质。人类肉毒中毒主要是由A、B和E型引起。本研究克隆了B型肉毒毒素重链C端片段(Hc),在大肠杆菌中进行了诱导表达,并用Ni离子亲和柱进行了纯化。Hc蛋白免疫后的BALB/c小鼠可以抵抗6×103LD50B型肉毒毒素攻击。     

Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths

A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5–6 months old Assaf lambs using Al(OH)₃ or Escherichia coli lipopolysaccharide + inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%–85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs.     

Multi-state epidemic processes on complex networks

Infectious diseases are practically represented by models with multiple states and complex transition rules corresponding to, for example, birth, death, infection, recovery, disease progression, and quarantine. In addition, networks underlying infection events are often much more complex than described by meanfield equations or regular lattices. In models with simple transition rules such as the SIS and SIR models, heterogeneous contact rates are known to decrease epidemic thresholds. We analyse steady states of various multi-state disease propagation models with heterogeneous contact rates. In many models, heterogeneity simply decreases epidemic thresholds. However, in models with competing pathogens and mutation, coexistence of different pathogens for small infection rates requires network-independent conditions in addition to heterogeneity in contact rates. Furthermore, models without spontaneous neighbor-independent state transitions, such as cyclically competing species, do not show heterogeneity effects.     

Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-Å X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.     

Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C–D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (∼two-third) and BoNT/D (∼one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.     

Synchrotron SAXS studies on the structural stability of Carcinus aestuarii hemocyanin in solution

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.